Novel human ion channel and transporter family members

BACKGROUND OF THE 52906, 33408, AND 12189 INVENTION

[0002] Potassium (K+) channels are ubiquitous proteins which are involved in the setting of the resting membrane potential as well as in the modulation of the electrical activity of cells. In excitable cells, K+ channels influence action potential waveforms, firing frequency, and neurotransmitter secretion (Rudy, B. (1988) Neuroscience, 25, 729-749; Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd Ed.). In non-excitable cells, they are involved in hormone secretion, cell volume regulation and potentially in cell proliferation and differentiation (Lewis et al. (1995) Annu. Rev. Immunol., 13, 623-653). Developments in electrophysiology have allowed the identification and the characterization of an astonishing variety of K+ channels that differ in their biophysical properties, pharmacology, regulation and tissue distribution (Rudy, B. (1988) Neuroscience, 25, 729-749; Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd Ed.). More recently, cloning efforts have shed considerable light on the mechanisms that determine this functional diversity. Furthermore, analyses of structure-function relationships have provided an important set of data concerning the molecular basis of the biophysical properties (selectivity, gating, assembly) and the pharmacological properties of cloned K+ channels.

[0003] Functional diversity of K+ channels arises mainly from the existence of a great number of genes coding for pore-forming subunits, as well as for other associated regulatory subunits. Two main structural families of pore-forming subunits have been identified. The first one consists of subunits with a conserved hydrophobic core containing six transmembrane domains (TMDs). These K+ channel α subunits participate in the formation of outward rectifier voltage-gated (Kv) and Ca2+-dependent K+ channels. The fourth TMD contains repeated positive charges involved in the voltage gating of these channels and hence in their outward rectification (Logothetis et al. (1992) Neuron, 8, 531-540; Bezanilla et al. (1994) Biophys. J. 66, 1011-1021).

[0004] The second family of pore-forming subunits have only two TMDs. They are essential subunits of inward-rectifying (IRK), G-protein-coupled (GIRK) and ATP-sensitive (KATP) K+ channels. The inward rectification results from a voltage-dependent block by cytoplasmic Mg2+ and polyamines (Matsuda, H. (1991) Annu. Rev. Physiol., 53, 289-298). A conserved domain, called the P domain, is present in all members of both families (Pongs, O. (1993) J. Membr. Biol., 136, 1-8; Heginbotham et al. (1994) Biophys. J. 66,1061-1067; Mackinnon, R. (1995) Neuron, 14, 889-892; Pascual et al., (1995) Neuron., and 14, 1055-1063). This domain is an essential element of the aqueous K+-selective pore. In both groups, the assembly of four subunits is necessary to form a functional K+ channel (Mackinnon, R. (1991) Nature, 350, 232-235; Yang et al., (1995) Neuron, 15, 1441-1447.

[0005] In both six TMD and two TMD pore-forming subunit families, different subunits coded by different genes can associate to form heterotetramers with new channel properties (Isacoff et al., (1990) Nature, 345, 530-534). A selective formation of heteropolymeric channels may allow each cell to develop the best K+ current repertoire suited to its function. Pore-forming α subunits of Kv channels are classified into different subfamilies according to their sequence similarity (Chandy et al. (1993) Trends Pharmacol. Sci., 14, 434). Tetramerization is believed to occur preferentially between members of each subgroup (Covarrubias et al. (1991) Neuron, 7, 763-773). The domain responsible for this selective association is localized in the N-terminal region and is conserved between members of the same subgroup. This domain is necessary for hetero but not homomultimeric assembly within a subfamily and prevents co-assembly between subfamilies. Recently, pore-forming subunits with two TMDs were also shown to co-assemble to form heteropolymers (Duprat et al. (1995) Biochem. Biophys. Res. Commun., 212, 657-663. This heteropolymerization seems necessary to give functional GIRKs. IRKs are active as homopolymers but also form heteropolymers.

SUMMARY OF THE 52906, 33408, AND 12189 INVENTION

[0006] The present invention is based, in part, on the discovery of novel potassium channel family members, referred to herein as “52906,” “33408,” and “12189.” The nucleotide sequence of a cDNA encoding 52906 is shown in SEQ ID NO: 1, and the amino acid sequence of a 52906 polypeptide is shown in SEQ ID NO: 2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 3. The nucleotide sequence of a cDNA encoding 33408 is shown in SEQ ID NO: 4, and the amino acid sequence of a 33408 polypeptide is shown in SEQ ID NO: 5. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 6. The nucleotide sequence of a cDNA encoding 12189 is shown in SEQ ID NO: 7, and the amino acid sequence of a 12189 polypeptide is shown in SEQ ID NO: 8. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 7.

[0007] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 52906, 33408, or 12189 protein or polypeptide, e.g., a biologically active portion of the 52906, 33408, or 12189 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In other embodiments, the invention provides isolated 52906, 33408, or 12189 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, the sequence of the DNA insert of the plasmid, ted with ATCC Accession Number ______, the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number _____. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 52906, 33408, or 12189 protein or an active fragment thereof.

[0008] In a related aspect, the invention further provides nucleic acid constructs that include a 52906, 33408, or 12189 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 52906, 33408, or 12189 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 52906, 33408, or 12189 nucleic acid molecules and polypeptides.

[0009] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 52906, 33408, or 12189-encoding nucleic acids.

[0010] In still another related aspect, isolated nucleic acid molecules that are antisense to a 52906, 33408, or 12189 encoding nucleic acid molecule are provided.

[0011] In another aspect, the invention features, 52906, 33408, or 12189 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 52906, 33408, or 12189-mediated or -related disorders. In another embodiment, the invention provides 52906, 33408, or 12189 polypeptides having a 52906, 33408, or 12189 activity. Preferred polypeptides are 52906, 33408, or 12189 proteins including at least one ion transport protein domain, and, preferably, having a 52906, 33408, or 12189 activity, e.g., a 52906, 33408, or 12189 activity as described herein.

[0012] In other embodiments, the invention provides 52906, 33408, or 12189 polypeptides, e.g., a 52906, 33408, or 12189 polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 52906, 33408, or 12189 protein or an active fragment thereof.

[0013] In a related aspect, the invention further provides nucleic acid constructs which include a 52906, 33408, or 12189 nucleic acid molecule described herein.

[0014] In a related aspect, the invention provides 52906, 33408, or 12189 polypeptides or fragments operatively linked to non-52906, 33408, or 12189 polypeptides to form fusion proteins.

[0015] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 52906, 33408, or 12189 polypeptides or fragments thereof, e.g., an ion transport protein domain, a cyclic nucleotide-binding domain, a potassium channel tetramerisation domain, a transmembrane domain, a cytoplasmic domain, an extracellular domain, a Pore-loop domain, or a PAS domain. In one embodiment, the antibodies or antigen-binding fragment thereof competitively inhibit the binding of a second antibody to a 52906, 33408, or 12189 polypeptide or a fragment thereof, e.g., an ion transport protein domain, a cyclic nucleotide-binding domain, a potassium channel tetramerisation domain, a transmembrane domain, a cytoplasmic domain, an extracellular domain, a Pore-loop domain, or a PAS domain.

[0016] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 52906, 33408, or 12189 polypeptides or nucleic acids.

[0017] In still another aspect, the invention provides a process for modulating 52906, 33408, or 12189 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 52906, 33408, or 12189 polypeptides or nucleic acids, such as conditions characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder.

[0018] The invention also provides assays for determining the activity of or the presence or absence of 52906, 33408, or 12189 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[0019] In yet another aspect, the invention provides methods for modulating (increasing or decreasing) the ion flux, e.g., the flow of K+ ions, in a 52906, 33408, or 12189-expressing cell. The method includes contacting the cell with a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 52906, 33408, or 12189 polypeptide or nucleic acid. In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol. In a preferred embodiment, the cell is an electrically excitable cell, e.g., a neuronal cell or a muscle cell (e.g., a heart cell). For example, the cell can be from brain or cardiac tissues.

[0020] In a preferred embodiment, the compound is an inhibitor of a 52906, 33408, or 12189 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety). In another preferred embodiment, the compound is an inhibitor of a 52906, 33408, or 12189 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.

[0021] In another aspect, the invention features methods for treating or preventing a disorder characterized by the abnormal ion flux of a 52906, 33408, or 12189-expressing cell, in a subject. Preferably, the method includes administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 52906, 33408, or 12189 polypeptide or nucleic acid. In a preferred embodiment, the disorder is a neurological disorder or a cardiac disorder.

[0022] In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., a disorder characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with a compound identified using the methods described herein); and evaluating the expression of a 52906, 33408, or 12189 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 52906, 33408, or 12189 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 52906, 33408, or 12189 nucleic acid or polypeptide expression can be detected by any method described herein.

[0023] In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 52906, 33408, or 12189 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.

[0024] B In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent. The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein) and, evaluating the expression of 52906, 33408, or 12189 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 52906, 33408, or 12189 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 52906, 33408, or 12189 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes neuronal cells or muscle cells.

[0025] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 52906, 33408, or 12189 polypeptide or nucleic acid molecule, including for disease diagnosis.

[0026] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 52906, 33408, or 12189 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 52906, 33408, or 12189 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 52906, 33408, or 12189 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[0027] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]
FIG. 1 depicts a hydropathy plot of human 52906. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 52906 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 785-800 of SEQ ID NO: 2; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 241-265 of SEQ ID NO: 2.

[0029]
FIG. 2 depicts an alignment of the ion transport protein domain of human 52906 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 9), while the lower amino acid sequence corresponds to amino acids 472 to 661 of SEQ ID NO: 2.

[0030]
FIG. 3 depicts a hydropathy plot of human 33408. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 33408 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 585-600 of SEQ ID NO: 5; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 710-740 of SEQ ID NO: 5.

[0031]
FIG. 4A depicts an alignment of the ion transport protein domain of human 33408 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 9), while the lower amino acid sequence corresponds to amino acids 247 to 467 of SEQ ID NO: 5.

[0032]
FIG. 4B depicts an alignment of the cyclic nucleotide-binding domain of human 33408 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 10), while the lower amino acid sequence corresponds to amino acids 565 to 655 of SEQ ID NO: 5.

[0033] FIGS. 4C-4D depict an alignment of the amino acid sequence of human 33408 (upper sequence) with the amino acid sequence of rat Eag2 (lower sequence; Accession Number AF185637; SEQ ID NO: 12).

[0034]
FIG. 5 depicts a hydropathy plot of human 12189. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 12189 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 75-95 of SEQ ID NO: 8; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 35-55 of SEQ ID NO: 8.

[0035]
FIG. 6A depicts an alignment of the potassium channel tetramerisation domain of human 12189 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 11), while the lower amino acid sequence corresponds to amino acids 3 to 101 of SEQ ID NO: 8.

[0036]
FIG. 6B depicts an alignment of the ion transport protein domain of human 12189 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 9), while the lower amino acid sequence corresponds to amino acids 198 to 383 of SEQ ID NO: 8.

[0037]
FIG. 6C depicts an alignment of the amino acid sequence of human 12189 (lower sequence) with the amino acid sequence of mouse Kv1.7 (upper sequence; Accession Number AF032099; SEQ ID NO: 13).

[0038]
FIG. 7 depicts a hydropathy plot of human 21784. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 21784 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g the sequence of from about amino acid residue 10 to 30, amino acid residue 810 to 820, and amino acid residue 1005 to 1031 of SEQ ID NO: 15; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence from about amino acid residues 61 to 78, amino acid residues 311 to 326, and amino acid residues 712 to 721 of SEQ ID NO: 15; or a sequence which includes a Cys or an N-glycosylation site.

[0039] FIGS. 8A-8C depicts alignment of the human dihydropyridine sensitive L-type calcium channel alpha-2/delta subunit, hCIC2.pep (SEQ ID NO: 17) and the human 21784 (SEQ ID NO: 15) amino acid sequences.

[0040]
FIG. 9 shows the amino acid sequence of mouse alpha-2 delta-3 subunit (GenBank Accession Number AJ010949) (SEQ ID NO: 18).

[0041]
FIG. 10 depicts a hydropathy plot of human 56201. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 56201 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 114 to 131, from about 175 to 199, and from about 246 to 269 of SEQ ID NO: 21; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 110 to 120, from about 205 to 215, and from about 230 to 240 of SEQ ID NO: 21.

[0042]
FIG. 11 depicts an alignment of the ion channel domain of human 56201 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 23), while the lower amino acid sequence corresponds to amino acids 46 to 267 of SEQ ID NO: 21.

[0043] FIGS. 12A-12E depicts an alignment of human 56201 with the human sodium channel, skeletal muscle alpha subunit. The upper sequence corresponds to amino acids 1 to 398 of SEQ ID NO: 21 and the lower sequence corresponds to the sequence of human sodium channel, skeletal muscle alpha subunit (SEQ ID NO: 24; Genbank accession number Q16447).

[0044]
FIG. 13 depicts a hydropathy plot of human 32620. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The numbers corresponding to the amino acid sequence of human 32620 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 29 to 45, from about 177 to 190, and from about 417 to 439, of SEQ ID NO: 27; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 46 to 54, from about 473 to 478, and from about 505 to 512, of SEQ ID NO: 27.

[0045]
FIG. 14 depicts an alignment of the sodium-sugar symporter domain of human 32620 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 29), while the lower amino acid sequence corresponds to amino acids 58 to 487 of SEQ ID NO: 27.

[0046]
FIG. 15 depicts an alignment of human 32620 with human SGLT2 using the Clustal W algorithm (Thompson et al. (1994) Nucleic Acids Res., 22:4673-4680). The lower sequence is the complete amino acid sequence of SGLT2 as recited in SwissProt entry P31639, SEQ ID NO: 30, while the upper amino acid sequence corresponds to the complete amino acid sequence of human 32620, i.e. amino acids 1 to 675 of SEQ ID NO: 27.

[0047]
FIG. 16 depicts a hydropathy plot of human 44589. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 44589 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 300 to 340 and from about 920 to 960 of SEQ ID NO: 34; and all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 45 to 65 and from about 485 to 510 of SEQ ID NO: 34.

[0048]
FIG. 17A depicts an alignment of the first ABC transporter ATP cassette domain of human 44589 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 36), while the lower amino acid sequence corresponds to amino acids 515 to 686 of SEQ ID NO: 34.

[0049]
FIG. 17B depicts an alignment of the second ABC transporter ATP cassette domain of human 44589 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 36), while the lower amino acid sequence corresponds to amino acids 1146 to 1329 of SEQ ID NO: 34.

[0050]
FIG. 17C depicts an alignment of the first ABC transporter transmembrane region of human 44589 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 37), while the lower amino acid sequence corresponds to amino acids 163 to 445 of SEQ ID NO: 34.

[0051]
FIG. 17D depicts an alignment of the second ABC transporter transmembrane region of human 44589 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 37), while the lower amino acid sequence corresponds to amino acids 784 to 1073 of SEQ ID NO: 34.

[0052] FIGS. 18A-18D depict an alignment of the amino acid sequence of the human multidrug resistance-associated protein-5 (MRP5; SEQ ID NO: 38) and human 44589 (SEQ ID NO: 34). The location of the transmembrane domains in the human MRP5 and 44589 amino acid sequences is indicated as “TM1-12”.

[0053]
FIG. 19 depicts a hydropathy plot of human 84226. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 84226 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 80 to 92, from about 140 to 152, from about 232 to 248, and from about 312 to 328 of SEQ ID NO: 40; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 45 to 75, from about 200 to 218, and from about 248 to 258 of SEQ ID NO: 40; a sequence which includes a Cys, or a glycosylation site.

[0054]
FIG. 20 depicts an alignment of the cation transporter domain of human 84226 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 42), while the lower amino acid sequence corresponds to amino acids 74 to 361 of SEQ ID NO: 40.

[0055]
FIG. 21 depicts a hydropathy plot of human 8105. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 8105 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, e.g., a sequence above the dashed line, e.g., the sequence from about amino acid residues 70 to 90, 98 to 121, 319 to 342, or 496 to 518 of SEQ ID NO: 44; all or part of a hydrophilic sequence, e.g., a sequence below the dashed line, e.g., the sequence from about amino acid residues 145 to 153, 223 to 240, 243 to 252, or 392 to 407 of SEQ ID NO: 44; a sequence which includes a Cys; or a glycosylation site.

[0056]
FIGS. 22A and 22B depict an alignment of the sugar transporter domain of human 8105 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 46), while the lower amino acid sequence corresponds to amino acids 31 to 533 of SEQ ID NO: 44.

DETAILED DESCRIPTION OF 52906, 33408, AND 12189

[0057] Human 52906

[0058] The human 52906 sequence (see SEQ ID NO: 1, as recited in Example 1), which is approximately 3525 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2544 nucleotides, including the termination codon. The coding sequence encodes a 847 amino acid protein (see SEQ ID NO: 2, as recited in Example 1). The hydropathy plot of 52906 is depicted in FIG. 1.

[0059] Human 52906 contains the following regions or structural features: an ion transport protein domain (PFAM Accession Number PF00520) located at about amino. acid residues 472 to 661 of SEQ ID NO: 2 (see FIG. 2); and a core membrane region consisting of six transmembrane domains, four cytoplasmic domains, three extracellular domains, and a Pore-loop domain. The core membrane region is located at about amino acid 402 to about amino acid 662 of SEQ ID NO: 2. The six transmembrane domains are located at about amino acid 402 (cytoplasmic end) to about amino acid 419 (extracellular end) of SEQ ID NO: 2, about amino acid 433 (extracellular end) to about amino acid 456 (cytoplasmic end) of SEQ ID NO: 2, about amino acid 482 (cytoplasmic end) to about amino acid 498 (extracellular end) of SEQ ID NO: 2, about amino acid 524 (extracellular end) to about amino acid 543 (cytoplasmic end) of SEQ ID NO: 2, about amino acid 573 (cytoplasmic end) to about amino acid 597 (extracellular end) of SEQ ID NO: 2, and about amino acid 641 (extracellular end) to about amino acid 662 (cytoplasmic end) of SEQ ID NO: 2. The four cytoplasmic domains are located at about amino acids 1 to 401 (amino terminus), 457 to 481, 544 to 572, and 663 to 847 (carboxy terminus) of SEQ ID NO: 2. The three extracellular domains are located at about amino acids 420 to 432, 499 to 523, and 598 to 640 of SEQ ID NO: 2. The extracellular domain located at about amino acids 598 to 640 includes a Pore-loop domain (P-loop domain) located at about amino acid residues 616 to 639 of SEQ ID NO: 2.

[0060] The 52906 protein also includes the following domains: six predicted N-glycosylation sites (PS00001) located at about amino acids 10-13, 141-144, 182-185, 284-287, 342-345, and 500-503 of SEQ ID NO: 2; one predicted glycosaminoglycan attachment site (PS00002) located at about amino acids 367-370 of SEQ ID NO: 2; four predicted cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acids 176-179, 258-261, 400-403, and 832-835 of SEQ ID NO: 2; 13 predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 9-11, 12-14, 174-176, 271-273, 288-290, 377-379, 506-508, 552-554, 596-598, 684-686, 732-734, 799-801, and 829-831 of SEQ ID NO: 2; seven predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 330-333, 337-340, 518-521, 668-671, 746-749, 780-783, and 842-845 of SEQ ID NO: 2; 15 predicted N-myristoylation sites (PS00008) located at about amino acids 21-26, 42-47, 118-123, 132-137, 153-158, 165-170, 178-183, 227-232, 309-314, 351-356, 359-364, 366-371, 374-379, 647-652, and 787-792 of SEQ ID NO: 2; and one predicted coiled coil located at about amino acids 719-791 of SEQ ID NO: 2.

[0061] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/ software/packages/pfam/pfam.html.

[0062] A plasmid containing the nucleotide sequence encoding human 52906 (clone “Fbh52906FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0063] An alignment of the human 52906 amino acid sequence with the rat SK2 amino acid sequence (Accession Number U69882) suggests that 52906 is a human ortholog of rat SK2, a calcium activated potassium channel (Kohler et al. (1996) Science 273:1709-1714).

[0064] Human 33408

[0065] The human 33408 sequence (see SEQ ID NO: 4, as recited in Example 1), which is approximately 3553 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2967 nucleotides, including the termination codon. The coding sequence encodes a 988 amino acid protein (see SEQ ID NO: 5, as recited in Example 1). The hydropathy plot of 33408 is depicted in FIG. 3.

[0066] Human 33408 contains the following regions or structural features: an ion transport protein domain (PFAM Accession Number PF00520) located at about amino acid residues 247 to 467 of SEQ ID NO: 5 (see FIG. 4A); a cyclic nucleotide-binding domain (PFAM Accession Number PF00027) located at about amino acid residues 565 to 655 of SEQ ID NO: 5 (see FIG. 4B); and a core membrane region consisting of six transmembrane domains, four cytoplasmic domains, three extracellular domains, a Pore-loop domain, and a PAS domain. The core membrane region is located at about amino acid 219 to about amino acid 471 of SEQ ID NO: 5. The six transmembrane domains are located at about amino acid 219 (cytoplasmic end) to about amino acid 236 (extracellular end) of SEQ ID NO: 5, about amino acid 245 (extracellular end) to about amino acid 264 (cytoplasmic end) of SEQ ID NO: 5, about amino acid 292 (cytoplasmic end) to about amino acid 309 (extracellular end) of SEQ ID NO: 5, about amino acid 320 (extracellular end) to about amino acid 337 (cytoplasmic end) of SEQ ID NO: 5, about amino acid 344 (cytoplasmic end) to about amino acid 368 (extracellular end) of SEQ ID NO: 5, and about amino acid 447 (extracellular end) to about amino acid 471 (cytoplasmic end) of SEQ ID NO: 5. The four cytoplasmic domains are located at about amino acids 1 to 218 (amino terminus), 265 to 291, 338 to 343, and 472 to 988 (carboxy terminus) of SEQ ID NO: 5. The three extracellular domains are located at about amino acids 237 to 244, 310 to 319, and 369 to 446 of SEQ ID NO: 5. The extracellular domain located at about amino acids 369 to 446 includes a Pore-loop domain (P-loop domain) located at about amino acid residues 420 to 440 of SEQ ID NO: 5. The cytoplasmic domain located at about amino acids 1 to 218 includes a PAS domain located at about amino acid residues 1-134 of SEQ ID NO: 5 and a PAC domain located at about amino acid residues 92-132 of SEQ ID NO: 5.

[0067] The 33408 protein also includes the following domains: seven predicted N-glycosylation sites (PS00001) located at about amino acids 170-173, 235-238, 403-406, 466-469, 663-666, 743-746, and 830-833 of SEQ ID NO: 5; two predicted cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acids 21-24 and 677-680 of SEQ ID NO: 5; 13 predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 73-75, 127-129, 142-144, 237-239, 322-324, 478-480, 502-504, 521-523, 773-775, 925-927, 943-945, 952-954, and 981-983 of SEQ ID NO: 5; 16 predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 14-17, 127-130, 215-218, 252-255, 369-372, 442-445, 634-637, 725-728, 832-835, 847-850, 869-872, 883-886, 909-912, 929-932, 974-977, and 981-984 of SEQ ID NO: 5; eight predicted N-myristoylation sites (PS00008) located at about amino acids 3-8, 407-412, 465-470, 557-562, 723-728, 744-749, 806-811, and 867-872 of SEQ ID NO: 5; one predicted amidation site (PS00009) located at about amino acids 3-6 of SEQ ID NO: 5; one predicted leucine zipper pattern (PS00029) located at about amino acids 910-931 of SEQ ID NO: 5; and one predicted coiled coil located at about amino acids 906-944 of SEQ ID NO: 5.

[0068] A plasmid containing the nucleotide sequence encoding human 33408 (clone “Fbh33408FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0069] An alignment of the human 33408 amino acid sequence with the rat Eag2 amino acid sequence (SEQ ID NO: 12; Accession Number AF185637) is depicted in FIGS. 4C-4D. 33408 appears to be a human ortholog of rat Eag2, a subthreshold activating potassium channel (Saganich et al. (1999) J. Neuroscience 19:10789-10802).

[0070] Human 12189

[0071] The human 12189 sequence (see SEQ ID NO: 7, as recited in Example 1), which is approximately 1341 nucleotides long, contains a predicted coding sequence, including a termination codon. The coding sequence encodes a 446 amino acid protein (see SEQ ID NO: 8, as recited in Example 1). The hydropathy plot of 12189 is depicted in FIG. 5.

[0072] Human 12189 contains the following regions or structural features: a potassium channel tetramerisation domain (PFAM Accession Number PF02214) located at about amino acid residues 3 to 101 of SEQ ID NO: 8 (see FIG. 6A); an ion transport protein domain (PFAM Accession Number PF00520) located at about amino acid residues 198 to 383 of SEQ ID NO: 8 (see FIG. 6B); and a core membrane region consisting of six transmembrane domains, four cytoplasmic domains, three extracellular domains, and a Pore-loop domain. The core membrane region is located at about amino acid 134 to about amino acid 384 of SEQ ID NO: 8. The six transmembrane domains are located at about amino acid 134 (cytoplasmic end) to about amino acid 152 (extracellular end) of SEQ ID NO: 8, about amino acid 200 (extracellular end) to about amino acid 222 (cytoplasmic end) of SEQ ID NO: 8, about amino acid 231 (cytoplasmic end) to about amino acid 248 (extracellular end) of SEQ ID NO: 8, about amino acid 266 (extracellular end) to about amino acid 286 (cytoplasmic end) of SEQ ID NO: 8, about amino acid 302 (cytoplasmic end) to about amino acid 323 (extracellular end) of SEQ ID NO: 8, and about amino acid 363 (extracellular end) to about amino acid 384 (cytoplasmic end) of SEQ ID NO: 8. The four cytoplasmic domains are located at about amino acids 1 to 133 (amino terminus), 223 to 230, 287 to 301, and 385 to 446 (carboxy terminus) of SEQ ID NO: 8. The three extracellular domains are located at about amino acids 153 to 199, 249 to 265, and 324 to 362 of SEQ ID NO: 8. The extracellular domain located at about amino acids 324 to 362 includes a Pore-loop domain (P-loop domain) located at about amino acid residues 339 to 355 of SEQ ID NO: 8.

[0073] The 12189 protein also includes the following domains: two predicted N-glycosylation sites (PS00001) located at about amino acids 181-184 and 386-389 of SEQ ID NO: 8; two predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 294-296 and 298-300 of SEQ ID NO: 8; five predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 154-157, 298-301, 334-337, 395-398, and 404-407 of SEQ ID NO: 8; one predicted tyrosine kinase phosphorylation site (PS00007) located at about amino acids 52-60 of SEQ ID NO: 8; five predicted N-myristoylation sites (PS00008) located at about amino acids 87-92, 164-169, 248-253, 365-370, and 421-426 of SEQ ID NO: 8; and one predicted leucine zipper pattern (PS00029) located at about amino acids 281-302 of SEQ ID NO: 8.

[0074] A plasmid containing the nucleotide sequence encoding human 12189 (clone “Fbh12189 FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0075] An alignment of the human 12189 amino acid sequence with the mouse Kv1.7 amino acid sequence (SEQ ID NO: 13; Accession Number AF032099) is depicted in FIG. 6C. 12189 appears to be a human ortholog of mouse Kv1.7, a voltage-gated potassium channel (Kalman et al. (1998) J. Biol. Chem. 273:5851-5857).

1TABLE 1Summary of Sequence Information for 52906, 33408, and 12189ATCCAccessionGenecDNAORFPolypeptideNumber52906SEQ ID NO: 1SEQ ID NO: 3SEQ ID NO: 233408SEQ ID NO: 4SEQ ID NO: 6SEQ ID NO: 512189SEQ ID NO: 7SEQ ID NO: 8

[0076]

2

TABLE 2

Summary of Domains of 52906, 33408, and 12189

Domain
52906
33408
12189

Transmembrane
amino acids 402-662
amino acids 219-471
amino acids 134-384

Region
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Transmembrane
amino acids 402-419
amino acids 219-236
amino acids 134-152

Domain 1
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Transmembrane
amino acids 433-456
amino acids 245-264
amino acids 200-222

Domain 2
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Transmembrane
amino acids 482-498
amino acids 292-309
amino acids 231-248

Domain 3
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Transmembrane
amino acids 524-543
amino acids 320-337
amino acids 266-286

Domain 4
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Transmembrane
amino acids 573-597
amino acids 344-368
amino acids 302-323

Domain 5
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Transmembrane
amino acids 641-662
amino acids 447-471
amino acids 363-384

Domain 6
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Cytoplasmic
amino acids 1-401 of
amino acids 1-218 of
amino acids 1-133 of

Domain 1
SEQ ID NO: 2
SEQ ID NO: 5
SEQ ID NO: 8

Cytoplasmic
amino acids 457-481
amino acids 265-291
amino acids 223-230

Domain 2
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Cytoplasmic
amino acids 544-572
amino acids 338-343
amino acids 287-301

Domain 3
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Cytoplasmic
amino acids 663-847
amino acids 472-988
amino acids 385-446

Domain 4
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Extracellular
amino acids 420-432
amino acids 237-244
amino acids 153-199

Domain 1
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Extracellular
amino acids 499-523
amino acids 310-319
amino acids 249-265

Domain 2
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Extracellular
amino acids 598-640
amino acids 369-446
amino acids 324-362

Domain 3
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

Pore-loop
amino acids 616-639
amino acids 420-440
amino acids 339-355

Domain
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

ion transport
amino acids 472-661
amino acids 247-467
amino acids 198-383

protein domain
of SEQ ID NO: 2
of SEQ ID NO: 5
of SEQ ID NO: 8

cyclic

amino acids 565-655

nucleotide

of SEQ ID NO: 5

binding domain

potassium

amino acids 3-101 of

channel

SEQ ID NO: 8

tetramerisation

domain

[0077] The 52906, 33408, and 12189 proteins contain a significant number of structural characteristics in common with members of the potassium channel family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[0078] As used herein, a “potassium channel” includes a protein or polypeptide which is involved in receiving, conducting, and transmitting signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell. Potassium channels are potassium ion selective, and can determine membrane excitability (the ability of, for example, a neuron to respond to a stimulus and convert it into an impulse). Potassium channels can also influence the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation. Potassium channels are typically expressed in electrically excitable cells, e.g., neurons, muscle, endocrine, and egg cells, and may form heteromultimeric structures, e.g., composed of pore-forming α and cytoplasmic β subunits. Potassium channels may also be found in nonexcitable cells (e.g., thymus cells), where they may play a role in, e.g., signal transduction. Potassium channel proteins contain six transmembrane helices, wherein the last two helices flank a loop (a P-loop) which determines potassium ion selectivity. Examples of potassium channels include: (1) the voltage-gated potassium channels, (2) the ligand-gated potassium channels, e.g., neurotransmitter-gated potassium channels, and (3) cyclic-nucleotide-gated potassium channels. Voltage-gated and ligand-gated potassium channels are expressed in the brain, e.g., in brainstem monoaminergic and forebrain cholinergic neurons, where they are involved in the release of neurotransmitters, or in the dendrites of hippocampal and neocortical pyramidal cells, where they are involved in the processes of learning and memory formation. For a detailed description of potassium channels, see Kandel E. R. et al., Principles of Neural Science, second edition, (Elsevier Science Publishing Co., Inc., N.Y. (1985)), the contents of which are incorporated herein by reference.

[0079] A 52906, 33408, or 12189 polypeptide can include a “transmembrane domain” or regions homologous with a “transmembrane domain”.

[0080] As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al., (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. Amino acid residues 402-419, 433-456, 482-498, 524-543, 573-597, and 641-662 of the 52906 protein (SEQ ID NO: 2) are predicted to comprise transmembrane domains (see FIG. 2). Accordingly, 52906 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human 52906 are within the scope of the invention. Amino acid residues 219-236, 245-264, 292-309, 320-337, 344-368, and 447-471 of the 33408 protein (SEQ ID NO: 5) are predicted to comprise transmembrane domains (see FIG. 5). Accordingly, 33408 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human 33408 are within the scope of the invention. Amino acid residues 134-152, 200-222, 231-248, 266-286, 302-323, and 363-384 of the 12189 protein. (SEQ ID NO: 8) are predicted to comprise transmembrane domains (see FIGS. 8A-8C). Accordingly, 12189 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human 12189 are within the scope of the invention.

[0081] A 52906, 33408, or 12189 polypeptide can further include a “Pore loop” or regions homologous with a “Pore loop domain”.

[0082] As used herein, the term “Pore loop” or “P-loop” includes amino acid sequence of about 15-45 amino acid residues in length, preferably about 15-35 amino acid residues in length, and most preferably about 15-25 amino acid residues in length, which is hydrophobic and which is involved in lining the potassium channel pore. A P-loop is typically found between transmembrane domains of potassium channels and is believed to be a major determinant of ion selectivity in potassium channels. Preferably, P-loops contain a G-[HYDROPHOBIC AMINO ACID]-G sequence, e.g., a GYG, GLG, or GFG sequence. P-loops are described in, for example, Warmke et al. (1991) Science 252:1560-1562; Zagotta W. N. et al., (1996) Annual Rev. Neuronsci. 19:235-63 (Pongs, O. (1993) J. Membr. Biol., 136, 1-8; Heginbotham et al. (1994) Biophys. J. 66,1061-1067; Mackinnon, R. (1995) Neuron, and 14, 889-892; Pascual et al., (1995) Neuron., 14, 1055-1063), the contents of which are incorporated herein by reference. Amino acid residues 616-639 of SEQ ID NO: 2, 420-440 of SEQ ID NO: 5, and 339-355 of SEQ ID NO: 8 comprise P-loop domains. Accordingly, proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a -loop domain of human 52906, 33408, or 12189 are within the scope of the invention.

[0083] In one embodiment, a 52906, 33408, or 12189 protein includes at least one cytoplasmic domain. When located at the N-terminal domain the cytoplasmic domain is referred to herein as an “N-terminal cytoplasmic domain”. As used herein, an “N-terminal cytoplasmic domain” includes an amino acid sequence having about 1-500, preferably about 1-450, preferably about 1-400, preferably about 1-380, more preferably about 1-350, more preferably about 1-300, more preferably about 1-220, or even more preferably about 1-135 amino acid residues in length and is located inside of a cell or intracellularly. The C-terminal amino acid residue of a “N-terminal cytoplasmic domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a 52906, 33408, or 12189 protein. For example, an N-terminal cytoplasmic domain is located at about amino acid residues 1-401 of SEQ ID NO: 2, 1-218 of SEQ ID NO: 5, or 1-133 of SEQ ID NO: 8.

[0084] In a preferred embodiment, a 52906, 33408, or 12189 polypeptide or protein has at least one cytoplasmic domain or a region which includes at least about 5, preferably about 5-10, and more preferably about 10-20 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “cytoplasmic domain,” e.g., at least one cytoplasmic domain of human 52906, 33408, or 12189 (e.g., residues 1-401, 457-481, 544-572, and 663-847 of SEQ ID NO: 2; residues 1-218, 265-291, 338-343, and 447-988 of SEQ ID NO: 5; and residues 1-133, 223-230, 287-301, and 385-446 of SEQ ID NO: 8).

[0085] In another embodiment, a 52906, 33408, or 12189 protein includes at least one extracellular loop. As used herein, the term “loop” includes an amino acid sequence having a length of at least about 4, preferably about 5-10, and more preferably about 10-20 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a 52906, 33408, or 12189 molecule, and the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a 52906, 33408, or 12189 molecule. As used herein, an “extracellular loop” includes an amino acid sequence located outside of a cell, or extracellularly. For example, an extracellular loop can be found at about amino acids 420-432, 499-523, and 598-640 of SEQ ID NO: 2; at about amino acids 237-244, 310-319, and 369-446 of SEQ ID NO: 5; and at about amino acids 153-199, 249-265, and 324-362 of SEQ ID NO: 8.

[0086] In a preferred embodiment, a 52906, 33408, or 12189 polypeptide or protein has at least one extracellular loop or a region which includes at least about 4, preferably about 5-10, preferably about 10-20, and more preferably about 20-30 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “extracellular loop,” e.g., at least one extracellular loop of human 52906, 33408, or 12189 (e.g., residues 420-432, 499-523, and 598-640 of SEQ ID NO: 2; residues 237-244, 310-319, and 369-446 of SEQ ID NO: 5; and residues 153-199, 249-265, and 324-362 of SEQ ID NO: 8).

[0087] In another embodiment, a 52906, 33408, or 12189 protein includes a “C-terminal cytoplasmic domain”, also referred to herein as a C-terminal cytoplasmic tail, in the sequence of the protein. As used herein, a “C-terminal cytoplasmic domain” includes an amino acid sequence having a length of at least about 50, preferably about 500-550, preferably about 150-200, more preferably about 50-70 amino acid residues and is located within a cell or within the cytoplasm of a cell. Accordingly, the N-terminal amino acid residue of a “C-terminal cytoplasmic domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a 52906, 33408, or 12189 protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 663-847 of SEQ ID NO: 2; at about amino acid residues 472-988 of SEQ ID NO: 5; and at about amino acid residues 385-446 of SEQ ID NO: 8.

[0088] In a preferred embodiment, a 52906, 33408, or 12189 polypeptide or protein has a C-terminal cytoplasmic domain or a region which includes at least about 50, preferably about 150-550, more preferably about 50-70 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “C-terminal cytoplasmic domain,” e.g., the C-terminal cytoplasmic domain of human 52906, 33408, or 12189 (e.g., residues 663-847 of SEQ ID NO: 2; residues 472-988 of SEQ ID NO: 5; and residues 385-446 of SEQ ID NO: 8).

[0089] A 52906, 33408, or 12189 polypeptide can include an “ion transport protein domain” or regions homologous with an “ion transport protein domain.”

[0090] As used herein, the term “ion transport protein domain” includes an amino acid sequence of about 100 to 300 amino acid residues in length and having a bit score for the alignment of the sequence to the ion transport protein domain profile (Pfam HMM) of at least 50. Preferably, a ion transport protein domain includes at least about 150 to 280 amino acids, more preferably about 170 to 260 amino acid residues, or about 180 to 230 amino acids and has a bit score for the alignment of the sequence to the ion transport protein domain (HMM) of at least 90 or greater. The ion transport protein domain (HMM) has been assigned the PFAM Accession Number PF00520 (http;//genome.wustl.edu/Pfam/.html). An alignment of the ion transport protein domain (amino acids 472-661 of SEQ ID NO: 2) of human 52906 with a consensus amino acid sequence (SEQ ID NO: 9) derived from a hidden Markov model is depicted in FIG. 2. An alignment of the ion transport protein domain (amino acids 247-467 of SEQ ID NO: 5) of human 33408 with a consensus amino acid sequence (SEQ ID NO: 9) derived from a hidden Markov model is depicted in FIG. 4A. An alignment of the ion transport protein domain (amino acids 198-383 of SEQ ID NO: 8) of human 12189 with a consensus amino acid sequence (SEQ ID NO: 9) derived from a hidden Markov model is depicted in FIG. 6B.

[0091] In a preferred embodiment, a 52906, 33408, or 12189 polypeptide or protein has an “ion transport protein domain” or a region which includes at least about 150 to 280 more preferably about 170 to 260 or 180 to 230 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “ion transport protein domain,” e.g., the ion transport protein domain of human 52906, 33408, or 12189 (e.g., residues 472-661 of SEQ ID NO: 2, 247-467 of SEQ ID NO: 5, or 198-383 of SEQ ID NO: 8).

[0092] A 33408 molecule can further include a cyclic nucleotide binding domain or regions homologous with a “cyclic nucleotide binding domain.”

[0093] As used herein, the term “cyclic nucleotide binding domain” includes an amino acid sequence of about 40-180 amino acid residues in length and having a bit score for the alignment of the sequence to the cyclic nucleotide binding domain (HMM) of at least 50. Preferably, a cyclic nucleotide binding domain is capable of binding a cyclic nucleotide. Preferably, a cyclic nucleotide binding domain includes at least about 50-150 amino acids, more preferably about 70-120 amino acid residues, or about 80-100 amino acids and has a bit score for the alignment of the sequence to the cyclic nucleotide binding domain (HMM) of at least 80 or greater. The cyclic nucleotide binding domain (HMM) has been assigned the PFAM Accession PF00027 (http://genome.wustl.edu/Pfam/html). An alignment of the cyclic nucleotide binding domain (amino acids 565 to 655 of SEQ ID NO: 5) of human 33408 with a consensus amino acid sequence (SEQ ID NO: 10) derived from a hidden Markov model is depicted in FIG. 4B.

[0094] In a preferred embodiment a 33408 polypeptide or protein has a “cyclic nucleotide binding domain” or a region which includes at least about 50-150, more preferably about 70-120 or 80-100 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “cyclic nucleotide binding domain,” e.g., the cyclic nucleotide binding domain of human 33408 (e.g., residues 565 to 655 of SEQ ID NO: 5).

[0095] A 12189 polypeptide can further include a “potassium channel tetramerisation domain” or regions homologous with a “potassium channel tetramerisation domain.”

[0096] As used herein, the term “potassium channel tetramerisation domain” includes an amino acid sequence of about 50 to 200 amino acid residues in length and having a bit score for the alignment of the sequence to the potassium channel tetramerisation domain profile (Pfam HMM) of at least 100. A “potassium channel tetramerisation domain” promotes the assembly of alpha-subunits into functional tetrameric channels. Preferably, a potassium channel tetramerisation domain includes at least about 60 to 150 amino acids, more preferably about 70 to 130 amino acid residues, or about 90 to 110 amino acids and has a bit score for the alignment of the sequence to the potassium channel tetramerisation domain (HMM) of at least 165 or greater. The potassium channel tetramerisation domain (HMM) has been assigned the PFAM Accession Number PF02214 (http;//genome.wustl.edu/Pfam/.html). An alignment of the potassium channel tetramerisation domain (amino acids 3-101 of SEQ ID NO: 8) of human 12189 with a consensus amino acid sequence (SEQ ID NO: 11) derived from a hidden Markov model is depicted in FIG. 6A.

[0097] In a preferred embodiment, a 12189 polypeptide or protein has a “potassium channel tetramerisation domain” or a region which includes at least about 60 to 150 more preferably about 70 to 130 or 90 to 110 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “potassium channel tetramerisation domain,” e.g., the potassium channel tetramerisation domain of human 12189 (e.g., residues 3-101 of SEQ ID NO: 8).

[0098] To identify the presence of an “ion transport protein” domain, a “cyclic nucleotide-binding” domain, or a “potassium channel tetramerisation” domain in a 52906, 33408, or 12189 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al.(1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

[0099] A 33408 polypeptide can further include a “PAS domain” or regions homologous with a “PAS domain”. As used herein, a “PAS domain” includes an amino acid sequence of about 100-200 amino acid residues in length that is involved in ligand and/or protein-protein interactions. Preferably, the PAS domain interacts with the body of the channel, affecting gating, inactivation, and/or voltage sensitivity. Preferably, the PAS domain is located at the N-terminal cytoplasmic region of the 33408 polypeptide.

[0100] In a preferred embodiment, a 33408 polypeptide or protein has a “PAS domain” or a region which includes at least about 50-220, more preferably about 100-200 or 120-140 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “PAS domain,” e.g., the PAS domain of human 33408 (e.g., residues 1-134 of SEQ ID NO: 5).

[0101] A 33408 polypeptide can further include a “PAC domain” or regions homologous with a “PAC domain”. As used herein, a “PAC domain” includes an amino acid sequence of about 30-50 amino acid residues in length. Preferably, the PAC domain contributes to the folding of the PAS domain. Preferably, the PAC domain is located at the C-terminal end of the PAS domain in a 33408 polypeptide.

[0102] In a preferred embodiment, a 33408 polypeptide or protein has a “PAC domain” or a region which includes at least about 20-70 or 30-50 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “PAC domain,” e.g., the PAC domain of human 33408 (e.g., residues 92-132 of SEQ ID NO: 5).

[0103] A 52906 family member can include at least one (preferably two, three, four, five, or six) transmembrane domain, at least one (preferably two or three) cytoplasmic domain, at least one (preferably two or three) extracellular domain, at least one P-loop domain, and at least one ion transport protein domain. Furthermore, a 52906, 33408, or 12189 family member can include: at least one, two, three, four, five, and preferably six predicted N-glycosylation sites (PS00001); at least one predicted glycosaminoglycan attachment site (PS00002); at least one, two, three, and preferably four predicted cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, and preferably 13 predicted Protein Kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, and preferably seven predicted Casein Kinase TI phosphorylation sites (PS00006); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, and preferably 15 predicted N-myristoylation sites (PS00008); and at least one predicted coiled coil domain.

[0104] A 33408 family member can include at least one (preferably two, three, four, five, or six) transmembrane domain, at least one (preferably two or three) cytoplasmic domain, at least one (preferably two or three) extracellular domain, at least one P-loop domain, and at least one ion transport protein domain. A 33408 family member can further include a cyclic nucleotide-binding domain. A 33408 family member can further include a PAS domain and a PAC domain. Furthermore, a 33408 family member can include: at least one, two, three, four, five, six, and preferably seven predicted N-glycosylation sites (PS00001); at least one and preferably two predicted cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, and preferably 13 predicted Protein Kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, and preferably 16 predicted Casein Kinase II phosphorylation sites (PS00006); at least one, two, three, four, five, six, seven, and preferably eight predicted N-myristoylation sites (PS00008); at least one predicted amidation site (PS00009); at least one predicted leucine zipper pattern (PS00029); and at least one predicted coiled coil domain.

[0105] A 12189 family member can include at least one (preferably two, three, four, five, or six) transmembrane domain, at least one (preferably two or three) cytoplasmic domain, at least one (preferably two or three) extracellular domain, at least one P-loop domain, and at least one ion transport protein domain. A 12189 family member can further include a potassium channel tetramerisation domain. Furthermore, a 12189 family member can include: at least one and preferably two predicted N-glycosylation sites (PS00001); at least one and preferably two predicted Protein Kinase C phosphorylation sites (PS00005); at least one, two, three, four, and preferably five predicted Casein Kinase II phosphorylation sites (PS00006); at least one predicted tyrosine kinase phosphorylation site (PS00007); at least one, two, three, four, and preferably five predicted N-myristoylation sites (PS00008); and at least one predicted leucine zipper pattern (PS00029).

[0106] As the 52906, 33408, or 12189 polypeptides of the invention may modulate 52906, 33408, or 12189-mediated activities, e.g., potassium channel mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 52906, 33408, or 12189-mediated or related disorders, e.g., potassium channel associated disorders, as described below.

[0107] As used herein, a “52906, 33408, or 12189 activity”, “biological activity of 52906, 33408, or 12189 ” or “functional activity of 52906, 33408, or 12189 ”, refers to an activity exerted by a 52906, 33408, or 12189 protein, polypeptide or nucleic acid molecule. For example, a 52906, 33408, or 12189 activity can be an activity exerted by 52906, 33408, or 12189 in a physiological milieu on, e.g., a 52906, 33408, or 12189-responsive cell or on a 52906, 33408, or 12189 substrate, e.g., a protein substrate. A 52906, 33408, or 12189 activity can be determined in vivo or in vitro. In one embodiment, a 52906, 33408, or 12189 activity is a direct activity, such as an association with a 52906, 33408, or 12189 target molecule. A “target molecule” or “binding partner” is a molecule with which a 52906, 33408, or 12189 protein binds or interacts in nature. In an exemplary embodiment, 52906, 33408, or 12189 is an ion channel, e.g., a potassium channel.

[0108] A 52906, 33408, or 12189 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 52906, 33408, or 12189 protein with a 52906, 33408, or 12189 ligand, e.g., a potassium ion. The features of the 52906, 33408, or 12189 molecules of the present invention can provide similar biological activities as potassium channel family members. For example, the 52906, 33408, or 12189 proteins of the present invention can have one or more of the following activities: (1) interacting with a non-52906, 33408, or 12189 protein molecule; (2) activating a 52906, 33408, or 12189-dependent signal transduction pathway; (3) modulating the release of neurotransmitters; (4) modulating membrane excitability; (5) influencing the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; (6) binding a cyclic nucleotide; (7) contributing to the formation of potassium channels; (8) contributing to the formation of calcium-activated, voltage independent potassium channels; (9) modulating repolarization of the neuronal cell membrane; (10) contributing to the formation of voltage-gated potassium channels; (11) contributing to the formation of cyclic nucleotide-gated potassium channels; (12) modulating the flow of K+ ions through a cell membrane; and (13) modulating processes which underlie learning and memory, such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials.

[0109] Based on the above-described sequence similarities, the 52906, 33408, or 12189 molecules of the present invention are predicted to have similar biological activities as potassium channel family members. In addition, 52906 and 33408 mRNA was found to be highly expressed in cells derived from brain and heart (see Tables 3 and 4). Thus, the 52906, 33408, or 12189 molecules can act as novel diagnostic targets and therapeutic agents for controlling potassium channel associated disorders. Examples of such disorders include neurological disorders and cardiac-related disorders.

[0110] As used herein, a “potassium channel associated disorder” includes a disorder, disease or condition which is characterized by a misregulation of a potassium channel mediated activity. Potassium channel associated disorders can detrimentally affect conveyance of sensory impulses from the periphery to the brain and/or conductance of motor impulses from the brain to the periphery; integration of reflexes; interpretation of sensory impulses; cellular proliferation, growth, differentiation, or migration, and emotional, intellectual (e.g., learning and memory), or motor processes. Examples of potassium channel associated disorders include CNS disorders such as cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Further CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

[0111] Further examples of potassium channel associated disorders include cardiac-related disorders. Cardiovascular system disorders in which the 52906, 33408, or 12189 molecules of the invention may be directly or indirectly involved include arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, Jervell syndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, and arrhythmia. 52906, 33408, or 12189-mediated or related disorders also include disorders of the musculoskeletal system such as paralysis and muscle weakness, e.g., ataxia, myotonia, and myokymia.

[0112] As used herein, a “potassium channel mediated activity” includes an activity which involves a potassium channel, e.g., a potassium channel in a neuronal cell, a muscle cell, or a thymus cell associated with receiving, conducting, and transmitting signals in, for example, the nervous system. Potassium channel mediated activities include release of neurotransmitters, e.g., dopamine or norepinephrine, from cells, e.g., neuronal cells; modulation of resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; participation in signal transduction pathways, and modulation of processes such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials in, for example, neuronal cells or muscle cells.

[0113] The presence of 52906, 33408, or 12189 RNA or protein can be used to identify a cell or tissue, or other biological sample, as being derived from the brain, e.g., cerebral cortex, from the heart, from a muscle, or of neuronal origin. Expression can be determined by evaluating RNA, e.g., by hybridization of a 52906, 33408, or 12189 specific probe, or with a 52906, 33408, or 12189 specific antibody.

[0114] The 52906, 33408, or 12189 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 thereof are collectively referred to as “polypeptides or proteins of the invention” or “52906, 33408, or 12189 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “52906, 33408, or 12189 nucleic acids.” 52906, 33408, or 12189 molecules refer to 52906, 33408, or 12189 nucleic acids, polypeptides, and antibodies.

[0115] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0116] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0117] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C, followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[0118] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, corresponds to a naturally-occurring nucleic acid molecule.

[0119] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include at least an open reading frame encoding a 52906, 33408, or 12189 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 52906, 33408, or 12189 protein or derivative thereof.

[0120] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 52906, 33408, or 12189 protein is at least 10% pure. In a preferred embodiment, the preparation of 52906, 33408, or 12189 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-52906, 33408, or 12189 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-52906, 33408, or 12189 chemicals. When the 52906, 33408, or 12189 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0121] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 52906, 33408, or 12189 without abolishing or substantially altering a 52906, 33408, or 12189 activity. Preferably the alteration does not substantially alter the 52906, 33408, or 12189 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 52906, 33408, or 12189, results in abolishing a 52906, 33408, or 12189 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 52906, 33408, or 12189 are predicted to be particularly unamenable to alteration.

[0122] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 52906, 33408, or 12189 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 52906, 33408, or 12189 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 52906, 33408, or 12189 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0123] As used herein, a “biologically active portion” of a 52906, 33408, or 12189 protein includes a fragment of a 52906, 33408, or 12189 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 52906, 33408, or 12189 molecule and a non-52906, 33408, or 12189 molecule or between a first 52906, 33408, or 12189 molecule and a second 52906, 33408, or 12189 molecule (e.g., a dimerization interaction). Biologically active portions of a 52906, 33408, or 12189 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 52906, 33408, or 12189 protein, e.g., the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, which include less amino acids than the full length 52906, 33408, or 12189 proteins, and exhibit at least one activity of a 52906, 33408, or 12189 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 52906, 33408, or 12189 protein, e.g., the ability to modulate the flow of K+ ions through a cell membrane and/or the ability to modulate the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell. A biologically active portion of a 52906, 33408, or 12189 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 52906, 33408, or 12189 protein can be used as targets for developing agents which modulate a 52906, 33408, or 12189 mediated activity, e.g., the ability to modulate the flow of K+ions through a cell membrane and/or the ability to modulate the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell.

[0124] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0125] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0126] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0127] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0128] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0129] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 52906, 33408, or 12189 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 52906, 33408, or 12189 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0130] Particularly preferred 52906, 33408, or 12189 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 are termed substantially identical.

[0131] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7 are termed substantially identical.

[0132] “Misexpression or aberrant expression”, as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[0133] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[0134] A “purified preparation of cells”, as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[0135] Various aspects of the invention are described in further detail below.

[0136] Isolated 52906. 33408, and 12189 Nucleic Acid Molecules

[0137] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 52906, 33408, or 12189 polypeptide described herein, e.g., a full-length 52906, 33408, or 12189 protein or a fragment thereof, e.g., abiologically active portion of 52906, 33408, or 12189 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 52906, 33408, or 12189 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[0138] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 52906, 33408, or 12189 protein (i.e., “the coding region” of SEQ ID NO: 1, as shown in SEQ ID NO: 3 or “the coding region” of SEQ ID NO: 4, as shown in SEQ ID NO: 6), as well as 5′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 (e.g., SEQ ID NO: 3 or SEQ ID NO: 6) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acids 472-661 of SEQ ID NO: 2, amino acids 247-467 of SEQ ID NO: 5, amino acids 565-655 of SEQ ID NO: 5, amino acids 3-101 of SEQ ID NO: 8, or amino acids 198-383 of SEQ ID NO: 8.

[0139] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO: I, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, thereby forming a stable duplex.

[0140] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, or a portion, preferably of the same length, of any of these nucleotide sequences.

[0141] 52906, 33408, or 12189 Nucleic Acid Fragments

[0142] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 52906, 33408, or 12189 protein, e.g., an immunogenic or biologically active portion of a 52906, 33408, or 12189 protein. A fragment can comprise those nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, which encode an ion transport protein domain of human 52906, 33408, or 12189. The nucleotide sequence determined from the cloning of the 52906, 33408, or 12189 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 52906, 33408, or 12189 family members, or fragments thereof, as well as 52906, 33408, or 12189 homologues, or fragments thereof, from other species.

[0143] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein (e.g., an ion transport protein domain, a cyclic nucleotide-binding domain, a potassium channel tetramerisation domain, a transmembrane domain, a cytoplasmic domain, an extracellular domain, a Pore-loop domain, or a PAS domain) or fragments thereof, particularly fragments thereof which are at least 100, 200, 300, 400, or 500 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[0144] A nucleic acid fragment can include a sequence corresponding to a domain, region, or finctional site described herein. A nucleic acid fragment can also include one or more domain, region, or fumctional site described herein. Thus, for example, a 52906, 33408, or 12189 nucleic acid fragment can include a sequence corresponding to an ion transport protein domain, a cyclic nucleotide-binding domain, a potassium channel tetramerisation domain, a transmembrane domain, a cytoplasmic domain, an extracellular domain, a Pore-loop domain, or a PAS domain.

[0145] 52906, 33408, or 12189 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7.

[0146] In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0147] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: an ion transport protein domain, a cyclic nucleotide-binding domain, a potassium channel tetramerisation domain, a transmembrane domain, a cytoplasmic domain, an extracellular domain, a Pore-loop domain, or a PAS domain. The locations of these domains in SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 8 are described in Table 2.

[0148] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 52906, 33408, or 12189 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: an ion transport protein domain, a cyclic nucleotide-binding domain, a potassium channel tetramerisation domain, a transmembrane domain, a cytoplasmic domain, an extracellular domain, a Pore-loop domain, or a PAS domain.

[0149] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[0150] A nucleic acid fragment encoding a “biologically active portion of a 52906, 33408, or 12189 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, which encodes a polypeptide having a 52906, 33408, or 12189 biological activity (e.g., the biological activities of the 52906, 33408, or 12189 proteins are described herein), expressing the encoded portion of the 52906, 33408, or 12189 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 52906, 33408, or 12189 protein. For example, a nucleic acid fragment encoding a biologically active portion of 52906, 33408, or 12189 includes ion transport protein domain, e.g., amino acids 472-661 of SEQ ID NO: 2, amino acids 247-467 of SEQ ID NO: 5, or amino acids 198-383 of SEQ ID NO: 8. A nucleic acid fragment encoding a biologically active portion of a 52906, 33408, or 12189 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

[0151] In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, 3300, 3400, 3500, or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7.

[0152] In preferred embodiments, the fragment includes at least one, and preferably at least 5, 10, 15, 25, 50, 100, 200, 300, 400, or 500 nucleotides from nucleotides 1-2962, 3437-3525, 1-1441, 3182-3525, or 1-2687 of SEQ ID NO: 1.

[0153] In preferred embodiments, the fragment includes at least one, and preferably at least 5, 10, 15, 25, 50, 75, 100, 200, 300, 500, 1000, or 1500 nucleotides encoding a protein including 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, or 500 amino acids from amino acids 1-775, 1-268, 1-683 of SEQ ID NO: 2.

[0154] In preferred embodiments, the nucleic acid fragment includes a nucleotide sequence that is other than the sequence of AA418096, V35457, Z51630, W63707, or W63702.

[0155] In preferred embodiments, the fragment comprises the coding region of 52906, e.g., the nucleotide sequence of SEQ ID NO: 3.

[0156] In preferred embodiments, the fragment includes at least one, and preferably at least 5, 10, 15, 25, 50, 100, 200, 300, 400, or 500 nucleotides from nucleotides 1-1844, 1-277, 1-252, or 3245-3553, of SEQ ID NO: 4.

[0157] In preferred embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 6 and at least one, and preferably at least 5, 10, 15, 25, 50, 75, 100, 200, 300, or 500 nucleotides, e.g., consecutive nucleotides, of SEQ ID NO: 4.

[0158] In preferred embodiments, the fragment includes at least one, and preferably at least 5, 10, 15, 25, 50, 75, 100, 200, 300, 500, 1000, or 1500 nucleotides encoding a protein including 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, or 500 amino acids from amino acids 1-522 of SEQ ID NO: 5.

[0159] In preferred embodiments, the nucleic acid fragment includes a nucleotide sequence that is other than the sequence of U69185 or a sequence described in WO01/04133 or WO01/29068.

[0160] In preferred embodiments, the fragment comprises the coding region of 33408, e.g., the nucleotide sequence of SEQ ID NO: 6.

[0161]

52906
, 33408, or 12189 Nucleic Acid Variants

[0162] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 52906, 33408, or 12189 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0163] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[0164] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[0165] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0166] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 52906, 33408, or 12189 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 52906, 33408, or 12189 gene.

[0167] Preferred variants include those that are correlated with the ability to modulate the flow of K+ ions through a cell membrane and/or the ability to modulate the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell.

[0168] Allelic variants of 52906, 33408, or 12189, e.g., human 52906, 33408, or 12189, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 52906, 33408, or 12189 protein within a population that maintain the ability to modulate the flow of K+ ions through a cell membrane and/or the ability to modulate the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 52906, 33408, or 12189, e.g., human 52906, 33408, or 12189, protein within a population that do not have the ability to modulate the flow of K+ ions through a cell membrane and/or the ability to modulate the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[0169] Moreover, nucleic acid molecules encoding other 52906, 33408, or 12189 family members and, thus, which have a nucleotide sequence which differs from the 52906, 33408, or 12189 sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7 are intended to be within the scope of the invention.

[0170] Antisense Nucleic Acid Molecules, Ribozvmes and Modified 52906, 33408. or 12189 Nucleic Acid Molecules

[0171] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 52906, 33408, or 12189. An “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 52906, 33408, or 12189 coding strand, or to only a portion thereof (e.g., the coding region of human 52906, 33408, or 12189 corresponding to SEQ ID NO: 3 or SEQ ID NO: 6). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 52906, 33408, or 12189 (e.g., the 5′ and 3′ untranslated regions).

[0172] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 52906, 33408, or 12189 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 52906, 33408, or 12189 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 52906, 33408, or 12189 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[0173] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0174] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 52906, 33408, or 12189 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0175] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0176] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 52906, 33408, or 12189 -encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 52906, 33408, or 12189 cDNA disclosed herein (i.e., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 7), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 52906, 33408, or 12189-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 52906, 33408, or 12189 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0177] 52906, 33408, or 12189 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 52906, 33408, or 12189 (e.g., the 52906, 33408, or 12189 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 52906, 33408, or 12189 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. (1992) Ann. N.Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[0178] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

[0179] A 52906, 33408, or 12189 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulmé(2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.

[0180] For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[0181] PNAs of 52906, 33408, or 12189 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 52906, 33408, or 12189 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0182] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0183] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 52906, 33408, or 12189 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 52906, 33408, or 12189 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. 5,876,930.

[0184] Isolated 52906, 33408, or 12189 Polypeptides

[0185] In another aspect, the invention features, an isolated 52906, 33408, or 12189 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-52906, 33408, or 12189 antibodies. 52906, 33408, or 12189 protein can be isolated from cells or tissue sources using standard protein purification techniques. 52906, 33408, or 12189 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[0186] Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[0187] In a preferred embodiment, a 52906, 33408, or 12189 polypeptide has one or more of the following characteristics:

[0188] (i) it has the ability to modulate the flow of K+ ions through a cell membrane, e.g., to allow for the flow of K+ ions in and/or out of a cell under certain conditions;

[0189] (ii) it has the ability to modulate the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell;

[0190] (iii) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of a 52906, 33408, or 12189 polypeptide, e.g., a polypeptide of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0191] (iv) it has an overall sequence similarity of at least 60%, more preferably at least 70, 80, 90, or 95%, with a polypeptide a of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8;

[0192] (v) it can be found in neuronal cells or muscle cells (e.g., heart cells);

[0193] (vi) it has the ability to modulate the resting potential of membranes;

[0194] (vii) it has a P-loop domain which is preferably about 70%, 80%, 90% or 95% similar with amino acids 616-639 of SEQ ID NO: 2, amino acids 420-440 of SEQ ID NO: 5, or amino acids 339-355 of SEQ ID NO: 8;

[0195] (viii) it has an ion transport protein domain which is preferably about 70%, 80%, 90% or 95% similar with amino acids 472-661 of SEQ ID NO: 2, amino acids 247-467 of SEQ ID NO: 5, or amino acids 198-383 of SEQ ID NO: 8;

[0196] (ix) it has a cyclic nucleotide-binding domain which is preferably about 70%, 80%, 90% or 95% similar with amino acids 565-655 of SEQ ID NO: 5;

[0197] (x) it has a potassium channel tetramerisation domain which is preferably about 70%, 80%, 90% or 95% similar with amino acids 3-101 of SEQ ID NO: 8; or

[0198] (xi) it has least 70%, preferably 80%, and most preferably 90% of the cysteines found amino acid sequence of the native protein.

[0199] In a preferred embodiment the 52906, 33408, or 12189 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 by at least one residue but less than 20%,15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the ion transport protein domain. In another preferred embodiment one or more differences are in the ion transport protein domain.

[0200] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 52906, 33408, or 12189 proteins differ in amino acid sequence from SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, yet retain biological activity.

[0201] In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ED NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.

[0202] A 52906 protein or fragment is provided which varies from the sequence of SEQ ID NO: 2 in regions defined by amino acids about 1-471 and/or 662-847 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 2 in regions defined by amino acids about 472-661. A 33408 protein or fragment is provided which varies from the sequence of SEQ ID NO: 5 in regions defined by amino acids about 1-246 and/or 468-988 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 2 in regions defined by amino acids about 247-467. A 12189 protein or fragment is provided which varies from the sequence of SEQ ID NO: 8 in regions defined by amino acids about 1-197 and/or 384-446 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 2 in regions defined by amino acids about 198-383. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[0203] In one embodiment, a biologically active portion of a 52906, 33408, or 12189 protein includes an ion transport protein domain, a cyclic nucleotide-binding domain, a potassium channel tetramerisation domain, a transmembrane domain, a cytoplasmic domain, an extracellular domain, a Pore-loop domain, or a PAS domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 52906, 33408, or 12189 protein.

[0204] In a preferred embodiment, the 52906, 33408, or 12189 protein has an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In other embodiments, the 52906, 33408, or 12189 protein is substantially identical to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In yet another embodiment, the 52906, 33408, or 12189 protein is substantially identical to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 and retains the functional activity of the protein of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, as described in detail in the subsections above.

[0205] 52906, 33408, or 12189 Chimeric or Fusion Proteins

[0206] In another aspect, the invention provides 52906, 33408, or 12189 chimeric or fusion proteins. As used herein, a 52906, 33408, or 12189 “chimeric protein” or “fusion protein” includes a 52906, 33408, or 12189 polypeptide linked to a non-52906, 33408, or 12189 polypeptide. A “non-52906, 33408, or 12189 polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 52906, 33408, or 12189 protein, e.g., a protein which is different from the 52906, 33408, or 12189 protein and which is derived from the same or a different organism. The 52906, 33408, or 12189 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a 52906, 33408, or 12189 amino acid sequence. In a preferred embodiment, a 52906, 33408, or 12189 fusion protein includes at least one (or two) biologically active portion of a 52906, 33408, or 12189 protein. The non-52906, 33408, or 12189 polypeptide can be fused to the N-terminus or C-terminus of the 52906, 33408, or 12189 polypeptide.

[0207] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-52906, 33408, or 12189 fusion protein in which the 52906, 33408, or 12189 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 52906, 33408, or 12189. Alternatively, the fusion protein can be a 52906, 33408, or 12189 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 52906, 33408, or 12189 can be increased through use of a heterologous signal sequence.

[0208] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[0209] The 52906, 33408, or 12189 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 52906, 33408, or 12189 fusion proteins can be used to affect the bioavailability of a 52906, 33408, or 12189 substrate. 52906, 33408, or 12189 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 52906, 33408, or 12189 protein; (ii) mis-regulation of the 52906, 33408, or 12189 gene; and (iii) aberrant post-translational modification of a 52906, 33408, or 12189 protein.

[0210] Moreover, the 52906, 33408, or 12189 -fusion proteins of the invention can be used as immunogens to produce anti-52906, 33408, or 12189 antibodies in a subject, to purify 52906, 33408, or 12189 ligands and in screening assays to identify molecules which inhibit the interaction of 52906, 33408, or 12189 with a 52906, 33408, or 12189 substrate.

[0211] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 52906, 33408, or 12189-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 52906, 33408, or 12189 protein.

[0212] Variants of 52906, 33408, or 12189 Proteins

[0213] In another aspect, the invention also features a variant of a 52906, 33408, or 12189 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 52906, 33408, or 12189 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 52906, 33408, or 12189 protein. An agonist of the 52906, 33408, or 12189 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a 52906, 33408, or 12189 protein. An antagonist of a 52906, 33408, or 12189 protein can inhibit one or more of the activities of the naturally occurring form of the 52906, 33408, or 12189 protein by, for example, competitively modulating a 52906, 33408, or 12189 -mediated activity of a 52906,33408, or 12189 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 52906, 33408, or 12189 protein.

[0214] Variants of a 52906, 33408, or 12189 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 52906, 33408, or 12189 protein for agonist or antagonist activity.

[0215] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 52906, 33408, or 12189 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 52906, 33408, or 12189 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[0216] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 52906, 33408, or 12189 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 52906, 33408, or 12189 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[0217] Cell based assays can be exploited to analyze a variegated 52906, 33408, or 12189 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 52906, 33408, or 12189 in a substrate-dependent manner. The transfected cells are then contacted with 52906, 33408, or 12189 and the effect of the expression of the mutant on signaling by the 52906, 33408, or 12189 substrate can be detected, e.g., by measuring potassium channel activity, e.g., ion flux through a potassium channel. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 52906, 33408, or 12189 substrate, and the individual clones further characterized.

[0218] In another aspect, the invention features a method of making a 52906, 33408, or 12189 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 52906, 33408, or 12189 polypeptide, e.g., a naturally occurring 52906, 33408, or 12189 polypeptide. The method includes: altering the sequence of a 52906, 33408, or 12189 polypeptide, e.g., altering the sequence e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[0219] In another aspect, the invention features a method of making a fragment or analog of a 52906, 33408, or 12189 polypeptide a biological activity of a naturally occurring 52906, 33408, or 12189 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 52906, 33408, or 12189 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[0220] Anti-52906, 33408, or 12189 Antibodies

[0221] In another aspect, the invention provides an anti-52906, 33408, or 12189 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0222] The anti-52906, 33408, or 12189 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0223] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[0224] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 52906, 33408, or 12189 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-52906, 33408, or 12189 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0225] The anti-52906, 33408, or 12189 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[0226] Phage display and combinatorial methods for generating anti-52906, 33408, or 12189 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[0227] In one embodiment, the anti-52906, 33408, or 12189 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art. Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0228] An anti-52906, 33408, or 12189 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[0229] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fe constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fe, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0230] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a 52906, 33408, or 12189 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[0231] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[0232] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 52906, 33408, or 12189 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[0233] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[0234] In preferred embodiments an antibody can be made by immunizing with purified 52906, 33408, or 12189 antigen, or a fragment thereof, e.g., a fragment described herein, membrane associated antigen, tissue, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g., membrane fractions.

[0235] A full-length 52906, 33408, or 12189 protein or, antigenic peptide fragment of 52906, 33408, or 12189 can be used as an immunogen or can be used to identify anti-52906, 33408, or 12189 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 52906, 33408, or 12189 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 and encompasses an epitope of 52906, 33408, or 12189. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0236] Fragments of 52906, 33408, or 12189 which include residues about 241-265 of SEQ ID NO: 2, 710-740 of SEQ ID NO: 5, or 35-55 of SEQ ID NO: 8 can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophilic regions of the 52906, 33408, or 12189 protein. Similarly, fragments of 52906, 33408, or 12189 which include residues about 785-800 of SEQ ID NO: 2, 585-600 of SEQ ID NO: 5, or 75-95 of SEQ ID NO: 8 can be used to make an antibody against a hydrophobic region of the 52906, 33408, or 12189 protein. Fragments of 52906, 33408, or 12189 which include residues 420-432 of SEQ ID NO: 2, 237-244 of SEQ ID NO: 5, or 153-199 of SEQ ID NO: 8 can be used to make an antibody against an extracellular region of the 52906, 33408, or 12189 protein. Fragments of 52906, 33408, or 12189 which include residues 1-401 of SEQ ID NO: 2, 1-218 of SEQ ID NO: 5, or 1-133 of SEQ ID NO: 8 can be used to make an antibody against an intracellular region of the 52906, 33408, or 12189 protein. Fragments of 52906, 33408, or 12189 which include residues 616-639 of SEQ ID NO: 2,420-440 of SEQ ID NO: 5, or 339-355 of SEQ ID NO: 8 can be used to make an antibody against the P-loop region of the 52906, 33408, or 12189 protein. Fragments of 52906, 33408, or 12189 which include amino acids 472-661 of SEQ ID NO: 2, amino acids 247-467 of SEQ ID NO: 5, or amino acids 198-383 of SEQ ID NO: 8 can be used to make an antibody against the ion transport protein domain of the 52906, 33408, or 12189 protein. Fragments of 33408 which include amino acids 565-655 of SEQ ID NO: 5 can be used to make an antibody against the cyclic nucleotide-binding domain of the 33408 protein. Fragments of 12189 which include amino acids 3-101 of SEQ ID NO: 8 can be used to make an antibody against the potassium channel tetramerisation domain of the 12189 protein.

[0237] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[0238] Antibodies which bind only native 52906, 33408, or 12189 protein, only denatured or otherwise non-native 52906, 33408, or 12189 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 52906, 33408, or 12189 protein.

[0239] Preferred epitopes encompassed by the antigenic peptide are regions of 52906, 33408, or 12189 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 52906, 33408, or 12189 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 52906, 33408, or 12189 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[0240] In a preferred embodiment the antibody can bind to the extracellular portion of the 52906, 33408, or 12189 protein, e.g., it can bind to a whole cell which expresses the 52906, 33408, or 12189 protein. In another embodiment, the antibody binds an intracellular portion of the 52906, 33408, or 12189 protein.

[0241] In preferred embodiments antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g., tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g., membrane fractions.

[0242] The anti-52906, 33408, or 12189 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 52906, 33408, or 12189 protein.

[0243] In a preferred embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement.

[0244] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example., it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0245] In a preferred embodiment, an anti-52906, 33408, or 12189 antibody alters (e.g., increases or decreases) an activity of a 52906, 33408, or 12189 polypeptide, e.g., the ability to modulate the flow of K+ ions through a cell membrane and/or the ability to modulate the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell. For example, the antibody can bind at or in proximity to a Pore loop domain, e.g., to an epitope that includes a residue located from about 616-639 of SEQ ID NO: 2, 420-440 of SEQ ID NO: 5, or 339-355 of SEQ ID NO: 8.

[0246] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[0247] An anti-52906, 33408, or 12189 antibody (e.g., monoclonal antibody) can be used to isolate 52906, 33408, or 12189 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-52906, 33408, or 12189 antibody can be used to detect 52906, 33408, or 12189 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-52906, 33408, or 12189 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[0248] The invention also includes a nucleic acids which encodes an anti-52906, 33408, or 12189 antibody, e.g., an anti-52906, 33408, or 12189 antibody described herein. Also included are vectors which include the nucleic acid and sells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[0249] The invention also includes cell lines, e.g., hybridomas, which make an anti-52906, 33408, or 12189 antibody, e.g., and antibody described herein, and method of using said cells to make a 52906, 33408, or 12189 antibody.

[0250] 52906, 33408, and 12189 Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[0251] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[0252] A vector can include a 52906, 33408, or 12189 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 52906, 33408, or 12189 proteins, mutant forms of 52906, 33408, or 12189 proteins, fusion proteins, and the like).

[0253] The recombinant expression vectors of the invention can be designed for expression of 52906, 33408, or 12189 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0254] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0255] Purified fusion proteins can be used in 52906, 33408, or 12189 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 52906, 33408, or 12189 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[0256] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0257] The 52906, 33408, or 12189 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[0258] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[0259] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

[0260] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0261] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[0262] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 52906, 33408, or 12189 nucleic acid molecule within a recombinant expression vector or a 52906, 33408, or 12189 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0263] A host cell can be any prokaryotic or eukaryotic cell. For example, a 52906, 33408, or 12189 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Cell I 23:175-182)). Other suitable host cells are known to those skilled in the art.

[0264] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[0265] A host cell of the invention can be used to produce (i.e., express) a 52906, 33408, or 12189 protein. Accordingly, the invention further provides methods for producing a 52906, 33408, or 12189 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a 52906, 33408, or 12189 protein has been introduced) in a suitable medium such that a 52906, 33408, or 12189 protein is produced. In another embodiment, the method further includes isolating a 52906, 33408, or 12189 protein from the medium or the host cell.

[0266] In another aspect, the invention features, a cell or purified preparation of cells which include a 52906, 33408, or 12189 transgene, or which otherwise misexpress 52906, 33408, or 12189. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a 52906, 33408, or 12189 transgene, e.g., a heterologous form of a 52906, 33408, or 12189, e.g., a gene derived from humans (in the case of a non-human cell). The 52906, 33408, or 12189 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 52906, 33408, or 12189, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 52906, 33408, or 12189 alleles or for use in drug screening.

[0267] In another aspect, the invention features, a human cell, e.g., a neuronal cell or a muscle cell, transformed with nucleic acid which encodes a subject 52906, 33408, or 12189 polypeptide.

[0268] Also provided are cells, preferably human cells, e.g., a neuronal cell, a muscle cell, a hematopoietic cell, or a fibroblast cell, in which an endogenous 52906, 33408, or 12189 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 52906, 33408, or 12189 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 52906, 33408, or 12189 gene. For example, an endogenous 52906, 33408, or 12189 gene which is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[0269] In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 52906, 33408, or 12189 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Production of 52906, 33408, or 12189 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 52906, 33408, or 12189 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

[0270] 52906. 33408, and 12189 Transgenic Animals

[0271] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 52906, 33408, or 12189 protein and for identifying and/or evaluating modulators of 52906, 33408, or 12189 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 52906, 33408, or 12189 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0272] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 52906, 33408, or 12189 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 52906, 33408, or 12189 transgene in its genome and/or expression of 52906, 33408, or 12189 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 52906, 33408, or 12189 protein can further be bred to other transgenic animals carrying other transgenes.

[0273] 52906, 33408, or 12189 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[0274] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[0275] Uses of 52906, 33408, and 12189

[0276] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharrnacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[0277] The isolated nucleic acid molecules of the invention can be used, for example, to express a 52906, 33408, or 12189 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a 52906, 33408, or 12189 mRNA (e.g., in a biological sample) or a genetic alteration in a 52906, 33408, or 12189 gene, and to modulate 52906, 33408, or 12189 activity, as described further below. The 52906, 33408, or 12189 proteins can be used to treat disorders characterized by insufficient or excessive production of a 52906, 33408, or 12189 substrate or production of 52906, 33408, or 12189 inhibitors. In addition, the 52906, 33408, or 12189 proteins can be used to screen for naturally occurring 52906, 33408, or 12189 substrates, to screen for drugs or compounds which modulate 52906, 33408, or 12189 activity, as well as to treat disorders characterized by insufficient or excessive production of 52906, 33408, or 12189 protein or production of 52906, 33408, or 12189 protein forms which have decreased, aberrant or unwanted activity compared to 52906, 33408, or 12189 wild type protein (e.g., disorders characterized by abnormal ion flux such as neurological disorders or cardiac disorders). Moreover, the anti-52906, 33408, or 12189 antibodies of the invention can be used to detect and isolate 52906, 33408, or 12189 proteins, regulate the bioavailability of 52906, 33408, or 12189 proteins, and modulate 52906, 33408, or 12189 activity.

[0278] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 52906, 33408, or 12189 polypeptide is provided. The method includes: contacting the compound with the subject 52906, 33408, or 12189 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 52906, 33408, or 12189 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 52906, 33408, or 12189 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 52906, 33408, or 12189 polypeptide. Screening methods are discussed in more detail below.

[0279] 52906 33408, and 12189 Screening Assays

[0280] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 52906, 33408, or 12189 proteins, have a stimulatory or inhibitory effect on, for example, 52906, 33408, or 12189 expression or 52906, 33408, or 12189 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 52906, 33408, or 12189 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 52906, 33408, or 12189 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[0281] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 52906, 33408, or 12189 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 52906, 33408, or 12189 protein or polypeptide or a biologically active portion thereof.

[0282] In one embodiment, an activity of a 52906, 33408, or 12189 protein can be assayed by measuring the flow of K+ ions through a cell membrane and/or by measuring the transmission of signals in an electrically excitable cell, e.g., a neuronal cell or a muscle cell. For example, an activity of a 52906, 33408, or 12189 protein can be assayed by measuring membrane currents as described in Köhler et al. (1996) Science 273:1709-1714, Saganich et al. (1999) J. Neuroscience 19:10789-10802, or Kalman et al. (1998) J. Biol. Chem. 273:5851-5857.

[0283] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[0284] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[0285] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[0286] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 52906, 33408, or 12189 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 52906, 33408, or 12189 activity is determined. Determining the ability of the test compound to modulate 52906, 33408, or 12189 activity can be accomplished by monitoring, for example, potassium channel activity, e.g., ion flux through a potassium channel. The cell, for example, can be of mammalian origin, e.g., human.

[0287] The ability of the test compound to modulate 52906, 33408, or 12189 binding to a compound, e.g., a 52906, 33408, or 12189 substrate, or to bind to 52906, 33408, or 12189 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 52906, 33408, or 12189 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 52906, 33408, or 12189 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 52906, 33408, or 12189 binding to a 52906, 33408, or 12189 substrate in a complex. For example, compounds (e.g., 52906, 33408, or 12189 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0288] The ability of a compound (e.g., a 52906, 33408, or 12189 substrate) to interact with 52906, 33408, or 12189 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 52906, 33408, or 12189 without the labeling of either the compound or the 52906, 33408, or 12189. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 52906, 33408, or 12189.

[0289] In yet another embodiment, a cell-free assay is provided in which a 52906, 33408, or 12189 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 52906, 33408, or 12189 protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the 52906, 33408, or 12189 proteins to be used in assays of the present invention include fragments which participate in interactions with non-52906, 33408, or 12189 molecules, e.g., fragments with high surface probability scores.

[0290] Soluble and/or membrane-bound forms of isolated proteins (e.g., 52906, 33408, or 12189 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Tritong® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPS O), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0291] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[0292] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[0293] In another embodiment, determining the ability of the 52906, 33408, or 12189 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[0294] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[0295] It maybe desirable to immobilize either 52906, 33408, or 12189, an anti-52906, 33408, or 12189 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 52906, 33408, or 12189 protein, or interaction of a 52906, 33408, or 12189 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/52906, 33408, or 12189 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigmna Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 52906, 33408, or 12189 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 52906, 33408, or 12189 binding or activity determined using standard techniques.

[0296] Other techniques for immobilizing either a 52906, 33408, or 12189 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 52906, 33408, or 12189 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[0297] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[0298] In one embodiment, this assay is performed utilizing antibodies reactive with 52906, 33408, or 12189 protein or target molecules but which do not interfere with binding of the 52906, 33408, or 12189 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 52906, 33408, or 12189 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 52906, 33408, or 12189 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 52906, 33408, or 12189 protein or target molecule.

[0299] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[0300] In a preferred embodiment, the assay includes contacting the 52906, 33408, or 12189 protein or biologically active portion thereof with a known compound which binds 52906, 33408, or 12189 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 52906, 33408, or 12189 protein, wherein determining the ability of the test compound to interact with a 52906, 33408, or 12189 protein includes determining the ability of the test compound to preferentially bind to 52906, 33408, or 12189 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[0301] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 52906, 33408, or 12189 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a 52906, 33408, or 12189 protein through modulation of the activity of a downstream effector of a 52906, 33408, or 12189 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[0302] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[0303] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[0304] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[0305] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[0306] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[0307] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[0308] In yet another aspect, the 52906, 33408, or 12189 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 52906, 33408, or 12189 (“52906, 33408, or 12189-binding proteins” or “52906, 33408, or 12189-bp”) and are involved in 52906, 33408, or 12189 activity. Such 52906, 33408, or 12189-bps can be activators or inhibitors of signals by the 52906, 33408, or 12189 proteins or 52906, 33408, or 12189 targets as, for example, downstream elements of a 52906, 33408, or 12189-mediated signaling pathway.

[0309] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 52906, 33408, or 12189 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 52906, 33408, or 12189 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 52906, 33408, or 12189-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 52906, 33408, or 12189 protein.

[0310] In another embodiment, modulators of 52906, 33408, or 12189 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 52906, 33408, or 12189 mRNA or protein evaluated relative to the level of expression of 52906, 33408, or 12189 mRNA or protein in the absence of the candidate compound. When expression of 52906, 33408, or 12189 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 52906, 33408, or 12189 mRNA or protein expression. Alternatively, when expression of 52906, 33408, or 12189 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 52906, 33408, or 12189 mRNA or protein expression. The level of 52906, 33408, or 12189 mRNA or protein expression can be determined by methods described herein for detecting 52906, 33408, or 12189 mRNA or protein.

[0311] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a 52906, 33408, or 12189 protein can be confirmed in vivo, e.g., in an animal such as an animal model for a disorder characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder.

[0312] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 52906, 33408, or 12189 modulating agent, an antisense 52906, 33408, or 12189 nucleic acid molecule, a 52906, 33408, or 12189-specific antibody, or a 52906, 33408, or 12189-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[0313] 52906, 33408, and 12189 Detection Assays

[0314] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 52906, 33408, or 12189 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[0315] 52906, 33408, and 12189 Chromosome Mapping

[0316] The 52906, 33408, or 12189 nucleotide sequences or portions thereof can be used to map the location of the 52906, 33408, or 12189 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 52906, 33408, or 12189 sequences with genes associated with disease.

[0317] Briefly, 52906, 33408, or 12189 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 52906, 33408, or 12189 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 52906, 33408, or 12189 sequences will yield an amplified fragment.

[0318] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[0319] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 52906, 33408, or 12189 to a chromosomal location.

[0320] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[0321] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[0322] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[0323] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 52906, 33408, or 12189 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[0324] 52906, 33408, and 12189 Tissue Typing

[0325] 52906, 33408, or 12189 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[0326] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 52906, 33408, or 12189 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[0327] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0328] If a panel of reagents from 52906, 33408, or 12189 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[0329] Use of Partial 52906. 33408, or 12189 Sequences in Forensic Biology

[0330] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[0331] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[0332] The 52906, 33408, or 12189 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 52906, 33408, or 12189 probes can be used to identify tissue by species and/or by organ type.

[0333] In a similar fashion, these reagents, e.g., 52906, 33408, or 12189 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[0334] Predictive Medicine of 52906. 33408, and 12189

[0335] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[0336] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 52906, 33408, or 12189.

[0337] Such disorders include, e.g., a disorder associated with the misexpression of 52906, 33408, or 12189 gene, or a disorder characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder.

[0338] The method includes one or more of the following:

[0339] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 52906, 33408, or 12189 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[0340] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 52906, 33408, or 12189 gene;

[0341] detecting, in a tissue of the subject, the misexpression of the 52906, 33408, or 12189 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[0342] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 52906, 33408, or 12189 polypeptide.

[0343] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 52906, 33408, or 12189 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[0344] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 52906, 33408, or 12189 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[0345] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 52906, 33408, or 12189 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 52906, 33408, or 12189.

[0346] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[0347] In preferred embodiments the method includes determining the structure of a 52906, 33408, or 12189 gene, an abnormal structure being indicative of risk for the disorder.

[0348] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 52906, 33408, or 12189 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[0349] Diagnostic and Prognostic Assays of 52906, 33408, and 12189

[0350] Diagnostic and prognostic assays of the invention include methods for assessing the expression level of 52906, 33408, or 12189 molecules and for identifying variations and mutations in the sequence of 52906, 33408, or 12189 molecules.

[0351] Expression Monitoring and Profiling:

[0352] The presence, level, or absence of 52906, 33408, or 12189 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 52906, 33408, or 12189 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 52906, 33408, or 12189 protein such that the presence of 52906, 33408, or 12189 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 52906, 33408, or 12189 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 52906, 33408, or 12189 genes; measuring the amount of protein encoded by the 52906, 33408, or 12189 genes; or measuring the activity of the protein encoded by the 52906, 33408, or 12189 genes.

[0353] The level of mRNA corresponding to the 52906, 33408, or 12189 gene in a cell can be determined both by in situ and by in vitro formats.

[0354] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 52906, 33408, or 12189 nucleic acid, such as the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 52906, 33408, or 12189 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[0355] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 52906, 33408, or 12189 genes.

[0356] The level of mRNA in a sample that is encoded by one of 52906, 33408, or 12189 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[0357] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 52906, 33408, or 12189 gene being analyzed.

[0358] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 52906, 33408, or 12189 mRNA, or genomic DNA, and comparing the presence of 52906, 33408, or 12189 mRNA or genomic DNA in the control sample with the presence of 52906, 33408, or 12189 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 52906, 33408, or 12189 transcript levels.

[0359] A variety of methods can be used to determine the level of protein encoded by 52906, 33408, or 12189. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[0360] The detection methods can be used to detect 52906, 33408, or 12189 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 52906, 33408, or 12189 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 52906, 33408, or 12189 protein include introducing into a subject a labeled anti-52906, 33408, or 12189 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-52906, 33408, or 12189 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[0361] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 52906, 33408, or 12189 protein, and comparing the presence of 52906, 33408, or 12189 protein in the control sample with the presence of 52906, 33408, or 12189 protein in the test sample.

[0362] The invention also includes kits for detecting the presence of 52906, 33408, or 12189 in a biological sample. For example, the kit can include a compound or agent capable of detecting 52906, 33408, or 12189 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 52906, 33408, or 12189 protein or nucleic acid.

[0363] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[0364] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[0365] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 52906, 33408, or 12189 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such disorders characterized by abnormal ion flux such as neurological disorders or cardiac disorders.

[0366] In one embodiment, a disease or disorder associated with aberrant or unwanted 52906, 33408, or 12189 expression or activity is identified. A test sample is obtained from a subject and 52906, 33408, or 12189 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 52906, 33408, or 12189 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 52906, 33408, or 12189 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[0367] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 52906, 33408, or 12189 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for disorders characterized by abnormal ion flux such as neurological disorders or cardiac disorders.

[0368] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 52906, 33408, or 12189 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 52906, 33408, or 12189 (e.g., other genes associated with a 52906, 33408, or 12189 -disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[0369] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 52906, 33408, or 12189 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose an ion flux-related disorder in a subject wherein a modulation (increase or decrease) in 52906, 33408, or 12189 expression is an indication that the subject has or is disposed to having a disorder characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder. The method can be used to monitor a treatment for an ion flux-related disorder in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[0370] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 52906, 33408, or 12189 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[0371] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 52906, 33408, or 12189 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[0372] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[0373] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 52906, 33408, or 12189 expression.

[0374] 52906, 33408, and 12189 Arrays and Uses Thereof

[0375] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a 52906, 33408, or 12189 molecule (e.g., a 52906, 33408, or 12189 nucleic acid or a 52906, 33408, or 12189 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm2, and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[0376] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a 52906, 33408, or 12189 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 52906, 33408, or 12189. Each address of the subset can include a capture probe that hybridizes to a different region of a 52906, 33408, or 12189 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for a 52906, 33408, or 12189 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 52906, 33408, or 12189 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 52906, 33408, or 12189 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[0377] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[0378] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a 52906, 33408, or 12189 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 52906, 33408, or 12189 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-52906, 33408, or 12189 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[0379] In another aspect, the invention features a method of analyzing the expression of 52906, 33408, or 12189. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 52906, 33408, or 12189-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[0380] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 52906, 33408, or 12189. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 52906, 33408, or 12189. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[0381] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 52906, 33408, or 12189 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[0382] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[0383] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 52906, 33408, or 12189-associated disease or disorder; and processes, such as a cellular transformation associated with a 52906, 33408, or 12189-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 52906, 33408, or 12189-associated disease or disorder

[0384] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 52906, 33408, or 12189 ) that could serve as a molecular target for diagnosis or therapeutic intervention.

[0385] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon a 52906, 33408, or 12189 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80, 85, 90, 95 or 99% identical to a 52906, 33408, or 12189 polypeptide or fragment thereof. For example, multiple variants of a 52906, 33408, or 12189 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[0386] The polypeptide array can be used to detect a 52906, 33408, or 12189 binding compound, e.g., an antibody in a sample from a subject with specificity for a 52906, 33408, or 12189 polypeptide or the presence of a 52906, 33408, or 12189-binding protein or ligand.

[0387] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 52906, 33408, or 12189 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[0388] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 52906, 33408, or 12189 or from a cell or subject in which a 52906, 33408, or 12189 mediated response has been elicited, e.g., by contact of the cell with 52906, 33408, or 12189 nucleic acid or protein, or administration to the cell or subject 52906, 33408, or 12189 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 52906, 33408, or 12189 (or does not express as highly as in the case of the 52906, 33408, or 12189 positive plurality of capture probes) or from a cell or subject which in which a 52906, 33408, or 12189 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 52906, 33408, or 12189 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[0389] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 52906, 33408, or 12189 or from a cell or subject in which a 52906, 33408, or 12189-mediated response has been elicited, e.g., by contact of the cell with 52906, 33408, or 12189 nucleic acid or protein, or administration to the cell or subject 52906, 33408, or 12189 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 52906, 33408, or 12189 (or does not express as highly as in the case of the 52906, 33408, or 12189 positive plurality of capture probes) or from a cell or subject which in which a 52906, 33408, or 12189 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[0390] In another aspect, the invention features a method of analyzing 52906, 33408, or 12189, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 52906, 33408, or 12189 nucleic acid or amino acid sequence; comparing the 52906, 33408, or 12189 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 52906, 33408, or 12189.

[0391] Detection of 52906, 33408, and 12189 Variations or Mutations

[0392] The methods of the invention can also be used to detect genetic alterations in a 52906, 33408, or 12189 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 52906, 33408, or 12189 protein activity or nucleic acid expression, such as a disorder characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 52906, 33408, or 12189 -protein, or the mis-expression of the 52906, 33408, or 12189 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 52906, 33408, or 12189 gene; 2) an addition of one or more nucleotides to a 52906, 33408, or 12189 gene; 3) a substitution of one or more nucleotides of a 52906, 33408, or 12189 gene, 4) a chromosomal rearrangement of a 52906, 33408, or 12189 gene; 5) an alteration in the level of a messenger RNA transcript of a 52906, 33408, or 12189 gene, 6) aberrant modification of a 52906, 33408, or 12189 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a 52906, 33408, or 12189 gene, 8) a non-wild type level of a 52906, 33408, or 12189 -protein, 9) allelic loss of a 52906, 33408, or 12189 gene, and 10) inappropriate post-translational modification of a 52906, 33408, or 12189-protein.

[0393] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 52906, 33408, or 12189-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 52906, 33408, or 12189 gene under conditions such that hybridization and amplification of the 52906, 33408, or 12189-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[0394] In another embodiment, mutations in a 52906, 33408, or 12189 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0395] In other embodiments, genetic mutations in 52906, 33408, or 12189 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 52906, 33408, or 12189 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 52906, 33408, or 12189 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 52906, 33408, or 12189 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[0396] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 52906, 33408, or 12189 gene and detect mutations by comparing the sequence of the sample 52906, 33408, or 12189 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[0397] Other methods for detecting mutations in the 52906, 33408, or 12189 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[0398] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 52906, 33408, or 12189 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[0399] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 52906, 33408, or 12189 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 52906, 33408, or 12189 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0400] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[0401] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[0402] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0403] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 52906, 33408, or 12189 nucleic acid.

[0404] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 or the complement of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[0405] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 52906, 33408, or 12189. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[0406] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the Tm of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[0407] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 52906, 33408, or 12189 nucleic acid.

[0408] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 52906, 33408, or 12189 gene.

[0409] Use of 52906, 33408, or 12189 Molecules as Surrogate Markers

[0410] The 52906, 33408, or 12189 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 52906, 33408, or 12189 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 52906, 33408, or 12189 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[0411] The 52906, 33408, or 12189 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 52906, 33408, or 12189 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-52906, 33408, or 12189 antibodies may be employed in an immune-based detection system for a 52906, 33408, or 12189 protein marker, or 52906, 33408, or 12189 -specific radiolabeled probes may be used to detect a 52906, 33408, or 12189 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[0412] The 52906, 33408, or 12189 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 52906, 33408, or 12189 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 52906, 33408, or 12189 DNA may correlate 52906, 33408, or 12189 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[0413] Pharmaceutical Compositions of 52906, 33408, and 12189

[0414] The nucleic acid and polypeptides, fragments thereof, as well as anti-52906, 33408, or 12189 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0415] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0416] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0417] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0418] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0419] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0420] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0421] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0422] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0423] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0424] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0425] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0426] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0427] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[0428] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0429] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0430] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0431] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[0432] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[0433] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0434] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0435] Methods of Treatment for 52906, 33408. and 12189

[0436] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 52906, 33408, or 12189 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[0437] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 52906, 33408, or 12189 molecules of the present invention or 52906, 33408, or 12189 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0438] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 52906, 33408, or 12189 expression or activity, by administering to the subject a 52906, 33408, or 12189 or an agent which modulates 52906, 33408, or 12189 expression or at least one 52906, 33408, or 12189 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 52906, 33408, or 12189 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 52906, 33408, or 12189 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 52906, 33408, or 12189 aberrance, for example, a 52906, 33408, or 12189, 52906, 33408, or 12189 agonist or 52906, 33408, or 12189 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[0439] It is possible that some 52906, 33408, or 12189 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[0440] The 52906, 33408, or 12189 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, liver disorders, viral diseases, pain or metabolic disorders.

[0441] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[0442] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[0443] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0444] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0445] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0446] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promycloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.

[0447] Aberrant expression and/or activity of 52906, 33408, or 12189 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 52906, 33408, or 12189 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 52906, 33408, or 12189 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 52906, 33408, or 12189 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[0448] The 52906, 33408, or 12189 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders. Examples of immune disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyclitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

[0449] Disorders which may be treated or diagnosed by methods described herein include, but are not limited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, Al-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

[0450] Additionally, 52906, 33408, or 12189 molecules may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 52906, 33408, or 12189 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 52906, 33408, or 12189 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

[0451] Additionally, 52906, 33408, or 12189 may play an important role in the regulation of metabolism or pain disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various formns of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[0452] As discussed, successful treatment of 52906, 33408, or 12189 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 52906, 33408, or 12189 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[0453] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[0454] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[0455] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 52906, 33408, or 12189 expression is through the use of aptamer molecules specific for 52906, 33408, or 12189 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 52906, 33408, or 12189 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[0456] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 52906, 33408, or 12189 disorders. For a description of antibodies, see the Antibody section above.

[0457] In circumstances wherein injection of an animal or a human subject with a 52906, 33408, or 12189 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 52906, 33408, or 12189 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chatteiee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 52906, 33408, or 12189 protein. Vaccines directed to a disease characterized by 52906, 33408, or 12189 expression may also be generated in this fashion.

[0458] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0459] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 52906, 33408, or 12189 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[0460] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0461] Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 52906, 33408, or 12189 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 52906, 33408, or 12189 can be readily monitored and used in calculations of IC50.

[0462] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[0463] Another aspect of the invention pertains to methods of modulating 52906, 33408, or 12189 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 52906, 33408, or 12189 or agent that modulates one or more of the activities of 52906, 33408, or 12189 protein activity associated with the cell. An agent that modulates 52906, 33408, or 12189 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a 52906, 33408, or 12189 protein (e.g., a 52906, 33408, or 12189 substrate or receptor), a 52906, 33408, or 12189 antibody, a 52906, 33408, or 12189 agonist or antagonist, a peptidomimetic of a 52906, 33408, or 12189 agonist or antagonist, or other small molecule.

[0464] In one embodiment, the agent stimulates one or 52906, 33408, or 12189 activities. Examples of such stimulatory agents include active 52906, 33408, or 12189 protein and a nucleic acid molecule encoding 52906, 33408, or 12189. In another embodiment, the agent inhibits one or more 52906, 33408, or 12189 activities. Examples of such inhibitory agents include antisense 52906, 33408, or 12189 nucleic acid molecules, anti-52906, 33408, or 12189 antibodies, and 52906, 33408, or 12189 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 52906, 33408, or 12189 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 52906, 33408, or 12189 expression or activity. In another embodiment, the method involves administering a 52906, 33408, or 12189 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 52906, 33408, or 12189 expression or activity.

[0465] Stimulation of 52906, 33408, or 12189 activity is desirable in situations in which 52906, 33408, or 12189 is abnormally downregulated and/or in which increased 52906, 33408, or 12189 activity is likely to have a beneficial effect. For example, stimulation of 52906, 33408, or 12189 activity is desirable in situations in which a 52906, 33408, or 12189 is downregulated and/or in which increased 52906, 33408, or 12189 activity is likely to have a beneficial effect. Likewise, inhibition of 52906, 33408, or 12189 activity is desirable in situations in which 52906, 33408, or 12189 is abnormally upregulated and/or in which decreased 52906, 33408, or 12189 activity is likely to have a beneficial effect.

[0466] 52906, 33408, and 12189 Pharmacogenomics

[0467] The 52906, 33408, or 12189 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 52906, 33408, or 12189 activity (e.g., 52906, 33408, or 12189 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 52906, 33408, or 12189 associated disorders (e.g., a disorder characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder) associated with aberrant or unwanted 52906, 33408, or 12189 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 52906, 33408, or 12189 molecule or 52906, 33408, or 12189 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 52906, 33408, or 12189 molecule or 52906, 33408, or 12189 modulator.

[0468] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitroflirans) and consumption of fava beans.

[0469] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[0470] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 52906, 33408, or 12189 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[0471] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 52906, 33408, or 12189 molecule or 52906, 33408, or 12189 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[0472] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 52906, 33408, or 12189 molecule or 52906, 33408, or 12189 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[0473] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 52906, 33408, or 12189 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 52906, 33408, or 12189 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[0474] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a 52906, 33408, or 12189 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 52906, 33408, or 12189 gene expression, protein levels, or upregulate 52906, 33408, or 12189 activity, can be monitored in clinical trials of subjects exhibiting decreased 52906, 33408, or 12189 gene expression, protein levels, or downregulated 52906, 33408, or 12189 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 52906, 33408, or 12189 gene expression, protein levels, or downregulate 52906, 33408, or 12189 activity, can be monitored in clinical trials of subjects exhibiting increased 52906, 33408, or 12189 gene expression, protein levels, or upregulated 52906, 33408, or 12189 activity. In such clinical trials, the expression or activity of a 52906, 33408, or 12189 gene, and preferably, other genes that have been implicated in, for example, a 52906, 33408, or 12189-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[0475] 52906, 33408. or 12189 Informatics

[0476] The sequence of a 52906, 33408, or 12189 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 52906, 33408, or 12189. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 52906, 33408, or 12189 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[0477] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[0478] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[0479] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[0480] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[0481] Thus, in one aspect, the invention features a method of analyzing 52906, 33408, or 12189, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 52906, 33408, or 12189 nucleic acid or amino acid sequence; comparing the 52906, 33408, or 12189 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 52906, 33408, or 12189. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[0482] The method can include evaluating the sequence identity between a 52906, 33408, or 12189 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[0483] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[0484] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[0485] Thus, the invention features a method of making a computer readable record of a sequence of a 52906, 33408, or 12189 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[0486] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 52906, 33408, or 12189 sequence, or record, in machine-readable form; comparing a second sequence to the 52906, 33408, or 12189 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 52906, 33408, or 12189 sequence includes a sequence being compared. In a preferred embodiment the 52906, 33408, or 12189 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 52906, 33408, or 12189 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[0487] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 52906, 33408, or 12189-associated disease or disorder or a pre-disposition to a 52906, 33408, or 12189-associated disease or disorder, wherein the method comprises the steps of determining 52906, 33408, or 12189 sequence information associated with the subject and based on the 52906, 33408, or 12189 sequence information, determining whether the subject has a 52906, 33408, or 12189-associated disease or disorder or a pre-disposition to a 52906, 33408, or 12189-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[0488] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 52906, 33408,,or 12189-associated disease or disorder or a pre-disposition to a disease associated with a 52906, 33408, or 12189 wherein the method comprises the steps of determining 52906, 33408, or 12189 sequence information associated with the subject, and based on the 52906, 33408, or 12189 sequence information, determining whether the subject has a 52906, 33408, or 12189-associated disease or disorder or a pre-disposition to a 52906, 33408, or 12189-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 52906, 33408, or 12189 sequence of the subject to the 52906, 33408, or 12189 sequences in the database to thereby determine whether the subject as a 52906, 33408, or 12189-associated disease or disorder, or a pre-disposition for such.

[0489] The present invention also provides in a network, a method for determining whether a subject has a 52906, 33408, or 12189 associated disease or disorder or a pre-disposition to a 52906, 33408, or 12189-associated disease or disorder associated with 52906, 33408, or 12189, said method comprising the steps of receiving 52906, 33408, or 12189 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 52906, 33408, or 12189 and/or corresponding to a 52906, 33408, or 12189-associated disease or disorder (e.g., a disorder characterized by abnormal ion flux such as a neurological disorder or a cardiac disorder), and based on one or more of the phenotypic information, the 52906, 33408, or 12189 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 52906, 33408, or 12189-associated disease or disorder or a pre-disposition to a 52906, 33408, or 12189-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0490] The present invention also provides a method for determining whether a subject has a 52906, 33408, or 12189-associated disease or disorder or a pre-disposition to a 52906, 33408, or 12189-associated disease or disorder, said method comprising the steps of receiving information related to 52906, 33408, or 12189 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 52906, 33408, or 12189 and/or related to a 52906, 33408, or 12189-associated disease or disorder, and based on one or more of the phenotypic information, the 52906, 33408, or 12189 information, and the acquired information, determining whether the subject has a 52906, 33408, or 12189-associated disease or disorder or a pre-disposition to a 52906, 33408, or 12189-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0491] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

BACKGROUND OF THE 21784 INVENTION

[0492] Calcium signaling has been implicated in the regulation of a variety of cellular responses, such as growth and differentiation. There are two general methods by which intracellular concentrations of calcium ions may be increased: calcium ions may be brought into the cell from the extracellular milieu through the use of specific channels in the cellular membrane, or calcium ions may be freed from intracellular stores, again being transported by specific membrane channels in the storage organelle. In the situation in which the intracellular stores of calcium have been depleted, a specific type of calcium channel, termed a ‘capacitative calcium channel’ or a ‘store-operated calcium channel’ (SOC), is activated in the plasma membrane to import calcium ions from the extracellular environment to the cytosol (for review, see Putney and McKay (1999) BioEssays 21:38-46).

[0493] Members of the capacitative calcium channel family include the calcium release-activated calcium current (CRAC) (Hoth and Penner (1992) Nature 355: 353-355), calcium release-activated nonselective cation current (CRANC) (Krause et al. (1996) J. Biol. Chem. 271: 32523-32528), and the transient receptor potential (TRP) proteins. There is no single electrophysological profile characteristic of the family; rather, a wide array of single channel conductances, cation selectivity, and current properties have been observed for different specific channels. Further, in several instances it has been demonstrated that homo- or heteropolymerization of the channel molecule may occur, further changing the channel properties from that of the single molecule. In general, though, these channels function similarly, in that they are calcium ion-permeable cation channels that become activated upon stimulation of phospholipase Cβ by a G protein-coupled receptor. Depletion of intracellular calcium stores activate these channels by a mechanism which is as yet undefined, but which has been demonstrated to involve a diffusible factor using studies in which calcium stores were artificially depleted (e.g., by the introduction of chelators into the cell, by activating phospholipase Cγ, or by inhibiting the those enzymes responsible for pumping calcium ions into the stores or those enzymes responsible for maintaining resting intracellular calcium ion concentrations) (Putney, J. W., (1986) Cell Calcium 7: 1-12; Putney, J. W. (1990) Cell Calcium 11:611-624).

[0494] The TRP channel family is one of the best characterized of the capacitative calcium channel group. These channels include transient receptor potential protein and homologues thereof (to date, seven homologs and splice variants have been identified in a variety of organisms), the vanilloid receptor subtype I (also known as the capsaicin receptor), stretch-inhibitable non-selective cation channel (SIC), olfactory, mechanosensitive channel, insulin-like growth factor I-regulated calcium channel, and vitamin D-responsive apical, epithelial calcium channel (ECaC) (see, e.g., Montell and Rubin (1989) Neuron 2:1313-1323; Caterina et al. (1997) Nature 389: 816-824; Suzuki et al. (1999) J. Biol. Chem. 274: 6330-6335; Kiselyov et al. (1998) Nature 396: 478-482; and Hoenderop et al. (1999) J. Biol. Chem. 274: 8375-8378). Each of these molecules is 700 or more amino acids (TRP and TRP homologs have 1300 or more amino acid residues), and shares certain conserved structural features. Predominant among these structural features are six transmembrane domains, with an additional hydrophobic loop present between the fifth and sixth transmembrane domains. It is believed that this loop is integral to the activity of the pore of the channel formed upon membrane insertion (Hardie and Minke (1993) Trends Neurosci 16: 371-376). TRP channel proteins also include one or more ankyrin domains and frequently display a proline-rich region at the N-terminus. Although found in disparate tissues and organisms, members of the TRP channel protein family all serve to transduce signals by means of calcium entry into cells, particularly pain (see, e.g., McClesky and Gold (1999) Annu. Rev. Physiol. 61: 835-856), light (Hardie and Minke, supra), or olfactory signals (Colbert et al. (1997) J. Neurosci 17(21): 8259-8269). Thus, this family of molecules may play important roles in sensory signal transduction in general.

SUMMARY OF THE 21784 INVENTION

[0495] The present invention is based, in part, on the discovery of a novel calcium channel family member, referred to herein as “21784”. The nucleotide sequence of a cDNA encoding 21784 is shown in SEQ ID NO: 14, and the amino acid sequence of a 21784 polypeptide is shown in SEQ ID NO: 15. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 16.

[0496] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 21784 protein or polypeptide, e.g., a biologically active portion of the 21784 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 15. In other embodiments, the invention provides isolated 21784 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 14, SEQ ID NO: 16, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 14, SEQ ID NO: 16, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 16, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 21784 protein or an active fragment thereof.

[0497] In a related aspect, the invention further provides nucleic acid constructs that include a 21784 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 21784 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 21784 nucleic acid molecules and polypeptides.

[0498] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 21784-encoding nucleic acids.

[0499] In still another related aspect, isolated nucleic acid molecules that are antisense to a 21784 encoding nucleic acid molecule are provided.

[0500] In another aspect, the invention features 21784 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 21784-mediated or -related disorders, e.g., a calcium channel associated disorder (e.g., a CNS disorder, such as a neurodegenerative disorder, e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, Jakob-Creutzfieldt disease, AIDS related dementia, familial infantile convulsions, paroxysmal choreoathetosis; a disorder of the conveyance of sensory impulses from the periphery to the brain and/or conductance of motor impulses from the brain to the periphery; a psychiatric disorder (e.g., depression, schizophrenic disorders, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders); a learning or memory disorder (e.g., amnesia or age-related memory loss; and migraine).

[0501] In other embodiments, the invention provides 21784 polypeptides, e.g., a 21784 polypeptide having the amino acid sequence shown in SEQ ID NO: 15 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NQ: 15 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 14, SEQ ID NO: 16, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 21784 protein or an active fragment thereof.

[0502] In a related aspect, the invention further provides nucleic acid constructs which include a 21784 nucleic acid molecule described herein.

[0503] In a related aspect, the invention provides 21784 polypeptides or fragments operatively linked to non-21784 polypeptides to form fusion proteins.

[0504] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 21784 polypeptides or fragments thereof, e.g., an extracellular domain of a 21784 polypeptide. In one embodiment, the antibodies or antigen-binding fragment thereof competitively inhibit the binding of a second antibody to a 21784 polypeptide or a fragment thereof, e.g., an extracellular domain of a 21784 polypeptide.

[0505] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 21784 polypeptides or nucleic acids.

[0506] In still another aspect, the invention provides a process for modulating 21784 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. For example, the screened compounds can be used to modulate a calcium channel mediated activity, including one or more of: membrane excitability, neurite outgrowth and synaptogenesis, signal transduction, cell proliferation, growth, differentiation, and migration, and nociception. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 21784 polypeptides or nucleic acids, such as conditions involving aberrant calcium channel activity, e.g., a neurodegenerative condition.

[0507] The invention also provides assays for determining the activity of or the presence or absence of 21784 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[0508] In yet another aspect, the invention provides methods for modulating the activity (e.g., inhibiting the proliferation, or inducing the differentiation) of a 21784-expressing cell, e.g., a neural, heart, skeletal muscle cell. The method includes contacting the cell with an agent, e.g., a compound, (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 21784 polypeptide or nucleic acid. In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol.

[0509] In a preferred embodiment, the agent, e.g., compound, is an inhibitor of a 21784 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety selected from a cytotoxin, a cytotoxic agent and a radioactive metal ion). In another preferred embodiment, the compound is an inhibitor of a 21784 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.

[0510] In a preferred embodiment, the agent, e.g., compound, is administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation.

[0511] In another aspect, the invention features methods for treating or preventing a disorder characterized by activity of a 21784-expressing cell, in a subject. Preferably, the method includes comprising administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 21784 polypeptide or nucleic acid. In a preferred embodiment, the disorder is a neural (e.g., neuronal or glial cell), cardiovascular, or skeletal muscular disorder. In other embodiments, the disorder is a cancer.

[0512] In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., a neural, cardiovascular, or skeletal muscular disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with a compound identified using the methods described herein); and evaluating the expression of a 21784 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 21784 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 21784 nucleic acid or polypeptide expression can be detected by any method described herein.

[0513] In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 21784 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.

[0514] In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent. The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein) and, evaluating the expression of 21784 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 21784 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 21784 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a cancerous tissue, or heart, vein, brain, kidney, skeletal muscle, adipose, skin, spinal cord, dorsal root ganglion, breast, ovary, prostate, salivary gland, colon, lung, spleen, tonsil, lymph node, small intestine or synovium cells or tissue.

[0515] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 21784 polypeptide or nucleic acid molecule, including for disease diagnosis.

[0516] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 21784 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 21784 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 21784 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[0517] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION OF 21784

[0518] The human 21784 sequence (Example 6; SEQ ID NO: 14), which is approximately 3690 nucleotides long, including untranslated regions, contains a predicted methionine-initiated coding sequence of about 3276 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 14 in Example 6; SEQ ID NO: 16). The coding sequence encodes a 1091 amino acid protein (SEQ ID NO: 15). The human 21784 includes a predicted signal peptide located at amino acid 1 to about amino acid 31 of SEQ ID NO: 15. The mature 21784 protein corresponds to amino acids 32 to 1091 of SEQ ID NO: 15.

[0519] Human 21784 contains the following regions or other structural features:

[0520] three predicted transmembrane regions located at about amino acids 455 to 475, 927 to 947, and 1072 to 1089, of SEQ ID NO: 15;

[0521] a predicted N-terminal extracellular domain located at about amino acids 1-454 of SEQ ID NO: 15;

[0522] a predicted extracellular loop located at about amino acids 948-1071 of SEQ ID NO: 15;

[0523] a predicted intracellular loop located at about amino acids 476-926 of SEQ ID NO: 15;

[0524] a predicted C-terminal extracellular domain located at about amino acids 1090-1091 of SEQ ID NO: 15;

[0525] nine predicted N-glycosylation sites (PS00001) located from about amino acids 166 to 169, 309 to 312, 353 to 356, 488 to 491, 553 to 556, 632 to 635, 714 to 717, 793 to 796, and 1035 to 1038, of SEQ ID NO: 15;

[0526] two predicted cAMP/cGMP protein kinase phosphorylation sites (PS00004) located at about amino acids 8 to 11 and 896 to 899 of SEQ ID NO: 15;

[0527] twelve predicted protein kinase C phosphorylation sites (PS00005) located at about amino acids 6 to 8,216 to 218, 253 to 255, 266 to 268, 318 to 320, 580 to 582, 719 to 721, 894 to 896, 956 to 958, 978 to 980, 981 to 983, and 1037 to 1039, of SEQ ID NO: 15;

[0528] sixteen predicted casein kinase II phosphorylation sites (PS00006) located at about amino acids 168 to 171, 253 to 256, 281 to 284, 285 to 288, 318 to 321, 423 to 426, 535 to 538, 560 to 563, 634 to 637, 648 to 651, 668 to 671, 747 to 750, 848 to 851, 899 to 902, and 981 to 984, of SEQ ID NO: 15;

[0529] thirteen predicted N-myristoylation sites (PS00008) located at about amino acids 188 to 193, 215 to 220, 265 to 270, 358 to 363, 371 to 376, 494 to 499, 611 to 616, 617 to 622, 722 to 727, 729 to 734, 883 to 888, 987 to 992, and 1068 to 1073, of SEQ ID NO: 15;

[0530] two predicted amidation sites (PS00009) at about amino acid residues 545 to 548 and 593 to 596 of SEQ ID NO: 15; and

[0531] a predicted ‘homeobox’ domain signature (PS00027) located at about amino acids 31 to 54 of SEQ ID NO: 15.

[0532] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[0533] A plasmid containing the nucleotide sequence encoding human 21784 (clone “Fbh21784FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0534] The 21784 protein contains a significant number of structural characteristics in common with members of the calcium channel family. In particular, 21784 protein shows homology to the mouse alpha-2 delta-3 calcium channel subunit. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[0535] As used herein, a “calcium channel” includes a protein or polypeptide that is involved in receiving, conducting, and transmitting signals in an electrically excitable cell, e.g., a neuronal or muscular cell. Calcium channels are calcium ion selective, and can determine membrane excitability (the ability of, for example, a muscle cell to respond to a stimulus and to convert it into an impulse resulting in a contraction). Calcium channels can also influence the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation. Calcium channels are typically expressed in electrically excitable cells, e.g., neuronal or muscle cells, and may form heteromultimeric structures (e.g., composed of more than one type of subunit). For example, skeletal muscle L-type calcium channels are composed of at least four glycosylated, membrane spanning- or membrane associated-subunits (α1, α2, δ and γ), and two β subunits (Dunlap (1995) Trends Neurosci 18: 89-98). Examples of calcium channels include the low-voltage-gated channels and the high-voltage-gated channels. Calcium channels are described in, for example, Davila et al. (1999) Annals New York Academy of Sciences 868:102-17 and McEnery, M. W. et al. (1998) J. Bioenergetics and Biomembranes 30(4): 409-418, the contents of which are incorporated herein by reference. As the 21784 molecules of the present invention may modulate calcium channel mediated activities, these molecules may be useful for developing novel diagnostic and therapeutic agents for calcium channel associated disorders.

[0536] The 21784 protein shows homology to the human and mouse alpha-2 delta-3 (α2δ3) calcium channel subunits (FIGS. 2-3). The term “alpha-2 delta” protein refers to a membrane-spanning, glycoprotein which is a component of a calcium channel. Typically, the alpha-2 delta protein is encoded by a single gene with the alpha-2 portion forming the N-terminal sequence and the delta portion forming the C-terminal sequence, and having a disulphide bridge linking the alpha and the delta portions (Dunlap (1995) supra). Preferably, the “alpha-2 delta” protein is an alpha-2 delta-3 ((α2δ3) polypeptide, e.g., a 21784 as described herein, and having at least one, preferably two and most preferably three transmembrane domains and at least one glycosylation site.

[0537] 21784 proteins include at least one or two, and preferably three, transmembrane domains. As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15-45, preferably 16-30, more preferably 12-25, and most preferably 17-20, amino acid residues in length that spans the plasma membrane. More preferably, a transmembrane domain includes about at least 15, 17, or 20 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. Amino acid residues 455-475, 927-947, and 1072-1089 of SEQ ID NO: 15 are transmembrane domains (see FIG. 6). Accordingly, proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, about 80-90%, or about 90-100% homology with amino acids 455-475, 927-947, and 1072-1089, of SEQ ID NO: 15 are within the scope of the invention.

[0538] A 21784 protein further includes a predicted N-terminal extracellular domain located at about amino acids 1-454 of SEQ ID NO: 15. As used herein, an “N-terminal extracellular domain” includes an amino acid sequence about 1-600, preferably about 100-400, and even more preferably about 425-454, amino acid residues in length and is located outside of a cell or extracellularly. The C-terminal amino acid residue of a “N-terminal extracellular domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring 21784 or 21784-like protein. For example, an N-terminal cytoplasmic domain is located at about amino acid residues 1-454 of SEQ ID NO: 15.

[0539] In a preferred embodiment 21784 polypeptide or protein has an “N-terminal extracellular domain” or a region which includes at least about 1-600, preferably about 100-400, and even more preferably about 425-454 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “N-terminal extracellular domain,” e.g., the N-terminal extracellular domain of human 21784 (e.g., residues 1-454 of SEQ ID NO: 15). Preferably, the N-terminal extracellular domain is capable of interacting (e.g., binding to) with an extracellular signal, and/or modulating ion channel activity.

[0540] In another embodiment, a 21784 protein include at least one extracellular loop. As defined herein, the term “loop” includes an amino acid sequence having a length of at least about 80, preferably about 100-150, more preferably about 110-130, and most preferably about 123 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring a 21784 or a 21784-like molecule, and the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring 21784 or a 21784-like molecule. As used herein, an “extracellular loop” includes an amino acid sequence located outside of a cell, or extracellularly. For example, an extracellular loop can be found at about amino acids 948-1071 of SEQ ID NO: 15.

[0541] In a preferred embodiment 21784 polypeptide or protein has at least one extracellular loop or a region which includes at least about 80, preferably about 100-150, more preferably about 110-130, and most preferably about 123 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “extracellular loop,” e.g., at least one extracellular loop of human 21784 (e.g., residues 948-1071 of SEQ ID NO: 15).

[0542] In another embodiment, a 21784 protein includes at least one cytoplasmic loop, also referred to herein as a cytoplasmic domain. As used herein, a “cytoplasmic loop” includes an amino acid sequence having a length of at least about 400, preferably about 425-475, and more preferably about 450 amino acid residues located within a cell or within the cytoplasm of a cell. For example, a cytoplasmic loop is found at about amino acids 476-926 of SEQ ID NO: 15.

[0543] In a preferred embodiment 21784 polypeptide or protein has at least one cytoplasmic loop or a region which includes at least about 400, preferably about 425-475, and more preferably about 450 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “cytoplasmic loop,” e.g., at least one cytoplasmic loop of human 21784 (e.g., residues 476-926 of SEQ ID NO: 15).

[0544] In another embodiment, a 21784 protein includes a “C-terminal cytoplasmic domain”, also referred to herein as a C-terminal cytoplasmic tail, in the sequence of the protein. As used herein, a “C-terminal cytoplasmic domain” includes an amino acid sequence having a length of at least about 2 amino acid residues and is located within a cell or within the cytoplasm of a cell. Accordingly, the N-terminal amino acid residue of a “C-terminal cytoplasmic domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally-occurring 21784 or 21784-like protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 1090-1091 of SEQ ID NO: 15.

[0545] In a preferred embodiment, a 21784 polypeptide or protein has a C-terminal cytoplasmic domain or a region which includes at least about 2 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “C-terminal cytoplasmic domain,” e.g., the C-terminal cytoplasmic domain of human 21784 (e.g., residues 1090-1091 of SEQ ID NO: 15).

[0546] Accordingly, in one embodiment of the invention, a 21784 includes at least one, preferably three, transmembrane domains and/or at least one cytoplasmic loop, and/or at least one extracellular loop. In another embodiment, the 21784 further includes an N-terminal extracellular domain and/or a C-terminal cytoplasmic domain. In another embodiment, the 21784 can include three transmembrane domains, one cytoplasmic loop, one extracellular loops and can further include an N-terminal extracellular domain and/or a C-terminal cytoplasmic domain.

[0547] The 21784 molecule further can include a signal sequence. As used herein, a “signal sequence” refers to a peptide of about 20-30 amino acid residues in length that occurs at the N-terminus of secretory and integral membrane proteins and that contains a majority of hydrophobic amino acid residues. For example, a signal sequence contains at least about 15-45 amino acid residues, preferably about 20-40 amino acid residues, more preferably about 21-33 amino acid residues, and more preferably about 23-31 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g., alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a “signal sequence”, also referred to in the art as a “signal peptide”, serves to direct a protein containing such a sequence to a lipid bilayer. For example, in one embodiment, a 21784 protein contains a signal sequence of about amino acids 1-31 of SEQ ID NO: 15. The “signal sequence” is cleaved during processing of the mature protein. The mature 21784 protein corresponds to amino acids 32 to 1091 of SEQ ID NO: 15.

[0548] In another embodiment, a 21784 molecule of the present invention is identified based on the presence of at least one N-glycosylation site, e.g., at least two, at least four, or at least eight N-glycosylation sites. As used herein, the term “N-glycosylation site” includes an amino acid sequence of about 4 amino acid residues in length that serves as a glycosylation site. More preferably, an N-glycosylation site has the consensus sequence Asn-Xaa-Ser/Thr (where Xaa may be any amino acid) (SEQ ID NO: 19). N-glycosylation sites are described in, for example, Prosite PDOC00001 (http://www.expasy.ch/cgi-bin/get-prodoc-entry?PDOC00001), the contents of which are incorporated herein by reference. Amino acid residues 166-169, 309-312, 353-356, 488-491, 553-556, 632-635, 714-717, 793-796, and 1035-1038 of SEQ ID NO: 15 comprise N-glycosylation sites. Accordingly, 21784 proteins having at least one N-glycosylation site are within the scope of the invention.

[0549] As the 21784 polypeptides of the invention may modulate 21784-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 21784-mediated or related disorders, as described below.

[0550] As used herein, a “21784 activity”, “biological activity of 21784” or “functional activity of 21784”, refers to an activity exerted by a 21784 protein, polypeptide or nucleic acid molecule. For example, a 21784 activity can be an activity exerted by 21784 in a physiological milieu on, e.g., a 21784-responsive cell or on a 21784 substrate, e.g., a protein substrate. A 21784 activity can be determined in vivo or in vitro. In one embodiment, a 21784 activity is a direct activity, such as an association with a 21784 target molecule. A “target molecule” or “binding partner” is a molecule with which a 21784 protein binds or interacts in nature.

[0551] A 21784 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 21784 protein with a second protein.

[0552] The features of the 21784 molecules of the present invention can provide similar biological activities as other calcium channel family members. For example, the 21784 proteins of the present invention can have one or more of the following activities: (1) modulation of calcium channel activity; (2) modulation of membrane excitability, (3) influence the resting potential of membranes, (4) modulation of wave forms and frequencies of action potentials, (5) modulation of thresholds of excitation, (6) modulation of neurite outgrowth and synaptogenesis, (7) modulation of signal transduction, (8) modulation of gene expression; or (9) modulation of cell proliferation, differentiation, or morphogenesis.

[0553] As used herein, a “calcium channel mediated activity” includes an activity that involves a calcium channel, e.g., a calcium channel in a neuronal cell or a muscular cell, associated with receiving, conducting, and transmitting signals, in, for example, the skeletal muscle or the nervous system. Calcium channel mediated activities include release of neurotransmitters or second messenger molecules (e.g., dopamine or norepinephrine), from cells, e.g., neuronal cells or muscle cells; modulation of resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; and modulation of processes such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials in, for example, neuronal cells or muscle cells (e.g., changes in those action potentials resulting in a morphological or differentiative response in the cell).

[0554] Thus, the 21784 molecules can act as novel diagnostic targets and therapeutic agents for controlling calcium channel associated disorders. As used herein, a “calcium channel associated disorder” includes a disorder, disease or condition that is characterized by a misregulation of calcium channel mediated activity. The 21784 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders (e.g., inflammatory disorders), cardiovascular disorders, liver disorders, viral diseases, pain or metabolic disorders.

[0555] Calcium channel disorders include cellular proliferation, growth, differentiation, or migration disorders. The 21784 molecules of the present invention are involved in signal transduction mechanisms, which are known to be involved in cellular growth, differentiation, and migration processes. Thus, the 21784 molecules may modulate cellular growth, differentiation, or migration, and may play a role in disorders characterized by aberrantly regulated growth, differentiation, or migration. Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; neuronal deficiencies resulting from impaired neural induction and patterning; hepatic disorders; cardiovascular disorders; and hematopoietic and/or myeloproliferative disorders.

[0556] Calcium channel associated disorders include central nervous system disorders, such as cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, Jakob-Creutzfieldt disease, or AIDS related dementia; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Further CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

[0557] 21784 mRNA was found to be expressed at high levels in the brain cortex and hypothalmus, and therefore may mediate disorders involving aberrant activities of the brain, for example brain disorders. Disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-bome (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyclination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin B1) deficiency and vitamin B12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (difflise) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.

[0558] 21784 mRNA was found to exhibit increased expression in skeletal muscle. Thus, further examples of calcium channel associated disorders can include muscular disorders such as muscular dystrophy (e.g., Duchenne muscular dystrophy or myotonic dystrophy), spinal muscular atrophy, congenital myopathies, central core disease, rod myopathy, central nuclear myopathy, Lambert-Eaton syndrome, denervation, paralysis, and muscle weakness (e.g., ataxia, myotonia, and myokymia) and infantile spinal muscular atrophy (Werdnig-Hoffinan disease).

[0559] As 21784 mRNA was found to be expressed in heart tissue, the molecules of the invention may mediate disorders involving aberrant activities of the heart tissue, for example heart disorders. Examples of disorders involving the heart or “cardiovascular disorder” include, but are not limited to, a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, congestive heart failure, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

[0560] Calcium channel disorders also include pain disorders. Pain disorders include those that affect pain signaling mechanisms. As used herein, the term “pain signaling mechanisms” includes the cellular mechanisms involved in the development and regulation of pain, e.g., pain elicited by noxious chemical, mechanical, or thermal stimuli, in a subject, e.g., a mammal such as a human. In mammals, the initial detection of noxious chemical, mechanical, or thermal stimuli, a process referred to as “nociception”, occurs predominantly at the peripheral terminals of specialized, small diameter sensory neurons. These sensory neurons transmit the information to the central nervous system, evoking a perception of pain or discomfort and initiating appropriate protective reflexes. The 21784 molecules of the present invention may be present on these sensory neurons and, thus, may be involved in detecting these noxious chemical, mechanical, or thermal stimuli and transducing this information into membrane depolarization events. Thus, the 21784 molecules by participating in pain signaling mechanisms, may modulate pain elicitation and act as targets for developing novel diagnostic targets and therapeutic agents to control pain. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[0561] 21784 mRNA was found to be expressed in kidney cells. Thus, the molecules of the invention may mediate disorders involving aberrant activities of these cells, for example kidney disorders. Disorders involving the kidney include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases of the kidney, that include but are not limited to, cystic renal dysplasia, autosomal dominant (adult) polycystic kidney disease, autosomal recessive (childhood) polycystic kidney disease, and cystic diseases of renal medulla, which include, but are not limited to, medullary sponge kidney, and nephronophthisis-uremic medullary cystic disease complex, acquired (dialysis-associated) cystic disease, such as simple cysts; glomerular diseases including pathologies of glomerular injury that include, but are not limited to, in situ immune complex deposition, that includes, but is not limited to, anti-GBM nephritis, Heyrnann nephritis, and antibodies against planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative complement pathway, epithelial cell injury, and pathologies involving mediators of glomerular injury including cellular and soluble mediators, acute glomerulonephritis, such as acute proliferative (poststreptococcal, postinfectious) glomerulonephritis, including but not limited to, poststreptococcal glomerulonephritis and nonstreptococcal acute glomerulonephritis, rapidly progressive (crescentic) glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, IgA nephropathy (Berger disease), focal proliferative and necrotizing glomerulonephritis (focal glomerulonephritis), hereditary nephritis, including but not limited to, Alport syndrome and thin membrane disease (benign familial hematuria), chronic glomerulonephritis, glomerular lesions associated with systemic disease, including but not limited to, systemic lupus erythematosus, Henoch-Schönlein purpura, bacterial endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary and immunotactoid glomerulonephritis, and other systemic disorders; diseases affecting tubules and interstitium, including acute tubular necrosis and tubulointerstitial nephritis, including but not limited to, pyelonephritis and urinary tract infection, acute pyelonephritis, chronic pyelonephritis and reflux nephropathy, and tubulointerstitial nephritis induced by drugs and toxins, including but not limited to, acute drug-induced interstitial nephritis, analgesic abuse nephropathy, nephropathy associated with nonsteroidal anti-inflammatory drugs, and other tubulointerstitial diseases including, but not limited to, urate nephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases of blood vessels including benign nephrosclerosis, malignant hypertension and accelerated nephrosclerosis, renal artery stenosis, and thrombotic microangiopathies including, but not limited to, classic (childhood) hemolytic-uremic syndrome, adult hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, and other vascular disorders including, but not limited to, atherosclerotic ischemic renal disease, atheroembolic renal disease, sickle cell disease nephropathy, diffuise cortical necrosis, and renal infarcts; urinary tract obstruction (obstructive uropathy); urolithiasis (renal calculi, stones); and tumors of the kidney including, but not limited to, benign tumors, such as renal papillary adenoma, renal fibroma or hamartoma (renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma, and malignant tumors, including renal cell carcinoma (hypemephroma, adenocarcinoma of kidney), which includes urothelial carcinomas of renal pelvis.

[0562] The 21784 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 15 thereof are collectively referred to as “polypeptides or proteins of the invention” or “21784 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “21784 nucleic acids.” 21784 molecules refer to 21784 nucleic acids, polypeptides, and antibodies.

[0563] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0564] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0565] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodiuim citrate (SSC) at about 45° C, followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[0566] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 14 or SEQ ID NO: 16, corresponds to a naturally-occurring nucleic acid molecule.

[0567] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include at least an open reading frame encoding a 21784 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 21784 protein or derivative thereof.

[0568] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 21784 protein is at least 10% pure. In a preferred embodiment, the preparation of 21784 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-21784 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-21784 chemicals. When the 21784 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0569] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 21784 without abolishing or substantially altering a 21784 activity. Preferably the alteration does not substantially alter the 21784 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 21784, results in abolishing a 21784 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 21784 are predicted to be particularly unamenable to alteration.

[0570] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 21784 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 21784 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 21784 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 14 or SEQ ID NO: 16, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0571] As used herein, a “biologically active portion” of a 21784 protein includes a fragment of a 21784 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 21784 molecule and a non-21784 molecule or between a first 21784 molecule and a second 21784 molecule (e.g., a dimerization interaction). Biologically active portions of a 21784 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 21784 protein, e.g., the amino acid sequence shown in SEQ ID NO: 15, which include less amino acids than the full length 21784 proteins, and exhibit at least one activity of a 21784 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 21784 protein, e.g., the ability to associate or attach to a cell membrane. A biologically active portion of a 21784 protein can be a polypeptide that is, for example, 10, 25, 50, 100, 200, 300, 400 or more amino acids in length. Biologically active portions of a 21784 protein can be used as targets for developing agents that modulate a 21784 mediated activity, e.g., a calcium channel mediated activity described herein.

[0572] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0573] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0574] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0575] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0576] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0577] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 21784 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 21784 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0578] Particularly preferred 21784 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 15. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 15 are termed substantially identical.

[0579] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 14 or 16 are termed substantially identical.

[0580] “Misexpression or aberrant expression”, as used herein, refers to a non-wild type pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[0581] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[0582] A “purified preparation of cells”, as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[0583] Various aspects of the invention are described in further detail below.

[0584] Isolated 21784 Nucleic Acid Molecules

[0585] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 21784 polypeptide described herein, e.g., a full-length 21784 protein or a fragment thereof, e.g., a biologically active portion of 21784 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 21784 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[0586] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 14, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 21784 protein (i.e., “the coding region” of SEQ ID NO: 14, as shown in SEQ ID NO: 16), as well as 5′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 14 (e.g., SEQ ID NO: 16) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to the mature protein from about amino acid 32 to amino acid 1089 of SEQ ID NO: 15.

[0587] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO: 14 or 16, thereby forming a stable duplex.

[0588] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16, or a portion, preferably of the same length, of any of these nucleotide sequences.

[0589] 21784 Nucleic Acid Fragments

[0590] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 14 or 16. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 21784 protein, e.g., an immunogenic or biologically active portion of a 21784 protein. A fragment can comprise those nucleotides of SEQ ID NO: 14, which encode a calcium channel domain of human 21784. The nucleotide sequence determined from the cloning of the 21784 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 21784 family members, or fragments thereof, as well as 21784 homologues, or fragments thereof, from other species.

[0591] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 300, 380, 400, 500, 600, 630, 650 or 700 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[0592] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 21784 nucleic acid fragment can include a sequence corresponding to transmembrane domain, at locations in the translated 21784 polypeptide described herein.

[0593] 21784 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 14 or SEQ ID NO: 16.

[0594] In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0595] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes, e.g., a transmembrane domain located from about amino acids 455 to about 475, amino acids 927-947, or amino acids 1072-1089 of SEQ ID NO: 15.

[0596] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 21784 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: a transmembrane domain located from about amino acids 455 to about 475, amino acids 927-947, or amino acids 1072-1089 of SEQ ID NO: 15.

[0597] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[0598] A nucleic acid fragment encoding a “biologically active portion of a 21784 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 14 or 16, which encodes a polypeptide having a 21784 biological activity (e.g., the biological activities of the 21784 proteins are described herein), expressing the encoded portion of the 21784 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 21784 protein. For example, a nucleic acid fragment encoding a biologically active portion of 21784 includes a transmembrane domain located from about amino acids 455 to about 475, amino acids 927-947, or amino acids 1072-1089 of SEQ ID NO: 15. A nucleic acid fragment encoding a biologically active portion of a 21784 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

[0599] In preferred embodiments, the nucleic acid fragment includes a nucleotide sequence that is other than the sequence of AA188635, AJ272268, AX098896, AX099316, AX098884, AX099304, AX098883, AX099303, AX098882, AX099302.

[0600] In preferred embodiments, the fragment comprises the sequence from 311 to 3304 plus at least 1, preferably 3, 15, 30, 45, 60, 90, 120, 180, 210, 240, 270, or 282 nucleotides from nucleotides 29 to 282 of SEQ ID NO: 14.

[0601] In preferred embodiments, the fragment comprises the coding region of 21784, e.g., the nucleotide sequence of SEQ ID NO: 16.

[0602] In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 14, or SEQ ID NO: 16.

[0603] 21784 Nucleic Acid Variants

[0604] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 21784 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 15. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0605] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[0606] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[0607] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 14 or 16, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0608] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 15 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO: 15 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 21784 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 21784 gene.

[0609] Preferred variants include those that are correlated with modulating cell proliferation, differentiation, or mophogenesis, modulating membrane excitability, influencing the resting potential of membranes, modulating wave forms and frequencies of action potentials, modulating thresholds of excitation, modulating neurite outgrowth and synaptogenesis, modulating signal transduction, and modulating gene expression.

[0610] Allelic variants of 21784, e.g., human 21784, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 21784 protein within a population that maintain the ability to interact with other calcium chalnnel subunits and form functional calcium channels. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 15, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 21784, e.g., human 21784, protein within a population that do not have the ability to interact with other calcium channel subunits. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 15, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[0611] Moreover, nucleic acid molecules encoding other 21784 family members and, thus, which have a nucleotide sequence which differs from the 21784 sequences of SEQ ID NO: 14 or SEQ ID NO: 16 are intended to be within the scope of the invention.

[0612] Antisense Nucleic Acid Molecules, Ribozymes and Modified 21784 Nucleic Acid Molecules

[0613] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 21784. An “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 21784 coding strand, or to only a portion thereof (e.g., the coding region of human 21784 corresponding to SEQ ID NO: 16). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 21784 (e.g., the 5′ and 3′ untranslated regions).

[0614] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 21784 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 21784 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 21784 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[0615] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0616] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 21784 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0617] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0618] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 21784-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 21784 cDNA disclosed herein (i.e., SEQ ID NO: 14 or SEQ ID NO: 16), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 21784-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 21784 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0619] 21784 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 21784 (e.g., the 21784 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 21784 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[0620] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

[0621] A 21784 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulmé(2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.

[0622] For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. NatL. Acad. Sci. 93: 14670-675.

[0623] PNAs of 21784 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 21784 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0624] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0625] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 21784 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 21784 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

[0626] Isolated 21784 Polypeptides

[0627] In another aspect, the invention features an isolated 21784 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-21784 antibodies. 21784 protein can be isolated from cells or tissue sources using standard protein purification techniques. 21784 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[0628] Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[0629] In a preferred embodiment, a 21784 polypeptide has one or more of the following characteristics:

[0630] (i) it has a signal peptide;

[0631] (ii) it associates or attaches to a cell membrane;

[0632] (iii) it associates with other calcium channel subunits (e.g., (α1, γ, and β subunits) to form a calcium channel, and/or to modulate calcium channel activity;

[0633] (iv) it has an amino acid composition of SEQ ID NO: 15;

[0634] (v) it has an overall sequence similarity of at least 60%, preferably at least 70%, more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% with a polypeptide of SEQ ID NO: 15;

[0635] (vi) it can be found in human tissue;

[0636] (vii) it has at least one, two, and preferably three transmembrane domains with a sequence similarity of about 70%, 80%, 90% or 95% with amino acid residues 455 to 475, 927 to 947, or 1072 to 1089 of SEQ ID NO: 15; or

[0637] (viii) it has at least 10, preferably at least 12, and most preferably at least 15 of the 20 cysteines found in the amino acid sequence of the native protein.

[0638] In a preferred embodiment the 21784 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID:2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 15 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 15. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In another preferred embodiment, one or more differences are in transmembrane or non-transmembrane domains.

[0639] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 21784 proteins differ in amino acid sequence from SEQ ID NO: 15, yet retain biological activity.

[0640] In one embodiment, the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 15.

[0641] A 21784 protein or fragment is provided which varies from the sequence of SEQ ID NO: 15 in regions defined by amino acids about 1 to about 454, 476 to about 926, and from amino acid 948 to about 1071 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 15 in regions defined by amino acids about 217 to about 443 of SEQ ID NO: 15. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[0642] Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 21784 protein.

[0643] In a preferred embodiment, the 21784 protein has an amino acid sequence shown in SEQ ID NO: 15. In other embodiments, the 21784 protein is substantially identical to SEQ ID NO: 15. In yet another embodiment, the 21784 protein is substantially identical to SEQ ID NO: 15 and retains the functional activity of the protein of SEQ ID NO: 15, as described in detail in the subsections above.

[0644] 21784 Chimeric or Fusion Proteins

[0645] In another aspect, the invention provides 21784 chimeric or fusion proteins. As used herein, a 21784 “chimeric protein” or “fusion protein” includes a 21784 polypeptide linked to a non-21784 polypeptide. A “non-21784 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 21784 protein, e.g., a protein which is different from the 21784 protein and which is derived from the same or a different organism. The 21784 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a 21784 amino acid sequence. In a preferred embodiment, a 21784 fusion protein includes at least one (or two) biologically active portion of a 21784 protein. The non-21784 polypeptide can be fused to the N-terminus or C-terminus of the 21784 polypeptide.

[0646] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-21784 fusion protein in which the 21784 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 21784. Alternatively, the fusion protein can be a 21784 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 21784 can be increased through use of a heterologous signal sequence.

[0647] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[0648] The 21784 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 21784 fusion proteins can be used to affect the bioavailability of a 21784 substrate. 21784 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 21784 protein; (ii) mis-regulation of the 21784 gene; and (iii) aberrant post-translational modification of a 21784 protein.

[0649] Moreover, the 21784-fusion proteins of the invention can be used as immunogens to produce anti-21784 antibodies in a subject, to purify 21784 ligands and in screening assays to identify molecules which inhibit the interaction of 21784 with a 21784 substrate.

[0650] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 21784-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 21784 protein.

[0651] Variants of 21784 Proteins

[0652] In another aspect, the invention also features a variant of a 21784 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 21784 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 21784 protein. An agonist of the 21784 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a 21784 protein. An antagonist of a 21784 protein can inhibit one or more of the activities of the naturally occurring form of the 21784 protein by, for example, competitively modulating a 21784-mediated activity of a 21784 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 21784 protein.

[0653] Variants of a 21784 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 21784 protein for agonist or antagonist activity.

[0654] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 21784 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 21784 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[0655] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 21784 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 21784 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[0656] Cell based assays can be exploited to analyze a variegated 21784 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 21784 in a substrate-dependent manner. The transfected cells are then contacted with 21784 and the effect of the expression of the mutant on signaling by the 21784 substrate can be detected. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 21784 substrate, and the individual clones further characterized.

[0657] In another aspect, the invention features a method of making a 21784 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 21784 polypeptide, e.g., a naturally occurring 21784 polypeptide. The method includes: altering the sequence of a 21784 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[0658] In another aspect, the invention features a method of making a fragment or analog of a 21784 polypeptide a biological activity of a naturally occurring 21784 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 21784 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[0659] Anti-21784 Antibodies

[0660] In another aspect, the invention provides an anti-21784 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0661] The anti-21784 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0662] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[0663] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 21784 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-21784 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0664] The anti-21784 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[0665] Phage display and combinatorial methods for generating anti-21784 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[0666] In one embodiment, the anti-21784 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art.

[0667] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0668] An anti-21784 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[0669] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0670] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a 21784 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[0671] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[0672] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 21784 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[0673] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[0674] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized imrunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[0675] In preferred embodiments an antibody can be made by immunizing with purified 21784 antigen, or a fragment thereof, e.g., a fragment described herein, or membrane associated antigen.

[0676] A full-length 21784 protein or, antigenic peptide fragment of 21784 can be used as an immunogen or can be used to identify anti-21784 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 21784 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 15 and encompasses an epitope of 21784. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0677] Fragments of 21784 which include residues about 61 to 78, about 311 to 326, or about 712 to 721 can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophilic regions of the 21784 protein. Similarly, fragments of 21784 which include residues about 10 to 30, about 810 to 820, or about 1005 to 1031 can be used to make an antibody against a hydrophobic region of the 21784 protein; fragments of 21784 which include, for example, residues 948 to 1071 can be used to make an antibody against an extracellular region of the 21784 protein; fragments of 21784 which include, for example, residues 476 to 926 can be used to make an antibody against an intracellular region of the 21784 protein.

[0678] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[0679] Antibodies which bind only native 21784 protein, only denatured or otherwise non-native 21784 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 21784 protein.

[0680] Preferred epitopes encompassed by the antigenic peptide are regions of 21784 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 21784 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 21784 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[0681] In a preferred embodiment the antibody can bind to the extracellular portion of the 21784 protein, e.g., it can bind to a whole cell which expresses the 21784 protein. In another embodiment, the antibody binds an intracellular portion of the 21784 protein. In preferred embodiments antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g., tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g., membrane fractions.

[0682] The anti-21784 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 21784 protein.

[0683] In a preferred embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement.

[0684] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example., it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0685] In a preferred embodiment, an anti-21784 antibody alters (e.g., increases or decreases) the activity of a 21784 polypeptide.

[0686] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[0687] An anti-21784 antibody (e.g., monoclonal antibody) can be used to isolate 21784 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-21784 antibody can be used to detect 21784 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-21784 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[0688] The invention also includes a nucleic acids which encodes an anti-21784 antibody, e.g., an anti-21784 antibody described herein. Also included are vectors which include the nucleic acid and sells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[0689] The invention also includes cell lines, e.g., hybridomas, which make an anti-21784 antibody, e.g., and antibody described herein, and method of using said cells to make a 21784 antibody.

[0690] 21784 Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[0691] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[0692] A vector can include a 21784 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 21784 proteins, mutant forms of 21784 proteins, fusion proteins, and the like).

[0693] The recombinant expression vectors of the invention can be designed for expression of 21784 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0694] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0695] Purified fusion proteins can be used in 21784 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 21784 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[0696] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0697] The 21784 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[0698] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[0699] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

[0700] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Baneiji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0701] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[0702] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 21784 nucleic acid molecule within a recombinant expression vector or a 21784 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0703] A host cell can be any prokaryotic or eukaryotic cell. For example, a 21784 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182)). Other suitable host cells are known to those skilled in the art.

[0704] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[0705] A host cell of the invention can be used to produce (i.e., express) a 21784 protein. Accordingly, the invention further provides methods for producing a 21784 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a 21784 protein has been introduced) in a suitable medium such that a 21784 protein is produced. In another embodiment, the method further includes isolating a 21784 protein from the medium or the host cell.

[0706] In another aspect, the invention features, a cell or purified preparation of cells which include a 21784 transgene, or which otherwise misexpress 21784. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a 21784 transgene, e.g., a heterologous form of a 21784, e.g., a gene derived from humans (in the case of a non-human cell). The 21784 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 21784, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 21784 alleles or for use in drug screening.

[0707] In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid which encodes a subject 21784 polypeptide.

[0708] Also provided are cells, preferably human cells, e.g., human hematopoietic or fibroblast cells, in which an endogenous 21784 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 21784 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 21784 gene. For example, an endogenous 21784 gene which is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[0709] In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 21784 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Production of 21784 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 21784 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

[0710] 21784 Transgenic Animals

[0711] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 21784 protein and for identifying and/or evaluating modulators of 21784 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 21784 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0712] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 21784 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 21784 transgene in its genome and/or expression of 21784 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 21784 protein can further be bred to other transgenic animals carrying other transgenes.

[0713] 21784 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[0714] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[0715] Uses of 21784

[0716] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[0717] The isolated nucleic acid molecules of the invention can be used, for example, to express a 21784 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a 21784 mRNA (e.g., in a biological sample) or a genetic alteration in a 21784 gene, and to modulate 21784 activity, as described further below. The 21784 proteins can be used to treat disorders characterized by insufficient or excessive production of a 21784 substrate or production of 21784 inhibitors. In addition, the 21784 proteins can be used to screen for naturally occurring 21784 substrates, to screen for drugs or compounds which modulate 21784 activity, as well as to treat disorders characterized by insufficient or excessive production of 21784 protein or production of 21784 protein forms which have decreased, aberrant or unwanted activity compared to 21784 wild type protein (e.g., a central nervous system or a muscular disorder). Moreover, the anti-21784 antibodies of the invention can be used to detect and isolate 21784 proteins, regulate the bioavailability of 21784 proteins, and modulate 21784 activity.

[0718] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 21784 polypeptide is provided. The method includes: contacting the compound with the subject 21784 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 21784 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 21784 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 21784 polypeptide. Screening methods are discussed in more detail below.

[0719] 21784 Screening Assays

[0720] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 21784 proteins, have a stimulatory or inhibitory effect on, for example, 21784 expression or 21784 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 21784 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 21784 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[0721] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 21784 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 21784 protein or polypeptide or a biologically active portion thereof.

[0722] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[0723] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[0724] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (I991) J. Mol. Biol. 222:301-310; Ladner supra.).

[0725] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 21784 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 21784 activity is determined. Determining the ability of the test compound to modulate 21784 activity can be accomplished by monitoring, for example, proteolytic activity. The cell, for example, can be of mammalian origin, e.g., human.

[0726] The ability of the test compound to modulate 21784 binding to a compound, e.g., a 21784 substrate, or to bind to 21784 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 21784 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 21784 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 21784 binding to a 21784 substrate in a complex. For example, compounds (e.g., 21784 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0727] The ability of a compound (e.g., a 21784 substrate) to interact with 21784 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 21784 without the labeling of either the compound or the 21784. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 21784.

[0728] In yet another embodiment, a cell-free assay is provided in which a 21784 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 21784 protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the 21784 proteins to be used in assays of the present invention include fragments which participate in interactions with non-21784 molecules, e.g., fragments with high surface probability scores.

[0729] Soluble and/or membrane-bound forms of isolated proteins (e.g., 21784 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1 -propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0730] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[0731] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[0732] In another embodiment, determining the ability of the 21784 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[0733] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[0734] It may be desirable to immobilize either 21784, an anti-21784 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 21784 protein, or interaction of a 21784 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/21784 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 21784 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 21784 binding or activity determined using standard techniques.

[0735] Other techniques for immobilizing either a 21784 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 21784 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[0736] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[0737] In one embodiment, this assay is performed utilizing antibodies reactive with 21784 protein or target molecules but which do not interfere with binding of the 21784 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 21784 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 21784 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 21784 protein or target molecule.

[0738] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[0739] In a preferred embodiment, the assay includes contacting the 21784 protein or biologically active portion thereof with a known compound which binds 21784 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 21784 protein, wherein determining the ability of the test compound to interact with a 21784 protein includes determining the ability of the test compound to preferentially bind to 21784 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[0740] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 21784 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a 21784 protein through modulation of the activity of a downstream effector of a 21784 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[0741] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[0742] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[0743] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[0744] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[0745] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[0746] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[0747] In yet another aspect, the 21784 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 21784 (“21784-binding proteins” or “21784-bp”) and are involved in 21784 activity. Such 21784-bps can be activators or inhibitors of signals by the 21784 proteins or 21784 targets as, for example, downstream elements of a 21784-mediated signaling pathway.

[0748] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 21784 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 21784 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 21784-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 21784 protein.

[0749] In another embodiment, modulators of 21784 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 21784 mRNA or protein evaluated relative to the level of expression of 21784 mRNA or protein in the absence of the candidate compound. When expression of 21784 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 21784 mRNA or protein expression. Alternatively, when expression of 21784 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 21784 mRNA or protein expression. The level of 21784 mRNA or protein expression can be determined by methods described herein for detecting 21784 mRNA or protein.

[0750] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a 21784 protein can be confirmed in vivo, e.g., in an animal such as an animal model for cancer.

[0751] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 21784 modulating agent, an antisense 21784 nucleic acid molecule, a 21784-specific antibody, or a 21784-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[0752] 21784 Detection Assays

[0753] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 21784 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[0754] 21784 Chromosome Mapping

[0755] The 21784 nucleotide sequences or portions thereof can be used to map the location of the 21784 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 21784 sequences with genes associated with disease.

[0756] Briefly, 21784 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 21784 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 21784 sequences will yield an amplified fragment.

[0757] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[0758] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 21784 to a chromosomal location.

[0759] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[0760] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[0761] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[0762] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 21784 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[0763] 21784 Tissue Typing

[0764] 21784 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[0765] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 21784 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[0766] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 14 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 16 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0767] If a panel of reagents from 21784 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[0768] Use of Partial 21784 Sequences in Forensic Biology

[0769] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[0770] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 14 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 14 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[0771] The 21784 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 21784 probes can be used to identify tissue by species and/or by organ type.

[0772] In a similar fashion, these reagents, e.g., 21784 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[0773] Predictive Medicine of 21784

[0774] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[0775] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 21784.

[0776] Such disorders include, e.g., a disorder associated with the misexpression of 21784 gene; a disorder of the central nervous system.

[0777] The method includes one or more of the following:

[0778] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 21784 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[0779] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 21784 gene;

[0780] detecting, in a tissue of the subject, the misexpression of the 21784 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[0781] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 21784 polypeptide.

[0782] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 21784 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[0783] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 14, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 21784 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[0784] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 21784 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 21784.

[0785] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[0786] In preferred embodiments the method includes determining the structure of a 21784 gene, an abnormal structure being indicative of risk for the disorder.

[0787] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 21784 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[0788] Diagnostic and Prognostic Assays of 21784

[0789] Diagnostic and prognostic assays of the invention include method for assessing the expression level of 21784 molecules and for identifying variations and mutations in the sequence of 21784 molecules.

[0790] Expression Monitoring and Profiling:

[0791] The presence, level, or absence of 21784 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 21784 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 21784 protein such that the presence of 21784 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 21784 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 21784 genes; measuring the amount of protein encoded by the 21784 genes; or measuring the activity of the protein encoded by the 21784 genes.

[0792] The level of mRNA corresponding to the 21784 gene in a cell can be determined both by in situ and by in vitro formats.

[0793] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 21784 nucleic acid, such as the nucleic acid of SEQ ID NO: 14, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 21784 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[0794] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 21784 genes.

[0795] The level of mRNA in a sample that is encoded by one of 21784 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[0796] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 21784 gene being analyzed.

[0797] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 21784 mRNA, or genomic DNA, and comparing the presence of 21784 mRNA or genomic DNA in the control sample with the presence of 21784 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 21784 transcript levels.

[0798] A variety of methods can be used to determine the level of protein encoded by 21784. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[0799] The detection methods can be used to detect 21784 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 21784 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 21784 protein include introducing into a subject a labeled anti-21784 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-21784 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[0800] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 21784 protein, and comparing the presence of 21784 protein in the control sample with the presence of 21784 protein in the test sample.

[0801] The invention also includes kits for detecting the presence of 21784 in a biological sample. For example, the kit can include a compound or agent capable of detecting 21784 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 21784 protein or nucleic acid.

[0802] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[0803] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[0804] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 21784 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as pain or deregulated cell proliferation.

[0805] In one embodiment, a disease or disorder associated with aberrant or unwanted 21784 expression or activity is identified. A test sample is obtained from a subject and 21784 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 21784 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 21784 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[0806] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 21784 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a cell proliferative or differentiative disorder.

[0807] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 21784 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 21784 (e.g., other genes associated with a 21784-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[0808] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 21784 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose a disorder in a subject wherein an increase or decrease in 21784 expression is an indication that the subject has or is disposed to having a disorder. The method can be used to monitor a treatment for a disorder in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[0809] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 21784 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[0810] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 21784 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[0811] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[0812] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 21784 expression.

[0813] 21784 Arrays and Uses Thereof

[0814] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a 21784 molecule (e.g., a 21784 nucleic acid or a 21784 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm2, and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[0815] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a 21784 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 21784. Each address of the subset can include a capture probe that hybridizes to a different region of a 21784 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for a 21784 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 21784 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 21784 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[0816] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[0817] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a 21784 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 21784 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-21784 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[0818] In another aspect, the invention features a method of analyzing the expression of 21784. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 21784-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[0819] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 21784. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 21784. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[0820] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 21784 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[0821] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[0822] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 21784-associated disease or disorder; and processes, such as a cellular transformation associated with a 21784-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 21784-associated disease or disorder

[0823] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 21784) that could serve as a molecular target for diagnosis or therapeutic intervention.

[0824] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon a 21784 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99 % identical to a 21784 polypeptide or fragment thereof For example, multiple variants of a 21784 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[0825] The polypeptide array can be used to detect a 21784 binding compound, e.g., an antibody in a sample from a subject with specificity for a 21784 polypeptide or the presence of a 21784-binding protein or ligand.

[0826] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 21784 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[0827] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 21784 or from a cell or subject in which a 21784 mediated response has been elicited, e.g., by contact of the cell with 21784 nucleic acid or protein, or administration to the cell or subject 21784 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 21784 (or does not express as highly as in the case of the 21784 positive plurality of capture probes) or from a cell or subject which in which a 21784 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 21784 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[0828] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 21784 or from a cell or subject in which a 21784-mediated response has been elicited, e.g., by contact of the cell with 21784 nucleic acid or protein, or administration to the cell or subject 21784 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 21784 (or does not express as highly as in the case of the 21784 positive plurality of capture probes) or from a cell or subject which in which a 21784 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[0829] In another aspect, the invention features a method of analyzing 21784, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 21784 nucleic acid or amino acid sequence; comparing the 21784 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 21784.

[0830] Detection of 21784 Variations or Mutations

[0831] The methods of the invention can also be used to detect genetic alterations in a 21784 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 21784 protein activity or nucleic acid expression, such as a neurodegenerative disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 21784-protein, or the mis-expression of the 21784 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 21784 gene; 2) an addition of one or more nucleotides to a 21784 gene; 3) a substitution of one or more nucleotides of a 21784 gene, 4) a chromosomal rearrangement of a 21784 gene; 5) an alteration in the level of a messenger RNA transcript of a 21784 gene, 6) aberrant modification of a 21784 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a 21784 gene, 8) a non-wild type level of a 21784-protein, 9) allelic loss of a 21784 gene, and 10) inappropriate post-translational modification of a 21784-protein.

[0832] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 21784-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 21784 gene under conditions such that hybridization and amplification of the 21784-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[0833] In another embodiment, mutations in a 21784 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0834] In other embodiments, genetic mutations in 21784 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 21784 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 21784 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 21784 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[0835] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 21784 gene and detect mutations by comparing the sequence of the sample 21784 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[0836] Other methods for detecting mutations in the 21784 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[0837] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 21784 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[0838] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 21784 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 21784 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0839] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[0840] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[0841] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0842] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 21784 nucleic acid.

[0843] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 14 or the complement of SEQ ID NO: 14. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[0844] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 21784. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[0845] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a. nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the Tm of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[0846] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 21784 nucleic acid.

[0847] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 21784 gene.

[0848] Use of 21784 Molecules as Surrogate Markers

[0849] The 21784 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 21784 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 21784 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[0850] The 21784 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 21784 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-21784 antibodies may be employed in an immune-based detection system for a 21784 protein marker, or 21784-specific radiolabeled probes may be used to detect a 21784 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[0851] The 21784 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 21784 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 21784 DNA may correlate 21784 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[0852] Pharmaceutical Compositions of 21784

[0853] The nucleic acid and polypeptides, fragments thereof, as well as anti-21784 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0854] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0855] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0856] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0857] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0858] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0859] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0860] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0861] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0862] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0863] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0864] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0865] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0866] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[0867] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0868] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0869] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactive ions include, but are not limited to iodine, yttrium and praseodymium.

[0870] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[0871] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0872] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0873] Methods of Treatment for 21784

[0874] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 21784 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[0875] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 21784 molecules of the present invention or 21784 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0876] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 21784 expression or activity, by administering to the subject a 21784 or an agent which modulates 21784 expression or at least one 21784 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 21784 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 21784 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 21784 aberrance, for example, a 21784, 21784 agonist or 21784 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[0877] It is possible that some 21784 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[0878] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[0879] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[0880] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0881] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0882] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0883] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[0884] Aberrant expression and/or activity of 21784 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 21784 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 21784 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 21784 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[0885] The 21784 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders. Examples of immune disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

[0886] Disorders which may be treated or diagnosed by methods described herein include, but are not limited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, A1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

[0887] Additionally, 21784 molecules may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 21784 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 21784 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

[0888] 21784 mRNA was found to be moderately expressed in the arteries and veins. Thus the molecules of the invention may mediate disorders involving aberrant activities of these cells, for example blood vessel disorders. Disorders involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease—the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis (microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis, thromboanglitis obliterans (Buerger disease), vasculitis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and aortic dissection (dissecting hematoma); disorders of veins and lymphatics, such as varicose veins, thrombophlebitis and phlebothrombosis, obstruction of superior vena cava (superior vena cava syndrome), obstruction of inferior vena cava (inferior vena cava syndrome), and lymphangitis and lymphedema; tumors, including benign tumors and tumor-like conditions, such as hemangioma, lymphangioma, glomus tumor (glomangioma), vascular ectasias, and bacillary angiomatosis, and intermediate-grade (borderline low-grade malignant) tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignant tumors, such as angiosarcoma and hemangiopericytoma; and pathology of therapeutic interventions in vascular disease, such as balloon angioplasty and related techniques and vascular replacement, such as coronary artery bypass graft surgery.

[0889] 21784 mRNA was found to be moderately expressed in ovary cells. Thus the molecules of the invention may mediate disorders involving aberrant activities of these cells, for example disorders of the ovary. Disorders involving the ovary include, for example, polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma peritonei and stromal hyperthecosis; ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.

[0890] As discussed, successful treatment of 21784 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 21784 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[0891] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[0892] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[0893] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 21784 expression is through the use of aptamer molecules specific for 21784 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 21784 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[0894] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 21784 disorders. For a description of antibodies, see the Antibody section above.

[0895] In circumstances wherein injection of an animal or a human subject with a 21784 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 21784 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chattejee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 21784 protein. Vaccines directed to a disease characterized by 21784 expression may also be generated in this fashion.

[0896] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0897] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 21784 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[0898] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 21784 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 21784 can be readily monitored and used in calculations of IC50.

[0899] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[0900] Another aspect of the invention pertains to methods of modulating 21784 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 21784 or agent that modulates one or more of the activities of 21784 protein activity associated with the cell. An agent that modulates 21784 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a 21784 protein (e.g., a 21784 substrate or receptor), a 21784 antibody, a 21784 agonist or antagonist, a peptidomimetic of a 21784 agonist or antagonist, or other small molecule.

[0901] In one embodiment, the agent stimulates one or 21784 activities. Examples of such stimulatory agents include active 21784 protein and a nucleic acid molecule encoding 21784. In another embodiment, the agent inhibits one or more 21784 activities. Examples of such inhibitory agents include antisense 21784 nucleic acid molecules, anti-21784 antibodies, and 21784 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 21784 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 21784 expression or activity. In another embodiment, the method involves administering a 21784 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 21784 expression or activity.

[0902] Stimulation of 21784 activity is desirable in situations in which 21784 is abnormally downregulated and/or in which increased 21784 activity is likely to have a beneficial effect. For example, stimulation of 21784 activity is desirable in situations in which a 21784 is downregulated and/or in which increased 21784 activity is likely to have a beneficial effect. Likewise, inhibition of 21784 activity is desirable in situations in which 21784 is abnormally upregulated and/or in which decreased 21784 activity is likely to have a beneficial effect.

[0903] Pharmacogenomics for 21784

[0904] The 21784 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 21784 activity (e.g., 21784 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 21784 associated disorders (e.g., central nervous system disorders or muscular disorders) associated with aberrant or unwanted 21784 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 21784 molecule or 21784 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 21784 molecule or 21784 modulator.

[0905] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0906] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[0907] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 21784 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[0908] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 21784 molecule or 21784 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[0909] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 21784 molecule or 21784 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[0910] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 21784 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 21784 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[0911] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a 21784 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 21784 gene expression, protein levels, or upregulate 21784 activity, can be monitored in clinical trials of subjects exhibiting decreased 21784 gene expression, protein levels, or downregulated 21784 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 21784 gene expression, protein levels, or downregulate 21784 activity, can be monitored in clinical trials of subjects exhibiting increased 21784 gene expression, protein levels, or upregulated 21784 activity. In such clinical trials, the expression or activity of a 21784 gene, and preferably, other genes that have been implicated in, for example, a 21784-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[0912] 21784 Informatics

[0913] The sequence of a 21784 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 21784. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 21784 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[0914] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[0915] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[0916] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[0917] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[0918] Thus, in one aspect, the invention features a method of analyzing 21784, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 21784 nucleic acid or amino acid sequence; comparing the 21784 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 21784. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[0919] The method can include evaluating the sequence identity between a 21784 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[0920] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[0921] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[0922] Thus, the invention features a method of making a computer readable record of a sequence of a 21784 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[0923] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 21784 sequence, or record, in machine-readable form; comparing a second sequence to the 21784 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 21784 sequence includes a sequence being compared. In a preferred embodiment the 21784 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 21784 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[0924] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 21784-associated disease or disorder or a pre-disposition to a 21784-associated disease or disorder, wherein the method comprises the steps of determining 21784 sequence information associated with the subject and based on the 21784 sequence information, determining whether the subject has a 21784-associated disease or disorder or a pre-disposition to a 21784-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[0925] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 21784-associated disease or disorder or a pre-disposition to a disease associated with a 21784 wherein the method comprises the steps of determining 21784 sequence information associated with the subject, and based on the 21784 sequence information, determining whether the subject has a 21784-associated disease or disorder or a pre-disposition to a 21784-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 21784 sequence of the subject to the 21784 sequences in the database to thereby determine whether the subject as a 21784-associated disease or disorder, or a pre-disposition for such.

[0926] The present invention also provides in a network, a method for determining whether a subject has a 21784 associated disease or disorder or a pre-disposition to a 21784-associated disease or disorder associated with 21784, said method comprising the steps of receiving 21784 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 21784 and/or corresponding to a 21784-associated disease or disorder (e.g., central nervous system disorder or muscular disorders), and based on one or more of the phenotypic information, the 21784 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 21784-associated disease or disorder or a pre-disposition to a 21784-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0927] The present invention also provides a method for determining whether a subject has a 21784-associated disease or disorder or a pre-disposition to a 21784-associated disease or disorder, said method comprising the steps of receiving information related to 21784 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 21784 and/or related to a 21784-associated disease or disorder, and based on one or more of the phenotypic information, the 21784 information, and the acquired information, determining whether the subject has a 21784-associated disease or disorder or a pre-disposition to a 21784-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0928] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

[0929] Background of the 56201 Invention

[0930] Ion channels are integral transmembrane proteins that facilitate the diffusion of ions across the lipid bilayer membrane in which they are embedded. Ion channels may be either non-selective, in which case several different types of ions can pass through the channel, or selective, in which case only a single type of ion, for example, sodium, potassium, or calcium ions, may pass through the channel. Sodium ion channels are typically composed of a large (e.g., 260 kilodalton in rat brain) pore-forming subunit designated α and one or more smaller subunits designated β (Catterall (2000), Neuron 26:13-25; Balser (1999), Cardiovascular Research 42:327-38). The α subunit of sodium ion channels contains four homologous domains that are arranged in a circle such that each subunit passes through the lipid bilayer and forms one quarter of the pore. Similarly, calcium and potassium ion channels are composed of four proteins that create a circular pore in the membrane. The proteins that make up the pores of calcium and potassium ion channels are homologous to the domains of the sodium ion channel a subunit, indicating that there is a conserved structure for certain types of selective ion channels. In the case of sodium ion channels, the association of the β subunit(s) with the α subunit influences the permeability of the channel proteins with regard to sodium ions.

[0931] Transmembrane flux of ions is important for the generation and maintenance of transmembrane action potentials, which are necessary for transmission of signals along the membranes of excitable cells such as muscle and nerve cells. Voltage-sensitive (sometimes referred to as voltage-gated) ion channels mediate the rapid influx of ions during distinct phases of an action potential, depending upon the type of ion that they are specific for, and they also mediate re-polarization of the membranes of excitable cells.

[0932] The voltage-sensitive sodium ion channels of excitable cells are believed to exist in three interchangeable forms (Catterall (2000), Neuron 26:13-25; Balser (1999), Cardiovascular Research 42:327-38). In a resting state, the sodium ion channel protein(s) inhibits passage of sodium ions from one side of the membrane to the other. As the membrane potential becomes less negative, the sodium ion channel is ‘activated.’ In its activated state, the sodium ion channel permits passage of sodium ions through the lipid bilayer at a much greater rate than in its resting state. Shortly after the sodium channel is activated, it becomes ‘inactivated,’ in which state passage of sodium ions is once again inhibited. The sodium channel remains in the inactivated state until the membrane becomes re-polarized. Thus, the sodium channel cannot be re-activated until the membrane potential returns to approximately the value it had when the channel was in the resting state.

[0933] Summary of the 56201 Invention

[0934] The present invention is based, in part, on the discovery of a novel sodium ion channel family member, referred to herein as “56201”. The nucleotide sequence of a cDNA encoding 56201 is recited in SEQ ID NO: 20, and the amino acid sequence of a 56201 polypeptide is recited in SEQ ID NO: 21 (see also Example 11, below). In addition, the nucleotide sequences of the coding region are recited in SEQ ID NO: 22.

[0935] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 56201 protein or polypeptide, e.g., a biologically active portion of the 56201 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 21. In other embodiments, the invention provides isolated 56201 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 20, SEQ ID NO: 22, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 20, SEQ ID NO: 22, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 20, SEQ ID NO: 22, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 56201 protein or an active fragment thereof.

[0936] In a related aspect, the invention further provides nucleic acid constructs that include a 56201 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included are vectors and host cells containing the 56201 nucleic acid molecules of the invention, e.g., vectors and host cells suitable for producing 56201 nucleic acid molecules and polypeptides.

[0937] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 56201-encoding nucleic acids.

[0938] In still another related aspect, isolated nucleic acid molecules that are antisense to a 56201 encoding nucleic acid molecule are provided.

[0939] In another aspect, the invention features, 56201 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 56201-mediated or -related disorders. In another embodiment, the invention provides 56201 polypeptides having a 56201 activity. Preferred polypeptides are 56201 proteins including at least one sodium ion channel domain and, preferably, having a 56201 activity, e.g., a 56201 activity as described herein.

[0940] In other embodiments, the invention provides 56201 polypeptides, e.g., a 56201 polypeptide having the amino acid sequence shown in SEQ ID NO: 21 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 21 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 20, SEQ ID NO: 22, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 56201 protein or an active fragment thereof.

[0941] In a related aspect, the invention provides 56201 polypeptides or fragments operatively linked to non-56201 polypeptides to form fusion proteins.

[0942] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 56201 polypeptides or fragments thereof, e.g., an extracellular region of a 56201 polypeptide. In one embodiment, the antibodies or antigen-binding fragment thereof competitively inhibit the binding of a second antibody to a 56201 polypeptide or a fragment thereof, e.g., an extracellular region of a 56201 polypeptide.

[0943] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 56201 polypeptides or nucleic acids.

[0944] In still another aspect, the invention provides a process for modulating 56201 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 56201 polypeptides or nucleic acids, such as conditions involving aberrant or deficient sodium channel kinetics, e.g., paramyotonia congenita and hyperkalemic periodic paralysis, or conditions involving abnormal generation or maintenance of membrane potential or transmembrane ion gradients which can lead to epilepsy, psychiatric diseases, or dementia.

[0945] The invention also provides assays for determining the activity of or the presence or absence of 56201 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[0946] In yet another aspect, the invention provides methods for inhibiting the abnormal flow of sodium ions across a lipid bilayer of a 56201-expressing cell, e.g., a neuron or muscle cell. The method includes contacting the cell with a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 56201 polypeptide or nucleic acid. In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol. In a preferred embodiment, the cell is a neuron or muscle cell.

[0947] In a preferred embodiment, the compound is an inhibitor of a 56201 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody. In another embodiment, the compound is an inhibitor of a 56201 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.

[0948] In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant sodium ion transport in a 56201-expressing cell, in a subject. Preferably, the method includes comprising administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 56201 polypeptide or nucleic acid. In a preferred embodiment, the disorder involves aberrant or deficient sodium channel kinetics, e.g., paramyotonia congenita and hyperkalemic periodic paralysis, or conditions involving abnormal generation or maintenance of membrane potential or transmembrane ion gradients which can lead to epilepsy, psychiatric diseases, or dementia.

[0949] In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., a neural or muscular disorder, e.g., paramyotonia congenita and hyperkalemic periodic paralysis, or conditions involving abnormal generation or maintenance of membrane potential or transmembrane ion gradients which can lead to epilepsy, psychiatric diseases, or dementia. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with a compound identified using the methods described herein); and evaluating the relative severity of the disorder before and after treatment. A change, e.g., a decrease or increase, in the severity of the disorder after treatment, relative to the severity before treatment, is indicative of the efficacy of the treatment of the disorder.

[0950] In another embodiment, the method for evaluating the efficacy of a treatment of a disorder, e.g., a neural or muscular disorder, e.g., paramyotonia congenita and hyperkalemic periodic paralysis, or conditions involving abnormal generation or maintenance of membrane potential or transmembrane ion gradients which can lead to epilepsy, psychiatric diseases, or dementia, includes: treating a subject, e.g., a patient or an animal, with a protocal under evaluation e.g., treating a subject with a compound identified using the methods described herein); and evaluating the expression of a 56201 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 56201 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 56201 nucleic acid or polypeptide expression can be detected by any method described herein.

[0951] In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 56201 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.

[0952] In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein) and, evaluating the expression of 56201 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 56201 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 56201 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a neural or muscular tissue.

[0953] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 56201 polypeptide or nucleic acid molecule, including for disease diagnosis.

[0954] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 56201 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 56201 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 56201 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[0955] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description of 56201

[0956] The human 56201 sequence (see SEQ ID NO: 20, as recited in Example 11), which is approximately 1356 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1197 nucleotides, including the termination codon. The coding sequence encodes a 398 amino acid protein (see SEQ ID NO: 21, as recited in Example 11).

[0957] Human 56201 contains the following regions or other structural features:

[0958] an ion channel domain (PFAM Accession Number PF00520) located at about amino acid residues 46 to 267 of SEQ ID NO: 21;

[0959] six predicted transmembrane domains located at about amino acids 46 to 70, 80 to 104, 114 to 131, 142 to 163, 175 to 199, and 246 to 269 of SEQ ID NO: 21;

[0960] one predicted pore-lining peptide located at about amino acid residues 210 to 231 of SEQ ID NO: 21;

[0961] two predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 28 to 30, and 274 to 276 of SEQ ID NO: 21;

[0962] four predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 16 to 19, 319 to 322, 332 to 335, and 354 to 357 of SEQ ID NO: 21; and

[0963] two predicted tyrosine kinase phosphorylation sites (PS00007) located at about amino acids 101 to 109, and 365 to 371 of SEQ ID NO: 21.

[0964] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[0965] A plasmid containing the nucleotide sequence encoding human 56201 (clone “Fbh56201FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0966] The 56201 protein contains a significant number of structural characteristics in common with members of the voltage-gated ion channel family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[0967] The voltage-gated ion channel family of proteins is characterized by a common fold, which includes six transmembrane domains and a pore-lining peptide located between the fifth and sixth transmembrane domains (Catterall (2000), supra; Balser (1999), supra). An assembly of four such folds (or domains) constitutes an ion channel. The four domains can be linked together as a single molecule, which is often the case for sodium ion channels, or they can be individual proteins. The N- and C-termini of each domain of a voltage-gated ion channel are located in the cytoplasm, along with the peptide regions that connect the second and third transmembrane domains and the forth and fifth transmembrane domains. The peptide regions that connect the first and second and the third and fourth transmembrane domains are located in the extracellular space. The pore-lining peptide is also located on the extracellular surface of the ion channel, but it is inserted into the lipid bilayer such that it lines the pore formed by the six transmembrane domains. The pore-lining peptide helps determine the ion selectivity of the channel. The fourth transmembrane domain contains several charged residues and functions to open the channel in response to an appropriate voltage gradient across the plasma membrane.

[0968] Protein kinase C (PKC) and tyrosine kinase phosphorylations sites are located in the cytoplasmic regions of some ion channels and are known to modulate various aspects of channel function, including peak ion current and the timing of channel inactivation (Catterall (2000), supra). For example, phosphorylation of sodium ion channels by PKC can reduce the speed of channel inactivation and reduce the magnitude of peak sodium ion currents. The cytopasmic peptide loop that connects domains III and IV of the sodium channel a subunit, which is known as the inactivation gate, is responsible for inactivation of the channel. Phosphorylation of the inactivation gate by PKC is responsible for reduction in the rate of sodium ion channel inactivation. Similarly, the intracellular peptide loop that connects domains I and II of the sodium channel a subunit can be phosphorylated by PKC, resulting in a reduction of the peak sodium ion current. Finally, phosphorylation of sodium ion channels by tyrosine kinases can produce a negative shift in the voltage dependence of channel inactivation (Catterall (2000), supra).

[0969] A 56201 polypeptide can include an “ion channel domain” or regions homologous with an “ion channel domain”.

[0970] As used herein, the term “ion channel domain” includes an amino acid sequence of about 150 to 300 amino acid residues in length and having a bit score for the alignment of the sequence to the ion channel profile (ion_trans, PFAM HMM) of at least 30. Preferably, a ion channel domain includes at least about 175 to 275 amino acids, more preferably about 200 to 275 amino acid residues, or about 210 to 250 amino acids and has a bit score for the alignment of the sequence to the ion channel domain (HMM) of at least 50 or greater. The ion channel domain (HMM) has been assigned the PFAM Accession Number PF00520 (http;//genome.wustl.edu/Pfam/.html). An alignment of the ion channel domain (amino acids 46 to 267 of SEQ ID NO: 21) of human 56201 with a consensus amino acid sequence (SEQ ID NO: 23) derived from a hidden Markov model is depicted in FIG. 11.

[0971] In a preferred embodiment 56201 polypeptide or protein has a “ion channel domain” or a region which includes at least about 150 to 300, more preferably about 200 to 275, or 210 to 250 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “ion channel domain,” e.g., the ion channel domain of human 56201 (e.g., residues 46 to 267 of SEQ ID NO: 21).

[0972] To identify the presence of a “ion channel” domain in a 56201 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the PFAM database of HMMs (e.g., the PFAM database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the PFAM database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a “ion channel” domain in the amino acid sequence of human 56201 at about residues 46 to 267 of SEQ ID NO: 21 (see FIG. 11).

[0973] In one embodiment, a 56201 protein includes at least one pore-lining peptide located between the fifth and sixth transmembrane domains, located at about amino acid residues 210 to 231 of SEQ ID NO: 21. As used herein, the term “pore-lining peptide” includes a sequence of at least 5 amino acid residues defined by the sequence: T-X-(D/E)-G-W (SEQ ID NO: 25). A pore-lining peptide, as defined, can be involved in the ion selectivity, e.g., sodium ion selectivity, of an ion channel. Pore-lining peptides have been described in Balser (1999), supra, the contents of which are incorporated herein by reference.

[0974] In a preferred embodiment, a 56201 polypeptide or protein has at least one pore-lining peptide, or a region which includes at least five amino acid residues and has at least about 60%, 80%, or 100% homology with a “pore-lining peptide”, a pore-lining peptide of human 56201 (e.g., amino acid residues 225 to 229 of SEQ ID NO: 21).

[0975] A 56201 molecule can further include several transmembrane regions. As used herein, the term “transmembrane domain” includes an amino acid sequence of at least about 14 amino acid residues in length that spans a phospholipid membrane. More preferably, a transmembrane domain includes at least about 14, 16, 18, 20, 22, or 24 amino acid residues and spans a phospholipid membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an α-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, valines, alanines, phenylalanines, methionines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al., (1996) Annual Rev. Neuronsci. 19: 235-63.

[0976] In a preferred embodiment, a 56201 polypeptide or protein has one, two, three, four, five, most preferably six transmembrane domains or regions which includes at least 18, 19, or 20 amino acid residues and have at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with a “transmembrane domain,” e.g., at least one transmembrane domain of human 56201 (e.g., from about amino acid residues 46 to 70, 80 to 104, 114 to 131, 142 to 163, 175 to 199, and 246 to 269 of SEQ ID NO: 21).

[0977] A 56201 family member can include at least one ion channel domain; at least one pore-lining peptide; and at least one, two, three, four, five, preferably six transmembrane domains. Furthermore, a 56201 family member can include at least one, preferably two predicted protein kinase C phosphorylation sites (PS00005); at least one, two, three, preferably four predicted casein kinase II phosphorylation sites (PS00006); and at least one, preferably two predicted tyrosine phosphorylation sites (PS00007).

[0978] As the 56201 polypeptides of the invention may modulate 56201-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 56201-mediated or related disorders, as described below.

[0979] As used herein, a “56201 activity”, “biological activity of 56201” or “functional activity of 56201”, refers to an activity exerted by a 56201 protein, polypeptide or nucleic acid molecule. For example, a 56201 activity can be an activity exerted by 56201 in a physiological milieu on, e.g., a 56201-responsive cell or on a 56201 substrate, e.g., a protein substrate. A 56201 activity can be determined in vivo or in vitro. In one embodiment, a 56201 activity is a direct activity, such as the opening of a pore in a lipid bilayer through which ions can pass. In an exemplary embodiment, 56201 is an ion channel, e.g., a voltage-gated sodium ion channel.

[0980] A 56201 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by the movement of ions, e.g. sodium ions, across a lipid bilayer, e.g., the plasma membrane. The features of the 56201 molecules of the present invention can provide similar biological activities as ion channel family members. For example, the 56201 proteins of the present invention can have one or more of the following activities: (1) mediate membrane permeability to ions; (2) mediate membrane permeability to sodium ions; (3) modulate the generation, alteration, and maintenance of transmembrane sodium ion gradients; (4) modulate transmission of electrochemical impulses along biological membranes; (5) modulate the rising phase of the action potential in the membranes of electrically excitable cells; (6) modulate smooth, cardiac, striated, and skeletal muscle contraction, including normal voluntary and involuntary movements, such as heartbeat, digestion, and vascular tone; or (7) modulate neuronal development and cell connectivity.

[0981] Analogous to sodium channel proteins, the 56201 protein contains the essential elements for ion conduction and voltage-dependent gating. At least eight human genes encoding sodium channel I subunits have been identified, e.g., in the central nervous system (CNS), peripheral nervous system (PNS), skeletal muscle and heart (Genbank accession numbers: X03638, X03639, Y00766, M26643, M27902, L39018, U79568, and X92148). More particularly, the 56201 protein mediates permeability of lipid bilayers, e.g., the plasma membrane, to sodium ions. Based on sequence homology, ligands of sodium channel I subunits are expected to function as ligands for 56201 protein. However, 56201 protein also has its own specific ligands and activities in addition to those reported for sodium channel I subunits.

[0982] Sodium channel proteins are involved in generation, alteration, and maintenance of transmembrane sodium ion gradients. Changes in transmembrane sodium ion gradients enable excitable cells (e.g. nerve cells, muscle cells, and neuronal cells of the central nervous system) to transmit impulses along their lengths. Sodium channel proteins are therefore implicated in a wide variety of normal and abnormal cellular processes, which involve transmission of electrochemical impulses along biological membranes. Such processes include, for example, normal and abnormal transmission of afferent and efferent nerve impulses and normal and abnormal transmission of voluntary and involuntary muscle contractile impulses, and any disorders which result from neuronal or muscular dysfunction.

[0983] Exemplary nerve and neuronal cellular processes with which sodium channel proteins such as the I subunit described herein are involved include generation and transmission of pain and other sensory or perceptive nerve impulses, generation and maintenance of epileptic seizures, and establishment and endurance of neurodegenerative and mood disorders. Sodium channel proteins also have a role in a variety of disorders of mixed neuronal and psychological etiology including, for example, sleep disorders such as insomnia, hiccup, disorders of smell and taste, vision and eye movement disorders, hearing loss, vertigo, motor weakness, ataxias, neuropathic arthropathy disorders of the neuronal motor unit, nerve root disorders, and peripheral and hereditary neuropathies.

[0984] Exemplary muscle cell processes with which sodium channel proteins such as the I subunit described herein are involved include smooth, cardiac, striated, and skeletal muscle contraction, including normal voluntary and involuntary movements, such as heartbeat, digestion, and vascular tone. Aberrant muscular processes in which the protein of the invention have a role include, for example, arterial and renovascular hypertension, shock, cardiac insufficiency, heart failure, cardiac arrhythmias, cardiomyopathy, cardiac arrest, and skeletal muscle disorders, e.g., perkalemic periodic paralysis and paramyotonia congenita.

[0985] Based on the above-described sequence similarities, the 56201 molecules of the present invention are predicted to have similar biological activities as sodium channel protein family members. Thus, the 56201 molecules can act as novel diagnostic targets and therapeutic agents for controlling disorders associated with abnormal transmission of afferent and efferent nerve impulses and abnormal transmission of voluntary and involuntary muscle contractile impulses, and any disorders which result from neuronal or muscular dysfunction like, for example, those described above.

[0986] The 56201 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 21 thereof are collectively referred to as “polypeptides or proteins of the invention” or “56201 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “56201 nucleic acids.” 56201 molecules refer to 56201 nucleic acids, polypeptides, and antibodies.

[0987] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0988] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0989] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6× SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[0990] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 20 or SEQ ID NO: 22, corresponds to a naturally-occurring nucleic acid molecule.

[0991] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein.

[0992] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include at least an open reading frame encoding a 56201 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 56201 protein or derivative thereof.

[0993] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 56201 protein is at least 10% pure. In a preferred embodiment, the preparation of 56201 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-56201 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-56201 chemicals. When the 56201 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0994] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 56201 without abolishing or substantially altering a 56201 activity. Preferably the alteration does not substantially alter the 56201 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 56201, results in abolishing a 56201 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 56201 are predicted to be particularly unamenable to alteration.

[0995] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 56201 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 56201 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 56201 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 20 or SEQ ID NO: 22, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0996] As used herein, a “biologically active portion” of a 56201 protein includes a fragment of a 56201 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 56201 molecule and a non-56201 molecule or between a first 56201 molecule and a second 56201 molecule (e.g., a dimerization interaction). Biologically active portions of a 56201 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 56201 protein, e.g., the amino acid sequence shown in SEQ ID NO: 21, which include less amino acids than the full length 56201 proteins, and exhibit at least one activity of a 56201 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 56201 protein, e.g., sodium ion transport across a lipid bilayer. A biologically active portion of a 56201 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 56201 protein can be used as targets for developing agents which modulate a 56201 mediated activity, e.g., sodium ion transport across a lipid bilayer.

[0997] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0998] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0999] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[1000] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[1001] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (1989), CABIOS 4:11-17, which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[1002] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 56201 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 56201 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[1003] Particular 56201 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 21. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 21 are termed substantially identical.

[1004] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 20 or 22 are termed substantially identical.

[1005] “Misexpression or aberrant expression”, as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[1006] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[1007] A “purified preparation of cells”, as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[1008] Various aspects of the invention are described in further detail below.

[1009] Isolated 56201 Nucleic Acid Molecules

[1010] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 56201 polypeptide described herein, e.g., a full-length 56201 protein or a fragment thereof, e.g., a biologically active portion of 56201 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 56201 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[1011] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 20, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 56201 protein (i.e., “the coding region” of SEQ ID NO: 20, as shown in SEQ ID NO: 22), as well as 5′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 20 (e.g., SEQ ID NO: 22) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid 46 to 267.

[1012] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 20 or SEQ ID NO: 22, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 20 or SEQ ID NO: 22, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NOS: 20 or 22, thereby forming a stable duplex.

[1013] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 20 or SEQ ID NO: 22, or a portion, preferably of the same length, of any of these nucleotide sequences.

[1014] 56201 Nucleic Acid Fragments

[1015] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NOS: 20 or 22. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 56201 protein, e.g., an immunogenic or biologically active portion of a 56201 protein. A fragment can comprise those nucleotides of SEQ ID NO: 20, which encode a sodium ion channel domain of human 56201. The nucleotide sequence determined from the cloning of the 56201 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 56201 family members, or fragments thereof, as well as 56201 homologues, or fragments thereof, from other species.

[1016] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100, 200, 250, 300, or 350 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention, e.g., AA527520, AI027609, AI219834, and AC004764.

[1017] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 56201 nucleic acid fragment can include a sequence corresponding to an ion channel domain, e.g., about nucleotides 205 to 876 of SEQ ID NO: 20. In addition, a 56201 nucleic acid could include a sequence corresponding to an N-terminal fragment of a 56201 molecule, e.g., about nucleotides 70 to 393 of SEQ ID NO: 20, or a C-terminal fragment of a 56201 molecule, e.g., about nucleotides 877 to 1263 of SEQ ID NO: 20. Additional nucleotide fragments can include about nucleotides 70 to 876 or 205 to 1263 of SEQ ID NO: 20.

[1018] 56201 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 20 or SEQ ID NO: 22, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 20 or SEQ ID NO: 22.

[1019] In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1020] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes:

[1021] an ion channel domain, e.g., about nucleotides 205 to 876 of SEQ ID NO: 20;

[1022] an N-terminal fragment of a 56201 molecule, e.g., about nucleotides 70 to 393 of SEQ ID NO: 20; or

[1023] a C-terminal fragment of a 56201 molecule, e.g., about nucleotides 877 to 1263 of SEQ ID NO: 20.

[1024] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 56201 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: a region that encodes an ion channel domain, e.g., from about nucleotides 205 to 876 of SEQ ID NO: 20; a region that encodes an N-terminal fragment of a 56201 molecule, e.g., from about nucleotides 70 to 393; or a region that encodes a C-terminal fragment of a 56201 molecule, e.g., from about nucleotides 877 to 1263.

[1025] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[1026] A nucleic acid fragment encoding a “biologically active portion of a 56201 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NOS: 20 or 22, which encodes a polypeptide having a 56201 biological activity (e.g., the biological activities of the 56201 proteins are described herein), expressing the encoded portion of the 56201 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 56201 protein. For example, a nucleic acid fragment encoding a biologically active portion of 56201 includes an ion channel domain, e.g., amino acid residues about 46 to 267 of SEQ ID NO: 21; an N-terminal sub-domain of an ion channel domain, e.g., about amino acid residues 1 to 74 of SEQ ID NO: 21; or a C-terminal sub-domain of an ion channel, e.g., about amino acid residues 210 to 398 of SEQ ID NO: 21. A nucleic acid fragment encoding a biologically active portion of a 56201 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

[1027] In preferred embodiments, a nucleic acid fragment includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 20, or SEQ ID NO: 22. In a preferred embodiment, a nucleic acid fragment includes at least one contiguous nucleotide from the region of about nucleotides 1 to 204, 70 to 300, 205 to 390, 301 to 675, 391 to 675, 490 to 690, 586 to 795, 676 to 876, 796 to 999, 877 to 1101, and 1000 to 1338.

[1028] In a preferred embodiment, a nucleic acid fragment differs, e.g., by at least one, two, or more nucleotides, from the sequence of AA527520, AI027609, and AI219834.

[1029] 56201 Nucleic Acid Variants

[1030] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 20 or SEQ ID NO: 22. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 56201 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 21. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1031] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[1032] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[1033] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 20 or 22, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1034] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 21 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO: 21 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 56201 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 56201 gene.

[1035] Preferred variants include those that are correlated with the ability to regulate transport of specific ions, e.g., sodium ions, across a lipid bilayer.

[1036] Allelic variants of 56201, e.g., human 56201, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 56201 protein within a population that maintain the ability to regulate transport of ions, e.g., sodium ions, across a lipid bilayer. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 21, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 56201, e.g., human 56201, protein within a population that do not have the ability to regulate transport of ions across a lipid bilayer. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 21, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[1037] Moreover, nucleic acid molecules encoding other 56201 family members and, thus, which have a nucleotide sequence which differs from the 56201 sequences of SEQ ID NO: 20 or SEQ ID NO: 22 are intended to be within the scope of the invention.

[1038] Antisense Nucleic Acid Molecules, Ribozymes and Modified 56201 Nucleic Acid Molecules

[1039] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 56201. An “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 56201 coding strand, or to only a portion thereof (e.g., the coding region of human 56201 corresponding to SEQ ID NO: 22). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 56201 (e.g., the 5′ and 3′ untranslated regions).

[1040] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 56201 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 56201 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 56201 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[1041] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[1042] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 56201 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[1043] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[1044] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 56201-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 56201 cDNA disclosed herein (i.e., SEQ ID NO: 20 or SEQ ID NO: 22), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 56201-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 56201 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[1045] 56201 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 56201 (e.g., the 56201 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 56201 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N.Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[1046] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

[1047] A 56201 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulmé (2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.

[1048] For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[1049] PNAs of 56201 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 56201 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[1050] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[1051] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 56201 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 56201 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al, U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

[1052] Isolated 56201 Polypeptides

[1053] In another aspect, the invention features, an isolated 56201 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-56201 antibodies. 56201 protein can be isolated from cells or tissue sources using standard protein purification techniques. 56201 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[1054] Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[1055] In a preferred embodiment, a 56201 polypeptide has one or more of the following characteristics:

[1056] (i) it has the ability to regulate transport of specific ions, e.g., sodium ions, across a lipid bilayer, e.g., the plasma membrane;

[1057] (ii) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of a 56201 polypeptide, e.g., a polypeptide of SEQ ID NO: 21;

[1058] (iii) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a polypeptide a of SEQ ID NO: 21;

[1059] (iv) it can be found in neurons or muscle cells;

[1060] (v) it has an ion channel domain which is preferably about 70%, 80%, 90% or 95% with amino acid residues about 46 to 267 of SEQ ID NO: 21;

[1061] (vi) it has a pore lining peptide that contains a T-X-[D/E]-G-W (SEQ ID NO: 25) motif;

[1062] (vii) it has at least one, preferable two Protein kinase C phosphorylation sites (PS00005) located in intracellular portions of the molecule;

[1063] (viii) it has at least one, preferably two, three, more preferably four Casein kinase II phosphorylation sites (PS00006) located in intracellular portions of the molecule; or

[1064] (ix) it has at least one, preferably two tyrosine kinase phosphorylation sites (PS00007) located in intracellular portions of the molecule.

[1065] In a preferred embodiment the 56201 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO: 2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 21 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 21. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the ion channel domain, e.g., about amino acids 46 to 267 of SEQ ID NO: 21. In another preferred embodiment one or more differences are in the ion channel domain, e.g., about amino acids 46 to 267 of SEQ ID NO: 21.

[1066] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 56201 proteins differ in amino acid sequence from SEQ ID NO: 21, yet retain biological activity.

[1067] In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 21.

[1068] A 56201 protein or fragment is provided which varies from the sequence of SEQ ID NO: 21 in regions defined by amino acids about 1 to 200 and 246 to 398 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 21 in regions defined by amino acids about 201 to 245 of SEQ ID NO: 21. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[1069] In one embodiment, a biologically active portion of a 56201 protein includes an ion channel domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 56201 protein.

[1070] In a preferred embodiment, the 56201 protein has an amino acid sequence shown in SEQ ID NO: 21. In other embodiments, the 56201 protein is substantially identical to SEQ ID NO: 21. In yet another embodiment, the 56201 protein is substantially identical to SEQ ID NO: 21 and retains the functional activity of the protein of SEQ ID NO: 21, as described in detail in the subsections above.

[1071] 56201 Chimeric or Fusion Proteins

[1072] In another aspect, the invention provides 56201 chimeric or fusion proteins. As used herein, a 56201 “chimeric protein” or “fusion protein” includes a 56201 polypeptide linked to a non-56201 polypeptide. A “non-56201 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 56201 protein, e.g., a protein which is different from the 56201 protein and which is derived from the same or a different organism. The 56201 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a 56201 amino acid sequence. In a preferred embodiment, a 56201 fusion protein includes at least one (or two) biologically active portion of a 56201 protein. The non-56201 polypeptide can be fused to the N-terminus or C-terminus of the 56201 polypeptide.

[1073] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-56201 fusion protein in which the 56201 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 56201. Alternatively, the fusion protein can be a 56201 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 56201 can be increased through use of a heterologous signal sequence.

[1074] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[1075] The 56201 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 56201 fusion proteins can be used to affect the bioavailability of a 56201 substrate. 56201 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 56201 protein; (ii) mis-regulation of the 56201 gene; and (iii) aberrant post-translational modification of a 56201 protein.

[1076] Moreover, the 56201-fusion proteins of the invention can be used as immunogens to produce anti-56201 antibodies in a subject, to purify 56201 ligands and in screening assays to identify molecules which inhibit the interaction of 56201 with a 56201 substrate.

[1077] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 56201-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 56201 protein.

[1078] Variants of 56201 Proteins

[1079] In another aspect, the invention also features a variant of a 56201 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 56201 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 56201 protein. An agonist of the 56201 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a 56201 protein. An antagonist of a 56201 protein can inhibit one or more of the activities of the naturally occurring form of the 56201 protein by, for example, competitively modulating a 56201-mediated activity of a 56201 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 56201 protein.

[1080] Variants of a 56201 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 56201 protein for agonist or antagonist activity.

[1081] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 56201 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 56201 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[1082] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 56201 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 56201 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[1083] Cell based assays can be exploited to analyze a variegated 56201 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 56201 in a substrate-dependent manner. The transfected cells are then contacted with 56201 and the effect of the expression of the mutant on signaling by the 56201 substrate can be detected, e.g., by measuring channel conductance. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 56201 substrate, and the individual clones further characterized.

[1084] In another aspect, the invention features a method of making a 56201 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 56201 polypeptide, e.g., a naturally occurring 56201 polypeptide. The method includes: altering the sequence of a 56201 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[1085] In another aspect, the invention features a method of making a fragment or analog of a 56201 polypeptide a biological activity of a naturally occurring 56201 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 56201 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[1086] Anti-56201 Antibodies

[1087] In another aspect, the invention provides an anti-56201 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[1088] The anti-56201 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[1089] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[1090] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 56201 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-56201 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[1091] The anti-56201 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[1092] Phage display and combinatorial methods for generating anti-56201 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[1093] In one embodiment, the anti-56201 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art.

[1094] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[1095] An anti-56201 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[1096] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fe, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[1097] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a 56201 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[1098] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[1099] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and US 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 56201 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[1100] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[1101] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[1102] In preferred embodiments an antibody can be made by immunizing with purified 56201 antigen, or a fragment thereof, e.g., a fragment described herein, membrane associated antigen, tissue, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g., membrane fractions.

[1103] A full-length 56201 protein or, antigenic peptide fragment of 56201 can be used as an immunogen or can be used to identify anti-56201 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 56201 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 21 and encompasses an epitope of 56201. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[1104] Fragments of 56201 which include residues about 14 to 131, about 175 to 199, or about 246 to 269 can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophilic regions of the 56201 protein. Similarly, fragments of 56201 which include residues about 110 to 120, about 205 to 215, or about 230 to 240 can be used to make an antibody against a hydrophobic region of the 56201 protein; fragments of 56201 which include residues about 71 to 79, about 131 to 141, or about 200 to 245 can be used to make an antibody against an extracellular region of the 56201 protein; fragments of 56201 which include residues about 1 to 45, about 164 to 174, or about 270 to 398 can be used to make an antibody against an intracellular region of the 56201 protein; a fragment of 56201 which include residues about 46 to 267 can be used to make an antibody against the ion channel region of the 56201 protein; a fragment of 56201 which includes residues 200 to 245 can be used to make an antibody against the pore-lining peptide of the ion channel; and fragments of 56201 which include residues 1 to 45, 142 to 163, or 270 to 398 can be used to make antibodies against sequences that regulate the properties of the ion channel, e.g., the conductance response to stimuli, e.g., voltage gradients or phosphorylation.

[1105] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[1106] Antibodies which bind only native 56201 protein, only denatured or otherwise non-native 56201 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 56201 protein.

[1107] Preferred epitopes encompassed by the antigenic peptide are regions of 56201 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 56201 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 56201 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[1108] In a preferred embodiment the antibody can bind to the extracellular portion of the 56201 protein, e.g., it can bind to a whole cell which expresses the 56201 protein. In another embodiment, the antibody binds an intracellular portion of the 56201 protein. In preferred embodiments antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g., tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g., membrane fractions.

[1109] The anti-56201 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 56201 protein.

[1110] In a preferred embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement.

[1111] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example., it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[1112] In a preferred embodiment, an anti-56201 antibody alters (e.g., increases or decreases) the ion transporting activity of a 56201 polypeptide. For example, the antibody can bind at or in proximity to the active site, e.g., to an epitope that includes a residue located from about 200 to 245 of SEQ ID NO: 21.

[1113] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[1114] An anti-56201 antibody (e.g., monoclonal antibody) can be used to isolate 56201 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-56201 antibody can be used to detect 56201 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-56201 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[1115] The invention also includes a nucleic acids which encodes an anti-56201 antibody, e.g., an anti-56201 antibody described herein. Also included are vectors which include the nucleic acid and sells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[1116] The invention also includes cell lines, e.g., hybridomas, which make an anti-56201 antibody, e.g., and antibody described herein, and method of using said cells to make a 56201 antibody.

[1117] 56201 Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[1118] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[1119] A vector can include a 56201 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 56201 proteins, mutant forms of 56201 proteins, fusion proteins, and the like).

[1120] The recombinant expression vectors of the invention can be designed for expression of 56201 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[1121] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[1122] Purified fusion proteins can be used in 56201 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 56201 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[1123] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[1124] The 56201 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[1125] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[1126] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

[1127] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[1128] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[1129] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 56201 nucleic acid molecule within a recombinant expression vector or a 56201 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[1130] A host cell can be any prokaryotic or eukaryotic cell. For example, a 56201 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182)). Other suitable host cells are known to those skilled in the art.

[1131] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[1132] A host cell of the invention can be used to produce (i.e., express) a 56201 protein. Accordingly, the invention further provides methods for producing a 56201 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a 56201 protein has been introduced) in a suitable medium such that a 56201 protein is produced. In another embodiment, the method further includes isolating a 56201 protein from the medium or the host cell.

[1133] In another aspect, the invention features, a cell or purified preparation of cells which include a 56201 transgene, or which otherwise misexpress 56201. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a 56201 transgene, e.g., a heterologous form of a 56201, e.g., a gene derived from humans (in the case of a non-human cell). The 56201 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 56201, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 56201 alleles or for use in drug screening.

[1134] In another aspect, the invention features, a human cell, e.g., a neuronal or muscle stem cell, transformed with nucleic acid which encodes a subject 56201 polypeptide.

[1135] Also provided are cells, preferably human cells, e.g., a neuron, a muscle cell, or fibroblast cells, in which an endogenous 56201 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 56201 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 56201 gene. For example, an endogenous 56201 gene which is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[1136] In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 56201 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Production of 56201 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 56201 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

[1137] 56201 Transgenic Animals

[1138] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 56201 protein and for identifying and/or evaluating modulators of 56201 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 56201 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[1139] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 56201 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 56201 transgene in its genome and/or expression of 56201 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 56201 protein can further be bred to other transgenic animals carrying other transgenes.

[1140] 56201 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[1141] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[1142] Uses of 56201

[1143] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[1144] The isolated nucleic acid molecules of the invention can be used, for example, to express a 56201 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a 56201 mRNA (e.g., in a biological sample) or a genetic alteration in a 56201 gene, and to modulate 56201 activity, as described further below. The 56201 proteins can be used to treat disorders characterized by insufficient or excessive production of a 56201 substrate or production of 56201 inhibitors. In addition, the 56201 proteins can be used to screen for naturally occurring 56201 substrates, to screen for drugs or compounds which modulate 56201 activity, as well as to treat disorders characterized by insufficient or excessive production of 56201 protein or production of 56201 protein forms which have decreased, aberrant or unwanted activity compared to 56201 wild type protein (e.g., a neural or muscular disorder, e.g., paramyotonia congenita and hyperkalemic periodic paralysis, or conditions involving abnormal generation or maintenance of membrane potential or transmembrane ion gradients which can lead to epilepsy, psychiatric diseases, or dementia.). Moreover, the anti-56201 antibodies of the invention can be used to detect and isolate 56201 proteins, regulate the bioavailability of 56201 proteins, and modulate 56201 activity.

[1145] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 56201 polypeptide is provided. The method includes: contacting the compound with the subject 56201 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 56201 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 56201 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 56201 polypeptide. Screening methods are discussed in more detail below.

[1146] 56201 Screening Assays

[1147] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 56201 proteins, have a stimulatory or inhibitory effect on, for example, 56201 expression or 56201 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 56201 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 56201 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[1148] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 56201 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 56201 protein or polypeptide or a biologically active portion thereof.

[1149] In one embodiment, an activity of a 56201 protein can be assayed by introducing a 56201 nucleic acid into a X. laevis oocyte, expression the nucleic acid such that 56201 protein is produced, and monitoring the conductance of the channel in response to specific stimuli, e.g., a voltage gradient. Assays of this type have been described in Goldin et al., (1986), Proc. Natl. Acad. Sci. USA 83(19):7503-7, Noda et al. (1986), Nature 320:188-92, and Noda et al. (1986), Nature 322:826-28, the contents of which are incorporated herein by reference.

[1150] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[1151] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[1152] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[1153] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 56201 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 56201 activity is determined. Determining the ability of the test compound to modulate 56201 activity can be accomplished by monitoring, for example, ion channel conductance. The cell, for example, can be of mammalian origin, e.g., human.

[1154] The ability of the test compound to modulate 56201 binding to a compound, e.g., a 56201 substrate, or to bind to 56201 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 56201 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 56201 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 56201 binding to a 56201 substrate in a complex. For example, compounds (e.g., 56201 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[1155] The ability of a compound (e.g., a 56201 substrate) to interact with 56201 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 56201 without the labeling of either the compound or the 56201. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 56201.

[1156] In yet another embodiment, a cell-free assay is provided in which a 56201 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 56201 protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the 56201 proteins to be used in assays of the present invention include fragments which participate in interactions with non-56201 molecules, e.g., fragments with high surface probability scores.

[1157] Soluble and/or membrane-bound forms of isolated proteins (e.g., 56201 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[1158] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[1159] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[1160] In another embodiment, determining the ability of the 56201 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[1161] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[1162] It may be desirable to immobilize either 56201, an anti-56201 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 56201 protein, or interaction of a 56201 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/56201 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 56201 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 56201 binding or activity determined using standard techniques.

[1163] Other techniques for immobilizing either a 56201 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 56201 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[1164] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[1165] In one embodiment, this assay is performed utilizing antibodies reactive with 56201 protein or target molecules but which do not interfere with binding of the 56201 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 56201 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 56201 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 56201 protein or target molecule.

[1166] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[1167] In a preferred embodiment, the assay includes contacting the 56201 protein or biologically active portion thereof with a known compound which binds 56201 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 56201 protein, wherein determining the ability of the test compound to interact with a 56201 protein includes determining the ability of the test compound to preferentially bind to 56201 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[1168] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment-are the 56201 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a 56201 protein through modulation of the activity of a downstream effector of a 56201 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[1169] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[1170] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[1171] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[1172] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[1173] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[1174] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[1175] In yet another aspect, the 56201 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 56201 (“56201-binding proteins” or “56201-bp”) and are involved in 56201 activity. Such 56201-bps can be activators or inhibitors of signals by the 56201 proteins or 56201 targets as, for example, downstream elements of a 56201-mediated signaling pathway.

[1176] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 56201 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 56201 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 56201-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 56201 protein.

[1177] In another embodiment, modulators of 56201 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 56201 mRNA or protein evaluated relative to the level of expression of 56201 mRNA or protein in the absence of the candidate compound. When expression of 56201 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 56201 mRNA or protein expression. Alternatively, when expression of 56201 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 56201 mRNA or protein expression. The level of 56201 mRNA or protein expression can be determined by methods described herein for detecting 56201 mRNA or protein.

[1178] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a 56201 protein can be confirmed in vivo, e.g., in an animal such as an animal model for a neural or muscular disorder, e.g., epilepsy or cardiac arrythmia.

[1179] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 56201 modulating agent, an antisense 56201 nucleic acid molecule, a 56201-specific antibody, or a 56201-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[1180] 56201 Detection Assays

[1181] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 56201 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[1182] 56201 Chromosome Mapping

[1183] The 56201 nucleotide sequences or portions thereof can be used to map the location of the 56201 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 56201 sequences with genes associated with disease.

[1184] Briefly, 56201 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 56201 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 56201 sequences will yield an amplified fragment.

[1185] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[1186] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 56201 to a chromosomal location.

[1187] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[1188] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[1189] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al (1987) Nature, 325:783-787.

[1190] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 56201 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[1191] 56201 Tissue Typing

[1192] 56201 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[1193] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 56201 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[1194] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 20 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 22 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[1195] If a panel of reagents from 56201 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[1196] Use of Partial 56201 Sequences in Forensic Biology

[1197] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[1198] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 20 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 20 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[1199] The 56201 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 56201 probes can be used to identify tissue by species and/or by organ type.

[1200] In a similar fashion, these reagents, e.g., 56201 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[1201] Predictive Medicine of 56201

[1202] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[1203] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 56201.

[1204] Such disorders include, e.g., a disorder associated with the misexpression of 56201 gene; or a disorder of the nervous system or muscular disorder.

[1205] The method includes one or more of the following:

[1206] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 56201 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[1207] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 56201 gene;

[1208] detecting, in a tissue of the subject, the misexpression of the 56201 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[1209] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 56201 polypeptide.

[1210] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 56201 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[1211] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 20, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 56201 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[1212] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 56201 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 56201.

[1213] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[1214] In preferred embodiments the method includes determining the structure of a 56201 gene, an abnormal structure being indicative of risk for the disorder.

[1215] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 56201 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[1216] Diagnostic and Prognostic Assays of 56201

[1217] Diagnostic and prognostic assays of the invention include method for assessing the expression level of 56201 molecules and for identifying variations and mutations in the sequence of 56201 molecules.

[1218] Expression Monitoring and Profiling:

[1219] The presence, level, or absence of 56201 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 56201 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 56201 protein such that the presence of 56201 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 56201 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 56201 genes; measuring the amount of protein encoded by the 56201 genes; or measuring the activity of the protein encoded by the 56201 genes.

[1220] The level of mRNA corresponding to the 56201 gene in a cell can be determined both by in situ and by in vitro formats.

[1221] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 56201 nucleic acid, such as the nucleic acid of SEQ ID NO: 20, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 56201 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[1222] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 56201 genes.

[1223] The level of mRNA in a sample that is encoded by one of 56201 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[1224] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 56201 gene being analyzed.

[1225] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 56201 mRNA, or genomic DNA, and comparing the presence of 56201 mRNA or genomic DNA in the control sample with the presence of 56201 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 56201 transcript levels.

[1226] A variety of methods can be used to determine the level of protein encoded by 56201. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[1227] The detection methods can be used to detect 56201 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 56201 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 56201 protein include introducing into a subject a labeled anti-56201 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-56201 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[1228] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 56201 protein, and comparing the presence of 56201 protein in the control sample with the presence of 56201 protein in the test sample.

[1229] The invention also includes kits for detecting the presence of 56201 in a biological sample. For example, the kit can include a compound or agent capable of detecting 56201 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 56201 protein or nucleic acid.

[1230] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[1231] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[1232] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 56201 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as epilepsy or cardiac arrhythmia.

[1233] In one embodiment, a disease or disorder associated with aberrant or unwanted 56201 expression or activity is identified. A test sample is obtained from a subject and 56201 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 56201 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 56201 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[1234] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 56201 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a neuron or muscle cell disorder.

[1235] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 56201 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 56201 (e.g., other genes associated with a 56201-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[1236] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 56201 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose a [disordera] disorder in a subject wherein a change in 56201 expression is an indication that the subject has or is disposed to having a neural or muscular disorder. The method can be used to monitor a treatment for a neural or muscular disorder in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[1237] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 56201 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[1238] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 56201 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[1239] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[1240] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 56201 expression.

[1241] 56201 Arrays and Uses Thereof

[1242] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a 56201 molecule (e.g., a 56201 nucleic acid or a 56201 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm2, and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[1243] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a 56201 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 56201. Each address of the subset can include a capture probe that hybridizes to a different region of a 56201 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for a 56201 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 56201 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 56201 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[1244] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[1245] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a 56201 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 56201 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-56201 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[1246] In another aspect, the invention features a method of analyzing the expression of 56201. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 56201-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[1247] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 56201. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 56201. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[1248] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 56201 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[1249] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[1250] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 56201-associated disease or disorder; and processes, such as a cellular transformation associated with a 56201-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 56201-associated disease or disorder

[1251] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 56201) that could serve as a molecular target for diagnosis or therapeutic intervention.

[1252] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon a 56201 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99% identical to a 56201 polypeptide or fragment thereof. For example, multiple variants of a 56201 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[1253] The polypeptide array can be used to detect a 56201 binding compound, e.g., an antibody in a sample from a subject with specificity for a 56201 polypeptide or the presence of a 56201-binding protein or ligand.

[1254] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 56201 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[1255] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 56201 or from a cell or subject in which a 56201 mediated response has been elicited, e.g., by contact of the cell with 56201 nucleic acid or protein, or administration to the cell or subject 56201 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 56201 (or does not express as highly as in the case of the 56201 positive plurality of capture probes) or from a cell or subject which in which a 56201 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 56201 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[1256] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 56201 or from a cell or subject in which a 56201-mediated response has been elicited, e.g., by contact of the cell with 56201 nucleic acid or protein, or administration to the cell or subject 56201 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 56201 (or does not express as highly as in the case of the 56201 positive plurality of capture probes) or from a cell or subject which in which a 56201 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[1257] In another aspect, the invention features a method of analyzing 56201, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 56201 nucleic acid or amino acid sequence; comparing the 56201 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 56201.

[1258] Detection of 56201 Variations or Mutations

[1259] The methods of the invention can also be used to detect genetic alterations in a 56201 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 56201 protein activity or nucleic acid expression, such as a neural or muscular disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 56201-protein, or the mis-expression of the 56201 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 56201 gene; 2) an addition of one or more nucleotides to a 56201 gene; 3) a substitution of one or more nucleotides of a 56201 gene, 4) a chromosomal rearrangement of a 56201 gene; 5) an alteration in the level of a messenger RNA transcript of a 56201 gene, 6) aberrant modification of a 56201 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a 56201 gene, 8) a non-wild type level of a 56201-protein, 9) allelic loss of a 56201 gene, and 10) inappropriate post-translational modification of a 56201-protein.

[1260] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 56201-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 56201 gene under conditions such that hybridization and amplification of the 56201-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[1261] In another embodiment, mutations in a 56201 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[1262] In other embodiments, genetic mutations in 56201 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 56201 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 56201 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 56201 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[1263] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 56201 gene and detect mutations by comparing the sequence of the sample 56201 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[1264] Other methods for detecting mutations in the 56201 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[1265] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 56201 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[1266] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 56201 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 56201 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[1267] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[1268] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[1269] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[1270] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 56201 nucleic acid.

[1271] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 20 or the complement of SEQ ID NO: 20. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[1272] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 56201. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[1273] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the Tm of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[1274] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 56201 nucleic acid.

[1275] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 56201 gene.

[1276] Use of 56201 Molecules as Surrogate Markers

[1277] The 56201 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 56201 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 56201 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[1278] The 56201 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 56201 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-56201 antibodies may be employed in an immune-based detection system for a 56201 protein marker, or 56201-specific radiolabeled probes may be used to detect a 56201 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[1279] The 56201 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 56201 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 56201 DNA may correlate 56201 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[1280] 56201 Pharmaceutical Compositions

[1281] The nucleic acid and polypeptides, fragments thereof, as well as anti-56201 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[1282] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[1283] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[1284] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[1285] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[1286] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[1287] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[1288] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[1289] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[1290] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[1291] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[1292] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[1293] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[1294] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[1295] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[1296] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated. An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[1297] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[1298] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[1299] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[1300] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[1301] 56201 Methods of Treatment

[1302] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 56201 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[1303] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 56201 molecules of the present invention or 56201 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[1304] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 56201 expression or activity, by administering to the subject a 56201 or an agent which modulates 56201 expression or at least one 56201 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 56201 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 56201 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 56201 aberrance, for example, a 56201, 56201 agonist or 56201 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[1305] It is possible that some 56201 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[1306] The 56201 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of neural disorders, muscle disorders, pain disorders, disorders associated with bone metabolism, immune disorders, cardiovascular disorders, viral disorders, and cellular proliferative and/or differentiative disorders.

[1307] Example of neural disorders not described above include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin B1) deficiency and vitamin B12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.

[1308] Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[1309] Aberrant expression and/or activity of 56201 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 56201 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 56201 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 56201 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[1310] The 56201 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders. Examples of immune disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

[1311] Examples of disorders involving the heart or “cardiovascular disorder” include, but are not limited to, a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, congestive heart failure, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

[1312] 56201 molecules may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 56201 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 56201 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

[1313] In addition, 56201 molecules may play an important role in the regulation of cellular proliferative and/or differentiative disorders. Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[1314] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[1315] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[1316] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[1317] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[1318] As discussed, successful treatment of 56201 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 56201 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[1319] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[1320] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[1321] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 56201 expression is through the use of aptamer molecules specific for 56201 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 56201 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[1322] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 56201 disorders. For a description of antibodies, see the Antibody section above.

[1323] In circumstances wherein injection of an animal or a human subject with a 56201 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 56201 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 56201 protein. Vaccines directed to a disease characterized by 56201 expression may also be generated in this fashion.

[1324] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[1325] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 56201 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[1326] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[1327] Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 56201 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 56201 can be readily monitored and used in calculations of IC50.

[1328] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[1329] Another aspect of the invention pertains to methods of modulating 56201 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 56201 or agent that modulates one or more of the activities of 56201 protein activity associated with the cell. An agent that modulates 56201 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a 56201 protein (e.g., a 56201 substrate or receptor), a 56201 antibody, a 56201 agonist or antagonist, a peptidomimetic of a 56201 agonist or antagonist, or other small molecule.

[1330] In one embodiment, the agent stimulates one or 56201 activities. Examples of such stimulatory agents include active 56201 protein and a nucleic acid molecule encoding 56201. In another embodiment, the agent inhibits one or more 56201 activities. Examples of such inhibitory agents include antisense 56201 nucleic acid molecules, anti-56201 antibodies, and 56201 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 56201 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 56201 expression or activity. In another embodiment, the method involves administering a 56201 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 56201 expression or activity.

[1331] Stimulation of 56201 activity is desirable in situations in which 56201 is abnormally downregulated and/or in which increased 56201 activity is likely to have a beneficial effect. For example, stimulation of 56201 activity is desirable in situations in which a 56201 is downregulated and/or in which increased 56201 activity is likely to have a beneficial effect. Likewise, inhibition of 56201 activity is desirable in situations in which 56201 is abnormally upregulated and/or in which decreased 56201 activity is likely to have a beneficial effect.

[1332] 56201 Pharmacogenomics

[1333] The 56201 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 56201 activity (e.g., 56201 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 56201 associated disorders (e.g.,neural or muscular disorder) associated with aberrant or unwanted 56201 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 56201 molecule or 56201 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 56201 molecule or 56201 modulator.

[1334] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[1335] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[1336] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 56201 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[1337] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 56201 molecule or 56201 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[1338] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 56201 molecule or 56201 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[1339] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 56201 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 56201 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[1340] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a 56201 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 56201 gene expression, protein levels, or upregulate 56201 activity, can be monitored in clinical trials of subjects exhibiting decreased 56201 gene expression, protein levels, or downregulated 56201 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 56201 gene expression, protein levels, or downregulate 56201 activity, can be monitored in clinical trials of subjects exhibiting increased 56201 gene expression, protein levels, or upregulated 56201 activity. In such clinical trials, the expression or activity of a 56201 gene, and preferably, other genes that have been implicated in, for example, a 56201-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[1341] 56201 Informatics

[1342] The sequence of a 56201 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 56201. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 56201 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[1343] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[1344] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[1345] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[1346] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[1347] Thus, in one aspect, the invention features a method of analyzing 56201, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 56201 nucleic acid or amino acid sequence; comparing the 56201 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 56201. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[1348] The method can include evaluating the sequence identity between a 56201 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[1349] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[1350] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[1351] Thus, the invention features a method of making a computer readable record of a sequence of a 56201 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[1352] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 56201 sequence, or record, in machine-readable form; comparing a second sequence to the 56201 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 56201 sequence includes a sequence being compared. In a preferred embodiment the 56201 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 56201 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[1353] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 56201-associated disease or disorder or a pre-disposition to a 56201-associated disease or disorder, wherein the method comprises the steps of determining 56201 sequence information associated with the subject and based on the 56201 sequence information, determining whether the subject has a 56201-associated disease or disorder or a pre-disposition to a 56201-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[1354] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 56201-associated disease or disorder or a pre-disposition to a disease associated with a 56201 wherein the method comprises the steps of determining 56201 sequence information associated with the subject, and based on the 56201 sequence information, determining whether the subject has a 56201-associated disease or disorder or a pre-disposition to a 56201-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 56201 sequence of the subject to the 56201 sequences in the database to thereby determine whether the subject as a 56201-associated disease or disorder, or a pre-disposition for such.

[1355] The present invention also provides in a network, a method for determining whether a subject has a 56201 associated disease or disorder or a pre-disposition to a 56201-associated disease or disorder associated with 56201, said method comprising the steps of receiving 56201 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 56201 and/or corresponding to a 56201-associated disease or disorder (e.g., neural or muscular disorders), and based on one or more of the phenotypic information, the 56201 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 56201-associated disease or disorder or a pre-disposition to a 56201-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1356] The present invention also provides a method for determining whether a subject has a 56201-associated disease or disorder or a pre-disposition to a 56201-associated disease or disorder, said method comprising the steps of receiving information related to 56201 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 56201 and/or related to a 56201-associated disease or disorder, and based on one or more of the phenotypic information, the 56201 information, and the acquired information, determining whether the subject has a 56201-associated disease or disorder or a pre-disposition to a 56201-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1357] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

[1358] Background of the 32620 Invention

[1359] Symporters are integral membranes that transport molecules across the lipid bilayer. Symporters couple the movement of one solute with another in order to mobilize the first solute across the membrane. Thus, symporters do not directly require ATP as an energy source unlike ATP-dependent transporters. The energy source that one class of symporters uses is the ion gradient that is maintained across the membrane. This ion gradient is manifest as an electric potential. A typical resting mammalian cell can have a transmembrane electric potential of about 50 to about 150 mV. ATP dependent transporters maintain this potential by extruding ions and protons from the cell. A Na+-K+ ATP dependent pump is largely responsible for the sodium gradient such that sodium concentrations are higher on the extracellular face of the membrane.

[1360] Sodium-sugar symporters are a particularly important family of symporters. These integral membrane proteins are typically believed to have twelve transmembrane spans, but can have eleven, twelve, thirteen, or fourteen transmembrane spans (Turk et al. (1996) J Biol Chem 271:1925-1934). They couple the movement of sodium ions down the sodium gradient into the cell with the movement of glucose into the cell. All cells require sugars as an energy source. Thus, this transport process is critical. Moreover, the process is especially critical in cells responsible for obtaining sugars for the rest of the body. In the digestive tract, intestinal brush border cells are responsible for acquiring sugars from digested foods and transporting them into the bloodstream. In the excretory system, glomerular cells in the proximal tubules of the kidney reabsorb sugars, especially glucose, from the glomerular filtrate in order to prevent their excretion. In humans, the key sodium-sugar symporters are SGLT1 and SGLT2. SGLT1 and SGLT2 are found in the S1 and S2 segments of kidney tubules.

[1361] Summary of the 32620 Invention

[1362] The present invention is based, in part, on the discovery of a novel sodium-sugar symporter family member, referred to herein as “32620”. The nucleotide sequence of a cDNA encoding 32620 is shown in SEQ ID NO: 26, and the amino acid sequence of a 32620 polypeptide is shown in SEQ ID NO: 27. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 28.

[1363] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 32620 protein or polypeptide, e.g., a biologically active portion of the 32620 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 27. In other embodiments, the invention provides isolated 32620 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 26, SEQ ID NO: 28, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 26, SEQ ID NO: 28, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 32620 protein or an active fragment thereof.

[1364] In a related aspect, the invention further provides nucleic acid constructs that include a 32620 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 32620 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 32620 nucleic acid molecules and polypeptides.

[1365] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 32620-encoding nucleic acids.

[1366] In still another related aspect, isolated nucleic acid molecules that are antisense to a 32620 encoding nucleic acid molecule are provided.

[1367] A nucleic acid of the invention can be attached to a solid support, e.g., a bead, matrix, or planar surface.

[1368] In another aspect, the invention features, 32620 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 32620-mediated or -related disorders. In another embodiment, the invention provides 32620 polypeptides having a 32620 activity. Preferred polypeptides are 32620 proteins including at least one sodium-sugar symporter domain, and, preferably, having a 32620 activity, e.g., the ability to transport sugars, e.g., D-glucose, D-fructose or D-galactose, into and out of a cell, e.g., a neuronal or glial cell (e.g., a brain cell (e.g., cortical or hypothalamic cell), a spinal cord cell).

[1369] In other embodiments, the invention provides 32620 polypeptides, e.g., a 32620 polypeptide having the amino acid sequence shown in SEQ ID NO: 27 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 27 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 26, SEQ ID NO: 28, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 32620 protein or an active fragment thereof. In one embodiment, a 32620 polypeptide is attached to a solid support.

[1370] In a related aspect, the invention further provides nucleic acid constructs which include a 32620 nucleic acid molecule described herein.

[1371] In a related aspect, the invention provides 32620 polypeptides or fragments operatively linked to non-32620 polypeptides to form fusion proteins.

[1372] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 32620 polypeptides or fragments thereof, e.g., an extracellular domain of a 32620 polypeptide. The antibody or antigen-binding fragment can be attached to a solid support, a label, a drug, or toxin.

[1373] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 32620 polypeptides or nucleic acids.

[1374] In still another aspect, the invention provides a process for modulating 32620 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 32620 polypeptides or nucleic acids, such as conditions involving aberrant or deficient, e.g., upregulated or downregulated, sugar transporter mediated activity. Sugar transporter associated disorders typically result in, e.g., upregulated or downregulated, sugar levels in a cell, e.g., a brain, spinal, glial, or nerve cell.

[1375] The invention also provides assays for determining the activity of or the presence or absence of 32620 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[1376] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 32620 polypeptide or nucleic acid molecule, including for disease diagnosis.

[1377] In another aspect, the invention provides methods of screening for agents, e.g., compounds, that modulate the expression or activity of the 32620 polypeptides or nucleic acids, e.g., compounds that modulate the activity of a 32620-expressing cell, e.g., a neuronal or glial cell, e.g., a brain (cortical or hypothalamic) cell, or a spinal cord cell.

[1378] In one embodiment, normal pain response, or aberrant or altered pain response is modulated. The effect of an agent, e.g., a compound, on the pain response can be evaluated by an analgesic test, e.g., the hot plate test, tail flick test, writhing test, paw pressure test, all electric stimulation test, tail withdrawal test, or formalin test. In one embodiment, the agent, e.g., compound, modulates (e.g., increases or decreases) 32620 activity. In a preferred embodiment, the agent, e.g., compound, modulates the endogenous levels of a 32620 substrate.

[1379] In still another aspect, the invention provides a process for modulating 32620 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant, e.g., decreased or increased expression of the 32620 polypeptides or nucleic acids, such as conditions involving pain response, aberrant or altered pain response, or pain related disorders.

[1380] In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) an activity of a cell, e.g., a 32620-expressing cell (e.g., a neural or glial cell), or a pain response in a subject. The method includes contacting the cell with, or administered to the subject, an agent, e.g., a compound, that modulates the activity or expression of a 32620 polypeptide or nucleic acid, in an amount effective to modulate the activity or the response. In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 32620 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.

[1381] In a preferred embodiment, the cell, e.g., the 32620-expressing cell, is a central or peripheral nervous system cell, e.g., a cortical or hypothalamic cell, or a cell in an area involved in pain control, e.g., a cell in the substantia gelatinosa of the spinal cord, or a cell in the periaqueductal gray matter.

[1382] In a preferred embodiment, the agent, e.g., the compound, and the 32620-polypeptide or nucleic acid are contacted in vitro or ex vivo. In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. The contacting or administering step(s) can be repeated.

[1383] Preferably, the subject is a human, e.g., a patient with a neurological disorder, or a patient suffering from pain or a pain-associated disorder, e.g., a disorder as disclosed herein. For example, the subject can be a neurological disorder, e.g., a patient with a neurological disorder as described herein, or a patient with pain elicited from tissue injury, e.g., inflammation, infection, ischemia; pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches, e.g., migrane; pain associated with surgery; pain related to inflammation, e.g., irritable bowel syndrome; or chest pain. The subject can be a patient with complex regional pain syndrome (CRPS), reflex sympathetic dystrophy (RSD), causalgia, neuralgia, central pain and dysesthesia syndrome, carotidynia, neurogenic pain, refractory cervicobrachial pain syndrome, myofascial pain syndrome, craniomandibular pain dysfunction syndrome, chronic idiopathic pain syndrome, Costen's pain-dysfunction, acute chest pain syndrome, gynecologic pain syndrome, patellofemoral pain syndrome, anterior knee pain syndrome, recurrent abdominal pain in children, colic, low back pain syndrome, neuropathic pain, phantom pain from amputation, phantom tooth pain, or pain asymbolia. The subject can be a cancer patient, e.g., a patient with brain cancer. In other embodiments, the subject is a non-human animal, e.g., an experimental animal, e.g., an arthritic rat model of chronic pain, a chronic constriction injury (CCI) rat model of neuropathic pain, or a rat model of unilateral inflammatory pain by intraplantar injection of Freund's complete adjuvant (FCA).

[1384] In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.

[1385] In additional preferred embodiments, the agent is an antisense molecule, a ribozyme, a triple helix molecule, or a 32620 nucleic acid, or any combination thereof. In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.

[1386] In another aspect, the invention features a method of treating or preventing, in a subject, a 32620-associated disorder. The method includes administering to the subject, e.g., a subject at risk of, or afflicted with, a 32620-associated disorder, an agent, e.g., a compound as described herein, that modulates the activity or expression of a 32620 polypeptide or nucleic acid, in an amount effective to treat or prevent the disorder. The agent can be administered by epidural or other route described herein.

[1387] In a preferred embodiment, the disorder is a neurological disorder, or a pain related disorder.

[1388] In a preferred embodiment, the subject is a subject as described herein, e.g., a human.

[1389] In still another aspect, the invention features a method for evaluating the efficacy of a treatment of a disorder, e.g., a disorder disclosed herein, in a subject. The method includes treating a subject with a protocol under evaluation; assessing the expression of a 32620 nucleic acid or 32620 polypeptide, such that a change in the level of 32620 nucleic acid or 32620 polypeptide after treatment, relative to the level before treatment, is indicative of the efficacy of the treatment of the disorder. Preferably, the subject is a human, e.g., a patient at risk of, or having, a neurological or a pain disorder.

[1390] The invention also features a method of diagnosing a disorder, e.g., a disorder disclosed herein, in a subject. The method includes evaluating the expression or activity of a 32620 nucleic acid or a 32620 polypeptide, such that, a difference in the level of 32620 nucleic acid or 32620 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of the disorder.

[1391] In a preferred embodiment, the disorder is a neurological or a pain-related disorder.

[1392] In a preferred embodiment, the subject is a human.

[1393] In a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample, biopsy sample, or cerebro-spinal fluid sample, is obtained from the subject. In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 32620 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 32620 nucleic acid or polypeptide.

[1394] The invention also provides assays for determining the activity of or the presence or absence of 32620 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[1395] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 32620 polypeptide or nucleic acid molecule, including for disease diagnosis.

[1396] In yet another aspect, the invention features a method for identifying an agent, e.g., a compound, which modulates the activity of a 32620 polypeptide, e.g., a 32620 polypeptide as described herein, or the expression of a 32620 nucleic acid, e.g., a 32620 nucleic acid as described herein, including contacting the 32620 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect of the test compound on the activity of the 32620 polypeptide or nucleic acid to thereby identify a compound which modulates the activity of the 32620 polypeptide or nucleic acid.

[1397] In a preferred embodiment, the activity of the 32620 polypeptide is modulation of neurological activity or pain response.

[1398] In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.

[1399] In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 32620 nucleic acid, or any combination thereof.

[1400] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 32620 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 32620 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 32620 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[1401] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[1402] Detailed Description of 32620

[1403] The human 32620 sequence (FIG. 13; SEQ ID NO: 26), which is approximately 2326 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2028 nucleotides, including the termination codon (nucleotides indicated as coding of SEQ ID NO: 26 in FIG. 13; SEQ ID NO: 28). The coding sequence encodes a 675 amino acid protein (SEQ ID NO: 27).

[1404] A human 32620 contains the following regions or other structural features:

[1405] a sodium-sugar symporter domain (PFAM Accession Number PF00474) located at about amino acid residues 58 to 487 of SEQ ID NO: 27;

[1406] twelve predicted transmembrane domains at about amino acids 28 to 48, 105 to 122, 136 to 155, 177 to 201, 209 to 229, 271 to 287, 376 to 400, 417 to 439, 447 to 471, 479 to 502, 521 to 542, and 651 to 669 of SEQ ID NO: 27;

[1407] two predicted extracellular domains at about amino acids 1 to 27, and 670 to 675 of SEQ ID NO: 27;

[1408] five predicted extracellular loops at about amino acids 123 to 135, 202 to 208, 288 to 375, 440 to 446, and 503 to 520 of SEQ ID NO: 27;

[1409] six predicted intracellular loops at about amino acids 49 to 104, 156 to 176, 230 to 270, 401 to 416, 472 to 478, and 543 to 650 of SEQ ID NO: 27;

[1410] seven predicted Protein Kinase C phosphorylation sites (PS00005) at about amino acids 47 to 49, 50 to 52, 54 to 56, 242 to 244, 413 to 415, 602 to 604, and 611 to 613 of SEQ ID NO: 27;

[1411] eight predicted Casein Kinase II phosphorylation sites (PS00006) located at about at amino acids 50 to 53, 99 to 102, 127 to 130, 173 to 176, 413 to 416, 558 to 561, 645 to 648, and 654 to 657 of SEQ ID NO: 27;

[1412] one predicted tyrosine kinase phosphorylation site (PS00007) located at about amino acids 494 to 501 of SEQ ID NO: 27;

[1413] one predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acid 51 to 54 of SEQ ID NO: 27;

[1414] four predicted N-glycosylation sites (PS00001) located at about amino acids 243 to 246, 247 to 250, 301 to 304, and 601 to 604; and

[1415] thirteen predicted N-myristylation sites (PS00008) from about amino acids 23 to 28, 43 to 48, 86 to 91, 95 to 100, 123 to 128, 195 to 200, 227 to 232, 273 to 278, 308 to 313, 375 to 380, 479 to 484, 487 to 492, and 583 to 588 of SEQ ID NO: 27.

[1416] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[1417] A plasmid containing the nucleotide sequence encoding human 32620 (clone Fbh32620FL1) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[1418] The 32620 protein contains a significant number of structural characteristics in common with members of the sodium-sugar symporter family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[1419] Sodium-sugar symporter family members are characterized by a common fold, having twelve predicted transmembrane spans. Proteins of this family can have eleven, twelve, thirteen, or fourteen actual transmembrane spans (Turk et al. (1996) 271:1925-1934). The sugar recognition domain is predicted to reside in the last about 150 to 110 amino acids of the protein (Panayotova-Heiermann, supra.). In addition, two conserved amino acids are implicated in sodium coupling, a glycine on the intracellular side of the first transmembrane span, and an arginine located in the extracellular loop between the sixth and seventh transmembrane spans (see annotation of SwissProt:P31639; Wells et al. (1992) Am. J. Physiol. 263:F459-F465).

[1420] A 32620 polypeptide can include a “sodium-sugar symporter domain” or regions homologous with a “sodium-sugar symporter domain”.

[1421] As used herein, the term “sodium-sugar symporter domain” includes an amino acid sequence of about 300 to 700 and having a bit score for the alignment of sequence to the sequence of the sodium-sugar symporters domain (HMM) of at least 300. In a preferred embodiment, a sodium-sugar symporters domain includes an amino acid sequence of about preferably about 350 to 600, more preferably about 400 to 500, even more preferably about 425 to 430, amino acid residues in length and having a bit score for the alignment of the sequence to the sodium-sugar symporter domain (HMM) of at least 400, preferably 500, and more preferably 600. The sodium-sugar symporter domain (HMM) has been assigned the PFAM Accession Number PF00474 (http;//genome.wustl.edu/Pfam/.html). By these criteria, human 32620 has a sodium-sugar symporter domain located at about residues 58 to 487 of SEQ ID NO: 27. An alignment of the sodium-sugar symporter domain (amino acids 58 to 487 of SEQ ID NO: 27) of human 32620 with a consensus amino acid sequence (SEQ ID NO: 29) derived from a hidden Markov model is depicted in FIG. 15.

[1422] A sodium-sugar symporter domain protein can include a perfect or imperfect match to the Prosite sodium:solute symporter signature 1 (PS00456; [GS]-x(2)-[LIY]-x(3)-[LIVMFYWSTAG](10)-[LIY]-[STAV]-x(2)-G-G-[LMF]-x-[SAP] wherein x is any amino acid, and a number in parenthesis indicates the amino acid is repeat that number of times; SEQ ID NO: 31). Preferably, a sodium-sugar symporter domain protein has three, two, one, or no mismatches relative to the signature. For example, human 32620 has a nearly perfect match (15 of 16) to the PS00456 signature from about amino acids 174 to 199 of SEQ ID NO: 27.

[1423] A sodium-sugar symporter domain protein can also include a perfect or imperfect match to the Prosite sodium:solute symporter signature 2 (PS00457; [GAST]-[LIVM]-x(3)-[KR]-x(4)-G-A-x(2)-[GAS]-[LIVMGS]-[LIVMW]-[LIVMGAT]-G-x-[LIVMGA]; wherein x is any amino acid, and a number in parenthesis indicates the amino acid is repeat that number of times; SEQ ID NO: 32). Preferably, a sodium-sugar symporter domain protein has three, two, one, or no mismatches relative to the signature. For example, human 32620 has a nearly perfect match (9 of 11) to the PS00457 signature from about amino acids 469 to 489 of SEQ ID NO: 27. Preferably, human 32620 has a conserved glycine at about amino acid 43 of SEQ ID NO: 27, and a conserved arginine at about amino acid 295 of SEQ ID NO: 27. These residues are implicated in the sodium coupling mechanism.

[1424] In a preferred embodiment 32620 polypeptide or protein has a “sodium-sugar symporter domain” or a region which includes at least about 350 to 800 more preferably about 400 to 500 or 420 to 450 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “sodium-sugar symporter domain,” e.g., the sodium-sugar symporter domain of human 32620 (e.g., residues 58 to 487 of SEQ ID NO: 27).

[1425] To identify the presence of a “sodium-sugar symporter” domain in a 32620 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al.(1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a “sodium-sugar symporter domain” in the amino acid sequence of human 32620 at about residues 58 to 487 of SEQ ID NO: 27 (see FIG. 13).

[1426] A 32620 molecule can further include: at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, thirteen, fourteen, and preferably twelve predicted transmembrane domains; at least one, preferably two predicted extracellular domains; at least one, two, three, four, and preferably five predicted extracellular loops; at least one, two, three, four, five and preferably six predicted intracellular loops; at least one, two, three, four, five, six, and preferably seven predicted protein kinase C phosphorylation sites; at least one, two, three, four, five, six, seven, and preferably eight predicted casein kinase II phosphorylation sites; at least one predicted tyrosine kinase phosphorylation sites; at least one predicted cAMP/cGMP-dependent protein kinase phosphorylation sites; at least one, two, three, and preferably four predicted N-glycosylation sites; and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, and preferably thirteen predicted N-myristylation sites.

[1427] In one embodiment, a 32620 protein includes at least one extracellular domain. When located at the N-terminal domain the extracellular domain is referred to herein as an “N-terminal extracellular domain” in the amino acid sequence of the protein. As used herein, an “N-terminal extracellular domain” includes an amino acid sequence having about 1-100, preferably about 1-50, more preferably about 1-40, even more preferably about 1-30 amino acid residues in length and is located outside of a cell or extracellularly. The C-terminal amino acid residue of a “N-terminal extracellular domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring 32620 or 32620-like protein. For example, an N-terminal extracellular domain is located at about amino acid residues 1-27 of SEQ ID NO: 27.

[1428] In a preferred embodiment, a 32620 polypeptide or protein has an “N-terminal extracellular domain” or a region which includes at least about 1-100, preferably about 1-50, more preferably about 1-40, even more preferably about 1-30 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “N-terminal extracellular domain,” e.g., the N-terminal extracellular domain of human 32620 (e.g., residues 1-27 of SEQ ID NO: 27).

[1429] In another embodiment, a 32620 protein includes at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or preferably, twelve transmembrane domains. As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15 amino acid residues in length that spans the plasma membrane. More preferably, a amino acid residues in length that spans the plasma membrane. More preferably, a transmembrane domain includes about at least 15, 16, 17, 19, 20, 22, 23, 24, 25, 30 or 35 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an α-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N. et al, (1996) Annual Rev. Neuronsci. 19: 235-63, the contents of which are incorporated herein by reference. Amino acid residues 28-48, 105-122, 136-155, 177-201, 209-229, 271-287, 376-400, 417-439, 447-471, 479-502, and 521-542 of SEQ ID NO: 27 comprise transmembrane domains in a 32620 protein.

[1430] In a preferred embodiment, a 32620 polypeptide or protein has at least one transmembrane domain or a region which includes at least 15, 16, 17, 19, 20, 22, 23, 24, 25, 30 or 35 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “transmembrane domain,” e.g., at least one transmembrane domain of human 32620 (e.g., residues 28-48, 105-122, 136-155, 177-201, 209-229, 271-287, 376-400, 417-439, 447-471, 479-502, and 521-542 of SEQ ID NO: 27). Preferably, the transmembrane domain interacts with a molecule traversing the plasma membrane, e.g., a sugar molecule, e.g., D-glucose, D-fructose or D-galactose.

[1431] In another embodiment, a 32620 protein include at least one, two, three, four and preferably five extracellular loops. As defined herein, the term “loop” includes an amino acid sequence having a length of at least about 4-100, preferably about 6-90, more preferably about 6, 15-87, and even more preferably about 6, 12, 17 or 87 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring 32620 or 32620-like molecule, and the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring 32620 or 32620-like molecule. As used herein, an “extracellular loop” includes an amino acid sequence located outside of a cell, or extracellularly. For example, an extracellular loop can be found at about amino acids 123-135, 202-208, 288-375, 440-446, and 503-520 of SEQ ID NO: 27.

[1432] In a preferred embodiment, a 32620 polypeptide or protein has at least one extracellular loop or a region which includes at least about 4-100, preferably about 6-90, more preferably about 6, 15-87, and even more preferably about 6, 12, 17 or 87 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “extracellular loop,” e.g., at least one extracellular loop of human 32620 (e.g., residues 123-135, 202-208, 288-375, 440-446, and 503-520 of SEQ ID NO: 27).

[1433] In another embodiment, a 32620 protein includes at least one, two, three, four, five and preferably six cytoplasmic loops. As used herein, a “cytoplasmic loop” includes an amino acid sequence having a length of at least about 4, preferably about 5-70, more preferably about 6-60, more preferably about 6, 7, 20-55, and most preferably about 6, 7, 20, 40 or 55 amino acid residues located within a cell or within the cytoplasm of a cell. For example, a cytoplasmic loop is found at about amino acids 49-104, 156-176, 230-270, 401-416, 472-478, and 543-650 of SEQ ID NO: 27.

[1434] In a preferred embodiment, a 32620 polypeptide or protein has at least one cytoplasmic loop or a region which includes at least about 4, preferably about 5-70, more preferably about 6-60, more preferably about 6, 7, 20-55, and most preferably about 6, 7, 20, 40 or 55 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “cytoplasmic loop,” e.g., at least one cytoplasmic loop of human 32620 (e.g., residues 49-104, 156-176, 230-270, 401-416, 472-478, and 543-650 of SEQ ID NO: 27).

[1435] In another embodiment, a 32620 protein includes a “C-terminal cytoplasmic domain”, also referred to herein as a C-terminal cytoplasmic tail, in the sequence of the protein. As used herein, a “C-terminal cytoplasmic domain” includes an amino acid sequence having a length of at least about 3, preferably about 4-10, more preferably about 5-6 amino acid residues, and is located outside a cell or extracellularly. Accordingly, the N-terminal amino acid residue of a “C-terminal cytoplasmic domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally-occurring 32620 or 32620-like protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 670-675 of SEQ ID NO: 27.

[1436] In a preferred embodiment, a 32620 polypeptide or protein has a C-terminal extracellular domain or a region which includes at least about 3, preferably about 4-10, more preferably about 5-6 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “C-terminal extracellular domain,” e.g., the C-terminal extracellular domain of human 32620 (e.g., residues 670-675 of SEQ ID NO: 27).

[1437] As the 32620 polypeptides of the invention may modulate 32620-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 32620-mediated or related disorders, as described below.

[1438] As used herein, a “32620 activity”, “biological activity of 32620” or “functional activity of 32620”, refers to an activity exerted by a 32620 protein, polypeptide or nucleic acid molecule on e.g., a 32620-responsive cell or on a 32620 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, a 32620 activity is a direct activity, such as an association with a 32620 target molecule. A “target molecule” or “binding partner” is a molecule with which a 32620 protein binds or interacts in nature, e.g., a sugar (e.g., monosaccharide, such as D-glucose, D-fructose, and/or D-galactose). A 32620 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 32620 protein with a 32620 receptor.

[1439] Based on the above-described structural features, the 32620 molecules of the present invention can have biological activities of sodium-sugar symporter family members. For example, the 32620 proteins of the present invention can have one or more of the following activities: (1) the ability to transport a sugar molecule (e.g., a monosaccharide, such as D-glucose, D-fructose, and/or D-galactose) across a cell membrane (e.g., a nerve, glial, liver, or kidney cell membrane); (2) the ability to transport an ion across a membrane, e.g., a sodium ion; (3) the ability to stimulate molecules that regulate glucose homeostasis (e.g., insulin and glucagon), from cells, e.g., nerve or glial cells; (4) the ability to participate in signal transduction pathways associated with sugar metabolism; (5) the ability to influence insulin and/or glucagon secretion; or (6) the ability to modulate sugar homeostasis in a cell, e.g., a neuronal or glial cell.

[1440] As the 32620 polypeptides of the invention may modulate 32620-mediated activities, they may be useful for developing novel diagnostic and therapeutic agents for 32620-mediated or related disorders. For example, the 32620 molecules can act as novel diagnostic targets and therapeutic agents controlling neurological disorders, as well as pain, pain disorders, and inflammatory disorders.

[1441] 32620 mRNA is abundantly expressed in the tissues of brain cortex and hypothalamus, thus, 32620 polypeptides can be associated with brain or other neurological disorders. Disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin B1) deficiency and vitamin B12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.

[1442] Examples of pain conditions include, but are not limited to, pain elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia; pain associated with musculoskeletal disorders, e.g., joint pain, or arthritis; tooth pain; headaches, e.g., migrane; pain associated with surgery; pain related to inflammation, e.g., irritable bowel syndrome; chest pain; or hyperalgesia, e.g., excessive sensitivity to pain (described in, for example, Fields (1987) Pain, New York:McGraw-Hill). Other examples of pain disorders or pain syndromes include, but are not limited to, complex regional pain syndrome (CRPS), reflex sympathetic dystrophy (RSD), causalgia, neuralgia, central pain and dysesthesia syndrome, carotidynia, neurogenic pain, refractory cervicobrachial pain syndrome, myofascial pain syndrome, craniomandibular pain dysfunction syndrome, chronic idiopathic pain syndrome, Costen's pain-dysfunction, acute chest pain syndrome, nonulcer dyspepsia, interstitial cystitis, gynecologic pain syndrome, patellofemoral pain syndrome, anterior knee pain syndrome, recurrent abdominal pain in children, colic, low back pain syndrome, neuropathic pain, phantom pain from amputation, phantom tooth pain, or pain asymbolia (the inability to feel pain). Other examples of pain conditions include pain induced by parturition, or post partum pain.

[1443] Agents that modulate 32620 polypeptide or nucleic acid activity or expression can be used to treat pain elicited by any medical condition. A subject receiving the treatment can be additionally treated with a second agent, e.g., an anti-inflammatory agent, an antibiotic, or a chemotherapeutic agent, to further ameliorate the condition.

[1444] The 32620 molecules can also act as novel diagnostic targets and therapeutic agents controlling pain caused by other disorders, e.g., cancer. Accordingly, the 32620 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, or pain therefrom.

[1445] The 32620 protein may be also associated with a sugar transporter associated disorder. As used herein, the term “sugar transporter associated disorder” includes a disorder, disease, or condition which is characterized by an aberrant, e.g., upregulated or downregulated, sugar transporter mediated activity. Sugar transporter associated disorders typically result in, e.g., upregulated or downregulated, sugar levels in a cell. Examples of sugar transporter associated disorders include disorders associated with sugar homeostasis, such as obesity, anorexia, type-1 diabetes, type-2 diabetes, hypoglycemia, glycogen storage disease (Von Gierke disease), type I glycogenosis, bipolar disorder, seasonal affective disorder, and cluster B personality disorders.

[1446] The 32620 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 27 thereof are collectively referred to as “polypeptides or proteins of the invention” or “32620 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “32620 nucleic acids.” 32620 molecules refer to 32620 nucleic acids, polypeptides, and antibodies.

[1447] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[1448] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[1449] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[1450] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 26 or SEQ ID NO: 28, corresponds to a naturally-occurring nucleic acid molecule.

[1451] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include at least an open reading frame encoding a 32620 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 32620 protein or derivative thereof.

[1452] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 32620 protein is at least 10% pure. In a preferred embodiment, the preparation of 32620 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-32620 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-32620 chemicals. When the 32620 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[1453] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 32620 without abolishing or substantially altering a 32620 activity. Preferably the alteration does not substantially alter the 32620 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 32620, results in abolishing a 32620 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 32620 are predicted to be particularly unamenable to alteration.

[1454] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 32620 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 32620 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 32620 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 26 or SEQ ID NO: 28, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[1455] As used herein, a “biologically active portion” of a 32620 protein includes a fragment of a 32620 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 32620 molecule and a non-32620 molecule or between a first 32620 molecule and a second 32620 molecule (e.g., a dimerization interaction). Biologically active portions of a 32620 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 32620 protein, e.g., the amino acid sequence shown in SEQ ID NO: 27, which include less amino acids than the full length 32620 proteins, and exhibit at least one activity of a 32620 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 32620 protein, e.g., sugar transport. A biologically active portion of a 32620 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 32620 protein can be used as targets for developing agents that modulate a 32620-mediated activity, e.g., sugar transport.

[1456] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[1457] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[1458] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[1459] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[1460] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[1461] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 32620 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 32620 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[1462] Particularly preferred 32620 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 27. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 27 are termed substantially identical.

[1463] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 26 or 28 are termed substantially identical.

[1464] “Misexpression or aberrant expression”, as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[1465] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[1466] A “purified preparation of cells”, as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[1467] Various aspects of the invention are described in further detail below.

[1468] Isolated 32620 Nucleic Acid Molecules

[1469] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 32620 polypeptide described herein, e.g., a full-length 32620 protein or a fragment thereof, e.g., a biologically active portion of 32620 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 32620 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[1470] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 26, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 32620 protein (i.e., “the coding region” of SEQ ID NO: 26, as shown in SEQ ID NO: 28), as well as 5′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 26 (e.g., SEQ ID NO: 28) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid 58 to 487 of SEQ ID NO: 27.

[1471] In a preferred embodiment, the nucleic acid molecule encodes a glutamic acid at the position corresponding to amino acid 62 of SEQ ID NO: 27. In another preferred embodiment, the nucleic acid encodes an asparagine at the position corresponding to amino acid 64 of SEQ ID NO: 27. In a much preferred embodiment, the nucleic acid encodes glutamic acid at the position corresponding to amino acid 62 and an asparagine at the position corresponding to amino acid 64 of SEQ ID NO: 27.

[1472] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 28, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 28, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NOS: 26 or 28, thereby forming a stable duplex.

[1473] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 28, or a portion, preferably of the same length, of any of these nucleotide sequences.

[1474] 32620 Nucleic Acid Fragments

[1475] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 26 or 28. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 32620 protein, e.g., an immunogenic or biologically active portion of a 32620 protein. A fragment can comprise those nucleotides of SEQ ID NO: 26, which encode a sodium-sugar symporter domain of human 32620. The nucleotide sequence determined from the cloning of the 32620 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 32620 family members, or fragments thereof, as well as 32620 homologues, or fragments thereof, from other species.

[1476] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[1477] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 32620 nucleic acid fragment can include a sequence corresponding to a sodium-sugar symporter domain.

[1478] 32620 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 26 or SEQ ID NO: 28, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 26 or SEQ ID NO: 28, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[1479] In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1480] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes a sodium-sugar symporter domain, e.g., amino acids about 58 to 487 of SEQ ID NO: 27. In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 32620 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: a nucleic acid encoding a sodium-sugar symporter domain from about amino acid 58 to 487 of SEQ ID NO: 27 (or a portion thereof, e.g., 58-70, 70-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-487 of SEQ ID NO: 27), an extracellular loop at about amino acids 123 to 135, 202 to 208, 288 to 375, 440 to 446, and 503 to 520 of SEQ ID NO: 27, or an intracellular loop at about amino acids 49 to 104, 156 to 176, 230 to 270, 401 to 416, 472 to 478, and 543 to 650 of SEQ ID NO: 27.

[1481] In one embodiment, the probe or primer is attached to a solid support, e.g., a solid support described herein.

[1482] One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g., the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 27. The reverse primer can anneal to the ultimate codon, e.g., the codon immediately before the stop codon, e.g., the codon encoding amino acid residue 675 of SEQ ID NO: 27. In a preferred embodiment, the annealing temperatures of the forward and reverse primers differ by no more than 5, 4, 3, or 2° C.

[1483] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[1484] A nucleic acid fragment encoding a “biologically active portion of a 32620 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NOS: 26 or 28, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, which encodes a polypeptide having a 32620 biological activity (e.g., the biological activities of the 32620 proteins are described herein), expressing the encoded portion of the 32620 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 32620 protein. For example, a nucleic acid fragment encoding a biologically active portion of 32620 includes a sodium-sugar symporter domain, e.g., amino acid residues about 58 to 487 of SEQ ID NO: 27 (or a fragment thereof). A nucleic acid fragment encoding a biologically active portion of a 32620 polypeptide may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

[1485] In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 750, 760, 775, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2100 or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 26, or SEQ ID NO: 28, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[1486] 32620 Nucleic Acid Variants

[1487] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 26 or SEQ ID NO: 28, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 32620 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 27. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1488] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[1489] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[1490] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NOS: 26 or 28, or the sequence in ATCC Accession Number ______, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the in the subject nucleic acid. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1491] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 27 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO: 27 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 32620 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 32620 gene.

[1492] Preferred variants include those that are correlated with sugar transport.

[1493] Allelic variants of 32620, e.g., human 32620, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 32620 protein within a population that maintain the ability to bind sugars. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 27, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 32620, e.g., human 32620, protein within a population that do not have the ability to transport sugars. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 27, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[1494] Moreover, nucleic acid molecules encoding other 32620 family members and, thus, which have a nucleotide sequence which differs from the 32620 sequences of SEQ ID NO: 26 or SEQ ID NO: 28, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ are intended to be within the scope of the invention.

[1495] Antisense Nucleic Acid Molecules, Ribozymes and Modified 32620 Nucleic Acid Molecules

[1496] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 32620. An “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 32620 coding strand, or to only a portion thereof (e.g., the coding region of human 32620 corresponding to SEQ ID NO: 28). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 32620 (e.g., the 5′ and 3′ untranslated regions).

[1497] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 32620 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 32620 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 32620 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[1498] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[1499] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 32620 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[1500] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[1501] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 32620-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 32620 cDNA disclosed herein (i.e., SEQ ID NO: 26 or SEQ ID NO: 28), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 32620-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 32620 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[1502] 32620 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 32620 (e.g., the 32620 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 32620 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N. Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[1503] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

[1504] A 32620 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[1505] PNAs of 32620 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 32620 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[1506] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[1507] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 32620 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 32620 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

[1508] Isolated 32620 Polypeptides

[1509] In another aspect, the invention features, an isolated 32620 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-32620 antibodies. 32620 protein can be isolated from cells or tissue sources using standard protein purification techniques. 32620 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically. A 32620 protein or fragment thereof can be attached to a solid support, e.g., a bead, matrix, or planar surface, e.g., a protein array.

[1510] Polypeptides of the invention include those that arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[1511] In a preferred embodiment, a 32620 polypeptide has one or more of the following characteristics:

[1512] (i) it has the ability to transport sugars, e.g., glucose or galactose, across the plasma membrane;

[1513] (ii) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of a 32620 polypeptide, e.g., a polypeptide of SEQ ID NO: 27;

[1514] (iii) it has an overall sequence similarity of at least 60%, more preferably at least 70, 80, 90, or 95%, with a polypeptide of SEQ ID NO: 27;

[1515] (iv) it can be found in tissue of the brain cortex, hypothalamus, and spinal cord;

[1516] (v) it has a sodium-sugar symporter domain which is preferably about 70%, 80%, 90% or 95% with amino acid residues about 58 to 487 of SEQ ID NO: 27;

[1517] (vi) it has a 9 of 11 amino acid match to the Prosite sodium:solute symporter signature 1 (PS00456) at about amino acids 174 to 199 of SEQ ID NO: 27;

[1518] (vi) it has a 15 of 16 amino acid match to the Prosite sodium:solute symporter signature 2 (PS00457) at about amino acids 469 to 489 of SEQ ID NO: 27;

[1519] (vii) it has a conserved glycine at about amino acid 43 of SEQ ID NO: 27, and a conserved arginine at about amino acid 295 of SEQ ID NO: 27; or

[1520] (viii) it has at least eleven, preferably greater than eleven, e.g., twelve, thirteen, or fourteen transmembrane domains.

[1521] In a preferred embodiment the 32620 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID:2. In one embodiment it differs by at least one but by less than 15,- 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 27 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 27. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the sodium-sugar symporter domain. In another preferred embodiment one or more differences are in the sodium-sugar symporter domain.

[1522] In a preferred embodiment, the protein includes a glutamic acid at the position corresponding to amino acid 62 of SEQ ID NO: 27. In another preferred embodiment, the protein includes an asparagine at the position corresponding to amino acid 64 of SEQ ID NO: 27. In a much preferred embodiment, the protein includes glutamic acid at the position corresponding to amino acid 62 and an asparagine at the position corresponding to amino acid 64 of SEQ ID NO: 27.

[1523] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 32620 proteins differ in amino acid sequence from SEQ ID NO: 27, yet retain biological activity.

[1524] In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 27. In other embodiments, the protein includes fragment of a 32620 polypeptide or a region homologous thereto (e.g., about 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous a fragment of SEQ ID NO: 27). Examples of fragments of 32620 polypeptide include a sodium-sugar symporter domain, e.g., from about amino acids 58 to 487 of SEQ ID NO: 27 or a portion thereof, e.g., 58-70, 70-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, or 400-487 of SEQ ID NO: 27; a transmembrane domain, e.g., at about amino acids 28 to 48, 105 to 122, 136 to 155, 177 to 201, 209 to 229, 271 to 287, 376 to 400, 417 to 439, 447 to 471, 479 to 502, 521 to 542, or 651 to 669 of SEQ ID NO: 27; an extracellular domains, e.g., at about amino acids 1 to 27, and 670 to 675 of SEQ ID NO: 27; an extracellular loop, e.g., at about amino acids 123 to 135, 202 to 208, 288 to 375, 440 to 446, or 503 to 520 of SEQ ID NO: 27; or an intracellular loop, e.g., at about amino acids 49 to 104, 156 to 176, 230 to 270, 401 to 416, 472 to 478, or 543 to 650 of SEQ ID NO: 27.

[1525] A 32620 protein or fragment is provided which varies from the sequence of SEQ ID NO: 27 in regions defined by amino acids about 58 to 487 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 27 in regions defined by amino acids about 58 to 487 of SEQ ID NO: 27. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[1526] In one embodiment, a biologically active portion of a 32620 protein includes a sodium-sugar symporter domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 32620 protein.

[1527] In a preferred embodiment, the 32620 protein has an amino acid sequence shown in SEQ ID NO: 27. In other embodiments, the 32620 protein is substantially identical to SEQ ID NO: 27. In yet another embodiment, the 32620 protein is substantially identical to SEQ ID NO: 27 and retains the functional activity of the protein of SEQ ID NO: 27, as described in detail in the subsections above.

[1528] 32620 Chimeric or Fusion Proteins

[1529] In another aspect, the invention provides 32620 chimeric or fusion proteins. As used herein, a 32620 “chimeric protein” or “fusion protein” includes a 32620 polypeptide linked to a non-32620 polypeptide. A “non-32620 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 32620 protein, e.g., a protein which is different from the 32620 protein and which is derived from the same or a different organism. The 32620 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a 32620 amino acid sequence. In a preferred embodiment, a 32620 fusion protein includes at least one (or two) biologically active portion of a 32620 protein. The non-32620 polypeptide can be fused to the N-terminus or C-terminus of the 32620 polypeptide.

[1530] The fusion protein can include a moiety that has a high affinity for a ligand. For example, the fusion protein can be a GST-32620 fusion protein in which the 32620 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 32620. Alternatively, the fusion protein can be a 32620 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 32620 can be increased through use of a heterologous signal sequence.

[1531] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[1532] The 32620 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 32620 fusion proteins can be used to affect the bioavailability of a 32620 substrate. 32620 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 32620 protein; (ii) mis-regulation of the 32620 gene; and (iii) aberrant post-translational modification of a 32620 protein.

[1533] Moreover, the 32620-fusion proteins of the invention can be used as immunogens to produce anti-32620 antibodies in a subject, to purify 32620 ligands and in screening assays to identify molecules which inhibit the interaction of 32620 with a 32620 substrate.

[1534] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 32620-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 32620 protein.

[1535] Variants of 32620 Proteins

[1536] In another aspect, the invention also features a variant of a 32620 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 32620 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 32620 protein. An agonist of the 32620 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a 32620 protein. An antagonist of a 32620 protein can inhibit one or more of the activities of the naturally occurring form of the 32620 protein by, for example, competitively modulating a 32620-mediated activity of a 32620 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 32620 protein.

[1537] Variants of a 32620 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 32620 protein for agonist or antagonist activity.

[1538] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 32620 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 32620 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[1539] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 32620 proteins. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 32620 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[1540] Cell based assays can be exploited to analyze a variegated 32620 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 32620 in a substrate-dependent manner. The transfected cells are then contacted with 32620 and the effect of the expression of the mutant on signaling by the 32620 substrate can be detected, e.g., by measuring sugar transport. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 32620 substrate, and the individual clones further characterized.

[1541] In another aspect, the invention features a method of making a 32620 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 32620 polypeptide, e.g., a naturally occurring 32620 polypeptide. The method includes: altering the sequence of a 32620 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[1542] In another aspect, the invention features a method of making a fragment or analog of a 32620 polypeptide a biological activity of a naturally occurring 32620 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 32620 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[1543] Anti-32620 Antibodies

[1544] In another aspect, the invention provides an anti-32620 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[1545] The anti-32620 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[1546] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[1547] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 32620 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-32620 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[1548] The anti-32620 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[1549] Phage display and combinatorial methods for generating anti-32620 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[1550] In one embodiment, the anti-32620 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods of producing rodent antibodies are known in the art.

[1551] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[1552] An anti-32620 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[1553] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fe constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fe constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[1554] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a 32620 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[1555] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[1556] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 32620 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[1557] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[1558] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[1559] A full-length 32620 protein or, antigenic peptide fragment of 32620 can be used as an immunogen or can be used to identify anti-32620 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 32620 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 27 and encompasses an epitope of 32620. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[1560] Fragments of 32620 which include residues about 46 to 54, about 473 to 478, or about 505 to 512 of SEQ ID NO: 27 can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophilic regions of the 32620 protein. Similarly, fragments of 32620 which include residues about 29 to 45, about 177 to 190, or about 417 to 439 of SEQ ID NO: 27 can be used to make an antibody against a hydrophobic region of the 32620 protein; fragments of 32620 which include residues 1 to 27, 123 to 135, 202 to 208, 288 to 375, 440 to 446, 503 to520, and 670 to 675 of SEQ ID NO: 27 can be used to make an antibody against an extracellular region of the 32620 protein; fragments of 32620 which include residues about 49 to 104, 156 to 176, 230 to 270, 401 to 416, 472 to 478, and 543 to 650 of SEQ ID NO: 27 can be used to make an antibody against an intracellular region of the 32620 protein; a fragment of 32620 which include residues about 58 to 487 of SEQ ID NO: 27 (or a portion thereof, e.g., 58-70, 70-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-487 of SEQ ID NO: 27) can be used to make an antibody against the sodium-sugar symporter region of the 32620 protein.

[1561] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[1562] Antibodies which bind only native 32620 protein, only denatured or otherwise non-native 32620 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies that bind to native but not denatured 32620 protein.

[1563] Preferred epitopes encompassed by the antigenic peptide are regions of 32620 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 32620 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 32620 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[1564] In a preferred embodiment the antibody can bind to the extracellular portion of the 32620 protein, e.g., it can bind to a whole cell which expresses the 32620 protein. In another embodiment, the antibody binds an intracellular portion of the 32620 protein.

[1565] In a preferred embodiment the antibody binds an epitope on any domain or region on 32620 proteins described herein.

[1566] Chimeric, humanized, but most preferably, completely human antibodies are desirable for applications which include repeated administration, e.g., therapeutic treatment (and some diagnostic applications) of human patients.

[1567] The anti-32620 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 32620 protein.

[1568] In a preferred embodiment the antibody has effector function and/or can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement.

[1569] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[1570] In a preferred embodiment, an anti-32620 antibody alters (e.g., increases or decreases) the transport activity of a 32620 polypeptide.

[1571] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e.g., ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels that produce detectable radioactive emissions or fluorescence are preferred.

[1572] An anti-32620 antibody (e.g., monoclonal antibody) can be used to isolate 32620 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-32620 antibody can be used to detect 32620 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-32620 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[1573] The invention also includes a nucleic acid which encodes an anti-32620 antibody, e.g., an anti-32620 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[1574] The invention also includes cell lines, e.g., hybridomas, which make an anti-32620 antibody, e.g., and antibody described herein, and method of using said cells to make a 32620 antibody.

[1575] 32620 Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[1576] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[1577] A vector can include a 32620 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 32620 proteins, mutant forms of 32620 proteins, fusion proteins, and the like).

[1578] The recombinant expression vectors of the invention can be designed for expression of 32620 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[1579] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[1580] Purified fusion proteins can be used in 32620 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 32620 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[1581] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[1582] The 32620 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[1583] When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[1584] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[1585] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

[1586] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 32620 nucleic acid molecule within a recombinant expression vector or a 32620 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[1587] A host cell can be any prokaryotic or eukaryotic cell. For example, a 32620 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) CellI23:175-182)). Other suitable host cells are known to those skilled in the art.

[1588] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[1589] A host cell of the invention can be used to produce (i.e., express) a 32620 protein. Accordingly, the invention further provides methods for producing a 32620 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a 32620 protein has been introduced) in a suitable medium such that a 32620 protein is produced. In another embodiment, the method further includes isolating a 32620 protein from the medium or the host cell.

[1590] In another aspect, the invention features, a cell or purified preparation of cells which include a 32620 transgene, or which otherwise misexpress 32620. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a 32620 transgene, e.g., a heterologous form of a 32620, e.g., a gene derived from humans (in the case of a non-human cell). The 32620 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that misexpresses an endogenous 32620, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed 32620 alleles or for use in drug screening.

[1591] In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid that encodes a subject 32620 polypeptide.

[1592] Also provided are cells, preferably human cells, e.g., human hematopoietic or fibroblast cells, in which an endogenous 32620 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 32620 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 32620 gene. For example, an endogenous 32620 gene that is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[1593] 32620 Transgenic Animals

[1594] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 32620 protein and for identifying and/or evaluating modulators of 32620 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 32620 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[1595] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 32620 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 32620 transgene in its genome and/or expression of 32620 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 32620 protein can further be bred to other transgenic animals carrying other transgenes.

[1596] 32620 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[1597] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[1598] Uses of 32620

[1599] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[1600] The isolated nucleic acid molecules of the invention can be used, for example, to express a 32620 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a 32620 mRNA (e.g., in a biological sample) or a genetic alteration in a 32620 gene, and to modulate 32620 activity, as described further below. The 32620 proteins can be used to treat disorders characterized by insufficient or excessive production of a 32620 substrate or production of 32620 inhibitors. In addition, the 32620 proteins can be used to screen for naturally occurring 32620 substrates, to screen for drugs or compounds which modulate 32620 activity, as well as to treat disorders characterized by insufficient or excessive production of 32620 protein or production of 32620 protein forms which have decreased, aberrant or unwanted activity compared to 32620 wild type protein (e.g., a kidney or an intestinal disorders). Moreover, the anti-32620 antibodies of the invention can be used to detect and isolate 32620 proteins, regulate the bioavailability of 32620 proteins, and modulate 32620 activity.

[1601] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 32620 polypeptide is provided. The method includes: contacting the compound with the subject 32620 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 32620 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 32620 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 32620 polypeptide. Screening methods are discussed in more detail below.

[1602] 32620 Screening Assays

[1603] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 32620 proteins, have a stimulatory or inhibitory effect on, for example, 32620 expression or 32620 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 32620 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 32620 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[1604] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 32620 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 32620 protein or polypeptide or a biologically active portion thereof.

[1605] In one embodiment, an activity of a 32620 protein can be assayed as follows. Xenopus laevis oocytes are injected with mRNA encoding the 32620 protein or a eukaryotic expression vector able to express such an mRNA, using a Drummond Nanoject (Drummond Scientific, Broomall, Pa. into the animal pole of defolliculated oocytes as described by Swick et al. ((1992) Proc. Natl. Acad. Sci. USA. 89:1812-1816). The injected oocytes are kept in MBS with 2.5 mM sodium pyruvate for 2-3 days, then transferred to microtitre wells about 12 to 24 hours prior to being assayed. Transport function of oocyte-expressed 32620 polypeptide is assessed by radiotracer uptakes from 50 μMD-[α-methyl-14C]glucopyranoside as described (Lostao et al. (1994) J. Membr. Biol. 142:161-170).

[1606] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[1607] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[1608] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (I991) J. Mol. Biol. 222:301-310; Ladner supra.).

[1609] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 32620 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 32620 activity is determined. Determining the ability of the test compound to modulate 32620 activity can be accomplished by monitoring, for example, sugar transport. The cell, for example, can be of mammalian origin, e.g., human.

[1610] The ability of the test compound to modulate 32620 binding to a compound, e.g., a 32620 substrate, or to bind to 32620 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 32620 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 32620 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 32620 binding to a 32620 substrate in a complex. For example, compounds (e.g., 32620 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[1611] The ability of a compound (e.g., a 32620 substrate) to interact with 32620 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 32620 without the labeling of either the compound or the 32620. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 32620.

[1612] In yet another embodiment, a cell-free assay is provided in which a 32620 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 32620 protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the 32620 proteins to be used in assays of the present invention include fragments which participate in interactions with non-32620 molecules, e.g., fragments with high surface probability scores.

[1613] Soluble and/or membrane-bound forms of isolated proteins (e.g., 32620 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[1614] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[1615] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[1616] In another embodiment, determining the ability of the 32620 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[1617] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[1618] It may be desirable to immobilize either 32620, an anti-32620 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 32620 protein, or interaction of a 32620 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/32620 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 32620 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 32620 binding or activity determined using standard techniques.

[1619] Other techniques for immobilizing either a 32620 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 32620 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[1620] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[1621] In one embodiment, this assay is performed utilizing antibodies reactive with 32620 protein or target molecules but which do not interfere with binding of the 32620 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 32620 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 32620 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 32620 protein or target molecule.

[1622] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11: 141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[1623] In a preferred embodiment, the assay includes contacting the 32620 protein or biologically active portion thereof with a known compound which binds 32620 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 32620 protein, wherein determining the ability of the test compound to interact with a 32620 protein includes determining the ability of the test compound to preferentially bind to 32620 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[1624] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 32620 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a 32620 protein through modulation of the activity of a downstream effector of a 32620 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[1625] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[1626] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[1627] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[1628] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[1629] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[1630] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[1631] In yet another aspect, the 32620 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 32620 (“32620-binding proteins” or “32620-bp”) and are involved in 32620 activity. Such 32620-bps can be activators or inhibitors of signals by the 32620 proteins or 32620 targets as, for example, downstream elements of a 32620-mediated signaling pathway.

[1632] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 32620 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 32620 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 32620-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 32620 protein.

[1633] In another embodiment, modulators of 32620 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 32620 mRNA or protein evaluated relative to the level of expression of 32620 mRNA or protein in the absence of the candidate compound. When expression of 32620 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 32620 mRNA or protein expression. Alternatively, when expression of 32620 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 32620 mRNA or protein expression. The level of 32620 mRNA or protein expression can be determined by methods described herein for detecting 32620 mRNA or protein.

[1634] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a 32620 protein can be confirmed in vivo, e.g., in an animal such as an animal model for pain disorder, e.g., e.g., an arthritic rat model of chronic pain, a chronic constriction injury (CCI) rat model of neuropathic pain, or a rat model of unilateral inflammatory pain by intraplantar injection of Freund's complete adjuvant (FCA).

[1635] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 32620 modulating agent, an antisense 32620 nucleic acid molecule, a 32620-specific antibody, or a 32620-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[1636] 32620 Detection Assays

[1637] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 32620 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[1638] 32620 Chromosome Mapping

[1639] The 32620 nucleotide sequences or portions thereof can be used to map the location of the 32620 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 32620 sequences with genes associated with disease.

[1640] Briefly, 32620 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 32620 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 32620 sequences will yield an amplified fragment.

[1641] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[1642] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 32620 to a chromosomal location.

[1643] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[1644] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[1645] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature 325:783-787.

[1646] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 32620 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[1647] 32620 Tissue Typing

[1648] 32620 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[1649] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 32620 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[1650] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 26 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 28 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[1651] If a panel of reagents from 32620 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[1652] Use of Partial 32620 Sequences in Forensic Biology

[1653] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[1654] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 26 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 26 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[1655] The 32620 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 32620 probes can be used to identify tissue by species and/or by organ type.

[1656] In a similar fashion, these reagents, e.g., 32620 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[1657] Predictive Medicine of 32620

[1658] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[1659] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 32620.

[1660] Such disorders include, e.g., a disorder associated with the misexpression of 32620 gene; a disorder of the renal or gastrointestinal system.

[1661] The method includes one or more of the following:

[1662] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 32620 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[1663] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 32620 gene;

[1664] detecting, in a tissue of the subject, the misexpression of the 32620 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[1665] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 32620 polypeptide.

[1666] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 32620 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[1667] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 26, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 32620 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[1668] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 32620 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 32620.

[1669] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[1670] In preferred embodiments the method includes determining the structure of a 32620 gene, an abnormal structure being indicative of risk for the disorder.

[1671] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 32620 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[1672] Diagnostic and Prognostic Assays of 32620

[1673] Diagnostic and prognostic assays of the invention include method for assessing the expression level of 32620 molecules and for identifying variations and mutations in the sequence of 32620 molecules.

[1674] Expression Monitoring and Profiling:

[1675] The presence, level, or absence of 32620 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 32620 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 32620 protein such that the presence of 32620 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 32620 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 32620 genes; measuring the amount of protein encoded by the 32620 genes; or measuring the activity of the protein encoded by the 32620 genes.

[1676] The level of mRNA corresponding to the 32620 gene in a cell can be determined both by in situ and by in vitro formats.

[1677] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a fill-length 32620 nucleic acid, such as the nucleic acid of SEQ ID NO: 26, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 32620 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[1678] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 32620 genes.

[1679] The level of mRNA in a sample that is encoded by one of 32620 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al, (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[1680] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 32620 gene being analyzed.

[1681] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 32620 mRNA, or genomic DNA, and comparing the presence of 32620 mRNA or genomic DNA in the control sample with the presence of 32620 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 32620 transcript levels.

[1682] A variety of methods can be used to determine the level of protein encoded by 32620. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[1683] The detection methods can be used to detect 32620 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 32620 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 32620 protein include introducing into a subject a labeled anti-32620 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-32620 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[1684] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 32620 protein, and comparing the presence of 32620 protein in the control sample with the presence of 32620 protein in the test sample.

[1685] The invention also includes kits for detecting the presence of 32620 in a biological sample. For example, the kit can include a compound or agent capable of detecting 32620 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 32620 protein or nucleic acid.

[1686] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[1687] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[1688] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 32620 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as pain or deregulated cell proliferation.

[1689] In one embodiment, a disease or disorder associated with aberrant or unwanted 32620 expression or activity is identified. A test sample is obtained from a subject and 32620 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 32620 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 32620 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[1690] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 32620 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a pain or solute transport disorder.

[1691] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 32620 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 32620 (e.g., other genes associated with a 32620-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[1692] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 32620 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose a pain disorder in a subject wherein an alteration in 32620 expression is an indication that the subject has or is disposed to having a pain. The method can be used to monitor a treatment for pain in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[1693] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 32620 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[1694] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 32620 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[1695] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[1696] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 32620 expression.

[1697] 32620 Arrays and Uses thereof

[1698] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a 32620 molecule (e.g., a 32620 nucleic acid or a 32620 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm2, and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[1699] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a 32620 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 32620. Each address of the subset can include a capture probe that hybridizes to a different region of a 32620 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for a 32620 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 32620 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 32620 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[1700] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[1701] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a 32620 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 32620 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-32620 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[1702] In another aspect, the invention features a method of analyzing the expression of 32620. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 32620-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[1703] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 32620. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 32620. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[1704] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 32620 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[1705] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[1706] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 32620-associated disease or disorder; and processes, such as a cellular transformation associated with a 32620-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 32620-associated disease or disorder

[1707] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 32620) that could serve as a molecular target for diagnosis or therapeutic intervention.

[1708] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon a 32620 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99% identical to a 32620 polypeptide or fragment thereof. For example, multiple variants of a 32620 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[1709] The polypeptide array can be used to detect a 32620 binding compound, e.g., an antibody in a sample from a subject with specificity for a 32620 polypeptide or the presence of a 32620-binding protein or ligand.

[1710] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 32620 expression on the expression of other genes). This provides, for example, for a selection-of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[1711] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 32620 or from a cell or subject in which a 32620 mediated response has been elicited, e.g., by contact of the cell with 32620 nucleic acid or protein, or administration to the cell or subject 32620 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 32620 (or does not express as highly as in the case of the 32620 positive plurality of capture probes) or from a cell or subject which in which a 32620 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 32620 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[1712] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 32620 or from a cell or subject in which a 32620-mediated response has been elicited, e.g., by contact of the cell with 32620 nucleic acid or protein, or administration to the cell or subject 32620 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 32620 (or does not express as highly as in the case of the 32620 positive plurality of capture probes) or from a cell or subject which in which a 32620 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[1713] In another aspect, the invention features a method of analyzing 32620, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 32620 nucleic acid or amino acid sequence; comparing the 32620 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 32620.

[1714] Detection of 32620 Variations or Mutations

[1715] The methods of the invention can also be used to detect genetic alterations in a 32620 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 32620 protein activity or nucleic acid expression, such as a neurological or a pain disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 32620-protein, or the mis-expression of the 32620 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 32620 gene; 2) an addition of one or more nucleotides to a 32620 gene; 3) a substitution of one or more nucleotides of a 32620 gene, 4) a chromosomal rearrangement of a 32620 gene; 5) an alteration in the level of a messenger RNA transcript of a 32620 gene, 6) aberrant modification of a 32620 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a 32620 gene, 8) a non-wild type level of a 32620-protein, 9) allelic loss of a 32620 gene, and 10) inappropriate post-translational modification of a 32620-protein.

[1716] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 32620-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 32620 gene under conditions such that hybridization and amplification of the 32620-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[1717] In another embodiment, mutations in a 32620 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[1718] In other embodiments, genetic mutations in 32620 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 32620 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 32620 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 32620 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[1719] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 32620 gene and detect mutations by comparing the sequence of the sample 32620 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[1720] Other methods for detecting mutations in the 32620 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[1721] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 32620 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[1722] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 32620 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 32620 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[1723] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1.987) Biophys Chem 265:12753).

[1724] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[1725] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[1726] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 32620 nucleic acid.

[1727] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 26 or the complement of SEQ ID NO: 26. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[1728] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 32620. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[1729] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the Tm of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[1730] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 32620 nucleic acid.

[1731] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 32620 gene.

[1732] Use of 32620 Molecules as Surrogate Markers

[1733] The 32620 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 32620 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 32620 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[1734] The 32620 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 32620 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-32620 antibodies may be employed in an immune-based detection system for a 32620 protein marker, or 32620-specific radiolabeled probes may be used to detect a 32620 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[1735] The 32620 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 32620 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 32620 DNA may correlate 32620 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[1736] Pharmaceutical Compositions of 32620

[1737] The nucleic acid and polypeptides, fragments thereof, as well as anti-32620 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[1738] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[1739] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[1740] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation-are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[1741] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[1742] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[1743] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[1744] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[1745] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[1746] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[1747] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[1748] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[1749] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[1750] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[1751] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[1752] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[1753] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1 065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactive ions include, but are not limited to iodine, yttrium and praseodymium.

[1754] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[1755] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[1756] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[1757] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[1758] Methods of Treatment for 32620

[1759] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 32620 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[1760] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 32620 molecules of the present invention or 32620 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[1761] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 32620 expression or activity, by administering to the subject a 32620 or an agent which modulates 32620 expression or at least one 32620 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 32620 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 32620 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 32620 aberrance, for example, a 32620, 32620 agonist or 32620 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[1762] It is possible that some 32620 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[1763] The 32620 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, cardiovascular disorders, liver disorders, viral diseases, pain or metabolic disorders.

[1764] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[1765] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[1766] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[1767] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

[1768] Aberrant expression and/or activity of 32620 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 32620 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 32620 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 32620 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[1769] The 32620 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders. Examples of immune disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

[1770] Examples of disorders involving the heart or “cardiovascular disorder” include, but are not limited to, a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, congestive heart failure, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

[1771] Disorders which may be treated or diagnosed by methods described herein include, but are not limited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, A1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

[1772] Additionally, 32620 molecules may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 32620 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 32620 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

[1773] Additionally, 32620 may play an important role in the regulation of metabolism or pain disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[1774] As discussed, successful treatment of 32620 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 32620 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[1775] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[1776] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[1777] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 32620 expression is through the use of aptamer molecules specific for 32620 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 32620 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[1778] Aberrant expression of 32620 protein may also be associated with renal or intestinal disorders. For example, 32620 proteins may modulate absorption sugars, such as glucose and galactose, from the intestinal lumen and from the glomerular filtrate. Thus, the 32620 molecules can act as novel diagnostic targets and therapeutic agents for controlling kidney or intestinal disorders.

[1779] Examples of kidney disorders can include chronic renal disease, acute renal failure, nephrotoxic renal failure, diabetes insipidus, autosomal dominant (adult) polycystic kidney disease, glomerular diseases, glomerulonephritis, and tumors of the kidney.

[1780] Examples of intestinal disorders can include ulcers, glucose-galactose malabsorption disease, other intestinal malabsorption disorders, enterocolitis, idiopathic inflammatory bowel disease, and tumors of the colon and stomach.

[1781] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 32620 disorders. For a description of antibodies, see the Antibody section above.

[1782] In circumstances wherein injection of an animal or a human subject with a 32620 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 32620 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 32620 protein. Vaccines directed to a disease characterized by 32620 expression may also be generated in this fashion.

[1783] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[1784] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 32620 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[1785] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[1786] Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 32620 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 32620 can be readily monitored and used in calculations of IC50.

[1787] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[1788] Another aspect of the invention pertains to methods of modulating 32620 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 32620 or agent that modulates one or more of the activities of 32620 protein activity associated with the cell. An agent that modulates 32620 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a 32620 protein (e.g., a 32620 substrate or receptor), a 32620 antibody, a 32620 agonist or antagonist, a peptidomimetic of a 32620 agonist or antagonist, or other small molecule.

[1789] In one embodiment, the agent stimulates one or 32620 activities. Examples of such stimulatory agents include active 32620 protein and a nucleic acid molecule encoding 32620. In another embodiment, the agent inhibits one or more 32620 activities. Examples of such inhibitory agents include antisense 32620 nucleic acid molecules, anti-32620 antibodies, and 32620 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 32620 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 32620 expression or activity. In another embodiment, the method involves administering a 32620 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 32620 expression or activity.

[1790] Stimulation of 32620 activity is desirable in situations in which 32620 is abnormally downregulated and/or in which increased 32620 activity is likely to have a beneficial effect. For example, stimulation of 32620 activity is desirable in situations in which a 32620 is downregulated and/or in which increased 32620 activity is likely to have a beneficial effect. Likewise, inhibition of 32620 activity is desirable in situations in which 32620 is abnormally upregulated and/or in which decreased 32620 activity is likely to have a beneficial effect.

[1791] 32620 Pharmacogenomics

[1792] The 32620 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 32620 activity (e.g., 32620 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 32620 associated disorders (e.g., kidney and gastrointestinal disorders) associated with aberrant or unwanted 32620 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 32620 molecule or 32620 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 32620 molecule or 32620 modulator.

[1793] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[1794] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[1795] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 32620 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[1796] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 32620 molecule or 32620 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[1797] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 32620 molecule or 32620 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[1798] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 32620 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 32620 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[1799] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a 32620 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 32620 gene expression, protein levels, or upregulate 32620 activity, can be monitored in clinical trials of subjects exhibiting decreased 32620 gene expression, protein levels, or downregulated 32620 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 32620 gene expression, protein levels, or downregulate 32620 activity, can be monitored in clinical trials of subjects exhibiting increased 32620 gene expression, protein levels, or upregulated 32620 activity. In such clinical trials, the expression or activity of a 32620 gene, and preferably, other genes that have been implicated in, for example, a 32620-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[1800] 32620 Informatics

[1801] The sequence of a 32620 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 32620. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 32620 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[1802] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[1803] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[1804] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[1805] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[1806] Thus, in one aspect, the invention features a method of analyzing 32620, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 32620 nucleic acid or amino acid sequence; comparing the 32620 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 32620. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[1807] The method can include evaluating the sequence identity between a 32620 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[1808] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[1809] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[1810] Thus, the invention features a method of making a computer readable record of a sequence of a 32620 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[1811] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 32620 sequence, or record, in machine-readable form; comparing a second sequence to the 32620 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 32620 sequence includes a sequence being compared. In a preferred embodiment the 32620 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 32620 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[1812] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 32620-associated disease or disorder or a pre-disposition to a 32620-associated disease or disorder, wherein the method comprises the steps of determining 32620 sequence information associated with the subject and based on the 32620 sequence information, determining whether the subject has a 32620-associated disease or disorder or a pre-disposition to a 32620-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[1813] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 32620-associated disease or disorder or a pre-disposition to a disease associated with a 32620 wherein the method comprises the steps of determining 32620 sequence information associated with the subject, and based on the 32620 sequence information, determining whether the subject has a 32620-associated disease or disorder or a pre-disposition to a 32620-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 32620 sequence of the subject to the 32620 sequences in the database to thereby determine whether the subject as a 32620-associated disease or disorder, or a pre-disposition for such.

[1814] The present invention also provides in a network, a method for determining whether a subject has a 32620 associated disease or disorder or a pre-disposition to a 32620-associated disease or disorder associated with 32620, said method comprising the steps of receiving 32620 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 32620 and/or corresponding to a 32620-associated disease or disorder (e.g., pain), and based on one or more of the phenotypic information, the 32620 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 32620-associated disease or disorder or a pre-disposition to a 32620-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1815] The present invention also provides a method for determining whether a subject has a 32620-associated disease or disorder or a pre-disposition to a 32620-associated disease or disorder, said method comprising the steps of receiving information related to 32620 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 32620 and/or related to a 32620-associated disease or disorder, and based on one or more of the phenotypic information, the 32620 information, and the acquired information, determining whether the subject has a 32620-associated disease or disorder or a pre-disposition to a 32620-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1816] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

[1817] Background of the 44589 Invention

[1818] The ATP-binding cassette (ABC) family comprises a group of structurally related proteins that typically contain one or two transmembrane regions (each region containing several membrane spanning domains) and one or two nucleotide binding domains characterized by Walker motifs A and B and an ATP-binding cassette signature. The majority of the members of the ABC family share a similar structure consisting of 12 transmembrane domains and two nucleotide binding domains, either joined in the same molecule or expressed as half molecules in the form of heterodimers. Members of the ABC family possess diverse biological functions such as transporters, channels, and receptors (Kast et. al (1996) J. Biol. Chem. 271:9240-9248).

[1819] Some members of the ABC family confer cellular resistance to toxic substances. This resistance is mediated by the ABC transporter's binding to a toxic substance and using the energy of ATP hydrolysis to reduce intracellular accumulation of the substance through an active efflux mechanism (Kast et. al (1996) J. Biol. Chem. 271:9240-9248). Examples of these ABC transporters include: members of the multidrug resistance-associated protein (MRP) family (MRP1, MRP2, MRP3, MRP4, and MRP5); members of the P-glycoprotein (Pgp) family (MDR1 and MDR2); BCRP; and MXR1 and MXR2. Cellular resistance to cytotoxic drugs (multidrug resistance) creates significant obstacles to the effective use of chemotherapeutic agents in treating many types of human tumors. Multidrug resistance of certain tumors is caused by the overexpression of members of Pgp and the MRP gene families.

[1820] Some members of the ATP family participate in ion channel formation and/or regulation. Examples of these proteins include: members of the sulfonylurea receptor (SUR) family (SUR1, SUR2A, and SUR2B; subunits of ATP-sensitive potassium channels); cystic fibrosis transmembrane conductance regulator (CFTR; chloride channel); and members of the MRP family (e.g., MRP-5; anion transporter and provides cellular resistance to CdCl2 and potassium antimonyl tartrate). ATP-sensitive potassium channels serve as a link between cellular metabolism and membrane electrical activity in excitable cells. The pharmacologic characteristics of ATP-sensitive potassium channels include blockade by the sulfonylurea class of agents, e.g., glibenclamide (McAleer et al. (1999) J. Biol. Chem. 274:23541-23548; Nasonkin et al. (1999) J. Biol. Chem. 274:29420-29425).

[1821] Summary of the 44589 Invention

[1822] The present invention is based, in part, on the discovery of a novel ABC Transporter family member, referred to herein as “44589”. The nucleotide sequence of a cDNA encoding 44589 is shown in SEQ ID NO: 33, and the amino acid sequence of a 44589 polypeptide is shown in SEQ ID NO: 34. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 35.

[1823] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 44589 protein or polypeptide, e.g., a biologically active portion of the 44589 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 34. In other embodiments, the invention provides isolated 44589 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 33, SEQ ID NO: 35, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 33, SEQ ID NO: 35, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 33, SEQ ID NO: 35, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 44589 protein or an active fragment thereof.

[1824] In a related aspect, the invention further provides nucleic acid constructs that include a 44589 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 44589 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 44589 nucleic acid molecules and polypeptides.

[1825] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 44589-encoding nucleic acids.

[1826] In still another related aspect, isolated nucleic acid molecules that are antisense to a 44589 encoding nucleic acid molecule are provided.

[1827] In another aspect, the invention features, 44589 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 44589-mediated or -related disorders. In another embodiment, the invention provides 44589 polypeptides having a 44589 activity. Preferred polypeptides are 44589 proteins including at least one ABC transporter ATP cassette domain and/or one ABC transporter transmembrane region and, preferably, having a 44589 activity, e.g., a 44589 activity as described herein.

[1828] In other embodiments, the invention provides 44589 polypeptides, e.g., a 44589 polypeptide having the amino acid sequence shown in SEQ ID NO: 34 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 34 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 33, SEQ ID NO: 35, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 44589 protein or an active fragment thereof.

[1829] In a related aspect, the invention further provides nucleic acid constructs which include a 44589 nucleic acid molecule described herein.

[1830] In a related aspect, the invention provides 44589 polypeptides or fragments operatively linked to non-44589 polypeptides to form fusion proteins.

[1831] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 44589 polypeptides or fragments thereof, e.g., an ABC transporter ATP cassette domain, an ABC transporter transmembrane region, an extracellular region, or an intracellular region of a 44589 polypeptide.

[1832] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 44589 polypeptides or nucleic acids.

[1833] In still another aspect, the invention provides a process for modulating 44589 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 44589 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular proliferation or differentiation, e.g., cancer.

[1834] The invention also provides assays for determining the activity of or the presence or absence of 44589 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[1835] In yet another aspect, the invention provides methods for inhibiting the proliferation or inducing the killing, of a 44589-expressing cell, e.g., a hyper-proliferative 44589-expressing cell. The method includes contacting the cell with a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 44589 polypeptide or nucleic acid. In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol. In a preferred embodiment, the cell is a hyperproliferative cell, e.g., a cell found in a solid tumor, a soft tissue tumor, or a metastatic lesion. For example, the hyperproliferative cell can be found in the breast, prostate, or liver.

[1836] In a preferred embodiment, the compound is an inhibitor of a 44589 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety selected from a cytotoxin, a cytotoxic agent arid a radioactive metal ion). In another preferred embodiment, the compound is an inhibitor of a 44589 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.

[1837] In a preferred embodiment, the compound is administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation.

[1838] In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant cellular proliferation or differentiation of a 44589-expressing cell, in a subject. Preferably, the method includes administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 44589 polypeptide or nucleic acid. In a preferred embodiment, the disorder is a cancerous or pre-cancerous condition.

[1839] In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., a proliferative disorder or a liver disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with one or more of: chemotherapy, radiation, and/or a compound identified using the methods described herein); and evaluating the expression of a 44589 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 44589 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 44589 nucleic acid or polypeptide expression can be detected by any method described herein.

[1840] In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 44589 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.

[1841] In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein, a cytotoxic agent) and, evaluating the expression of 44589 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 44589 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 44589 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a cancerous tissue, e.g. a cancerous breast tissue, or a liver tissue.

[1842] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 44589 polypeptide or nucleic acid molecule, including for disease diagnosis.

[1843] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 44589 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 44589 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 44589 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[1844] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[1845] Detailed Description of 44589

[1846] The human 44589 sequence (see SEQ ID NO: 33, as recited in Example 21), which is approximately 4638 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 4083 nucleotides, including the termination codon. The coding sequence encodes a 1360 amino acid protein (see SEQ ID NO: 34, as recited in Example 21).

[1847] 44589 contains the following regions or structural features: a first ABC transporter ATP cassette domain (FIG. 17A; PFAM Accession PF00005) located at about amino acid residues 515-686 of SEQ ID NO: 34; a second ABC transporter ATP cassette domain (FIG. 17B; PFAM Accession PF00005) located at about amino acid residues 1146-1329 of SEQ ID NO: 34; a first ABC transporter transmembrane region (FIG. 17C; PFAM Accession PF00664) located at about amino acid residues 163-445 of SEQ ID NO: 34; and a second ABC transporter transmembrane region (FIG. 17D; PFAM Accession PF00664) located at about amino acid residues 784-1073 of SEQ ID NO: 34. Each of the ABC transporter transmembrane regions of 44589 contains a unit of six transmembrane helices. Thus, 44589 has a total of 12 transmembrane helices. The six transmembrane domains of the first ABC transporter transmembrane region are located at about amino acids 163-185, 199-215, 283-303, 310-333, 353-369, and 396-416 of SEQ ID NO: 34. The six transmembrane domains of the second ABC transporter transmembrane region are located at about amino acids 781-805, 842-863, 919-935, 942-958, 1030-1047, and 1052-1069 of SEQ ID NO: 34. 44589 also contains seven predicted cytoplasmic regions (located at about amino acids 1-162, 216-282, 334-352, 417-780, 864-918, 959-1029, and 1070-1360 of SEQ ID NO: 34) and six predicted extracellular regions (located at about amino acids 186-198, 304-309, 370-395, 806-841, 936-941, and 1048-1051 of SEQ ID NO: 34).

[1848] The 44589 protein also includes the following domains: eight predicted N-glycosylation sites (PS00001) at about amino acids 11-14, 611-614, 691-694, 816-819, 822-825, 970-973, 1140-1143, and 1255-1258 of SEQ ID NO: 34; one predicted glycosaminoglycan attachment site (PS00002) at about amino acids 257-260 of SEQ ID NO: 34; three predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acids 3-6, 867-870, and 1004-1007 of SEQ ID NO: 34; nineteen predicted Protein Kinase C phosphorylation sites (PS00005) at about amino acids 2-4, 107-109, 126-128, 142-144, 526-528, 628-630, 658-660, 723-725, 767-769, 817-819, 866-868, 1020-1022, 1053-1055, 1072-1074, 1142-1144, 1157-1159, 1209-1211, 1297-1299, and 1358-1360 of SEQ ID NO: 34; sixteen predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 37-40, 44-47, 110-113, 121-124, 256-259, 444-447, 640-643, 693-696, 739-742, 756-759, 817-820, 824-827, 887-890, 1084-1087, 1257-1260, and 1297-1300 of SEQ ID NO: 34; twenty predicted N-myristoylation sites (PS00008) from about amino acids 14-19, 20-25, 145-150, 170-175, 202-207, 260-265, 393-398, 418-423, 467-472, 515-520, 522-527, 547-552, 609-614, 812-817, 855-860, 1138-1143, 1156-1161, 1181-1186, 1253-1258, and 1345-1350 of SEQ ID NO: 34; two predicted ATP/GTP-binding site motif A (P-loop) sites (PS00017) located at about amino acids 522-529 and 1153-1160 of SEQ ID NO: 34; and two predicted ABC transporter family signatures (PS00185) located at about amino acids 616-626 and 1256-1270 of SEQ ID NO: 34.

[1849] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[1850] A plasmid containing the nucleotide sequence encoding human 44589 (clone “Fbh44589FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[1851] The 44589 protein contains a significant number of structural characteristics in common with members of the ABC Transporter family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[1852] Members of the ABC transporter family of proteins are characterized by a common structure and related functions. ABC transporters form a large family of proteins responsible for translocation of a variety of compounds across biological membranes. ABC transporters share a conserved domain of approximately 200 amino acids, known as an ABC transporter ATP cassette domain, which includes an ATP-binding site. Many eukaryotic proteins of medical significance belong to the ABC transporter family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1-Tap2).

[1853] ABC transporters are typically composed of two copies of an ABC transporter ATP cassette domain and two copies of a ABC transporter transmembrane region. These four domains may be contained in a single polypeptide or may be present in different polypeptide chains. Many members of the ATP Transporter family are involved in the active transport of small hydrophilic molecules across the cytoplasmic membrane.

[1854] The nucleotide binding domain of ATP Transporters contains two characteristic Walker consensus motifs, designated ATP-binding motif A and motif B. The ATP binding motif A is also referred to as a P-loop. The conserved P-loop is a glycine-rich region, which typically forms a flexible loop between a beta-strand and an alpha-helix. The P-loop interacts with one of the phosphate groups of the nucleotide. A consensus P-loop sequence is as follows: [AG]-x(4)-G-K-[ST]. The two P-loops of 44589 are located at about amino acid residues 522-529 and 1153-1160 of SEQ ID NO: 34.

[1855] A 44589 polypeptide can include an “ABC transporter ATP cassette domain” or regions homologous with an “ABC transporter ATP cassette domain”.

[1856] As used herein, the term “ABC transporter ATP cassette domain” includes an amino acid sequence of about 70 to 300 amino acid residues in length and having a bit score for the alignment of the sequence to the ABC transporter ATP cassette domain profile (Pfam HMM) of at least 100. Preferably, an ABC transporter ATP cassette domain includes at least about 100 to 250 amino acids, more preferably about 150 to 200 amino acid residues, or about 165 to 185 amino acids and has a bit score for the alignment of the sequence to the ABC transporter ATP cassette domain (HMM) of at least 315 or greater. The ABC transporter ATP cassette domain (HMM) has been assigned the PFAM Accession Number PF00005 (http;//genome.wustl.edu/Pfam/.html). Alignments of the ABC transporter ATP cassette domains (amino acids 515 to 686 and 1146 to 1329 of SEQ ID NO: 34) of human 44589 with a consensus amino acid sequence (SEQ ID NO: 36) derived from a hidden Markov model are depicted in FIG. 17A (515 to 686 of SEQ ID NO: 34) and FIG. 17B (1146 to 1329 of SEQ ID NO: 34).

[1857] In a preferred embodiment, a 44589 polypeptide or protein has a “ABC transporter ATP cassette domain” or a region which includes at least about 100 to 250, more preferably about 150 to 200 or 165 to 185 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “ABC transporter ATP cassette domain,” e.g., the ABC transporter ATP cassette domain of human 44589 (e.g., residues 515 to 686 or 1146 to 1329 of SEQ ID NO: 34).

[1858] A 44589 molecule can further include an “ABC transporter transmembrane region” or regions homologous with a “ABC transporter transmembrane region”.

[1859] As used herein, the term “ABC transporter transmembrane region” includes an amino acid sequence of about 200 to 400 amino acid residues in length and having a bit score for the alignment of the sequence to the ABC transporter transmembrane region (HMM) of at least 40. Preferably, an ABC transporter transmembrane region includes at least about 250 to 330 amino acids, more preferably about 260 to 320 amino acid residues, or about 280 to 300 amino acids and has a bit score for the alignment of the sequence to the ABC transporter transmembrane region (HMM) of at least 60 or greater. The ABC transporter transmembrane region (HMM) has been assigned the PFAM Accession Number PF00664. Alignments of the ABC transporter transmembrane regions (amino acids 163 to 445 and 784 to 1073 of SEQ ID NO: 34) of human 44589 with a consensus amino acid sequence (SEQ ID NO: 37) derived from a hidden Markov model are depicted in FIG. 17C (163 to 445 of SEQ ID NO: 34) and FIG. 17D (784 to 1073 of SEQ ID NO: 34).

[1860] In a preferred embodiment, a 44589 polypeptide or protein has an “ABC transporter transmembrane region” or a region which includes at least about 250 to 330, more preferably about 260 to 320 or 280 to 300 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “ABC transporter transmembrane region,” e.g., an ABC transporter transmembrane region of human 44589 (e.g., residues 163 to 445 or 784 to 1073 of SEQ ID NO: 34).

[1861] To identify the presence of an “ABC transporter ATP cassette domain” or an “ABC transporter transmembrane region” in a 44589 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of two “ABC transporter ATP cassette” domains in the amino acid sequence of human 44589 at about residues 515 to 686 and 1146 to 1329 of SEQ ID NO: 34 (see FIGS. 17A and 17B). Two “ABC transporter transmembrane” regions in the amino acid sequence of human 44589 were identified at about residues 163 to 445 and 784 to 1073 of SEQ ID NO: 34 (see FIGS. 17C and 17D).

[1862] In one embodiment, a 44589 protein includes at least one cytoplasmic domain. When located at the N-terminal domain the cytoplasmic domain is referred to herein as an “N-terminal cytoplasmic domain” in the amino acid sequence of the protein. As used herein, an “N-terminal cytoplasmic domain” includes an amino acid sequence having about 1-250, preferably about 1-225, more preferably about 1-200, even more preferably about 1-180 amino acid residues in length and is located inside of a cell or intracellularly. The C-terminal amino acid residue of a “N-terminal cytoplasmic domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring 44589 or 44589-like protein. For example, an N-terminal cytoplasmic domain is located at about amino acid residues 1-162 of SEQ ID NO: 34.

[1863] In a preferred embodiment, a 44589 polypeptide or protein has an “N-terminal cytoplasmic domain” or a region which includes at least about 1-250, more preferably about 1-225, 1-200, 1-180 or 1-162 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “N-terminal cytoplasmic domain,” e.g., the N-terminal cytoplasmic domain of human 44589 (e.g., residues 1-162 of SEQ ID NO: 34).

[1864] In another embodiment, a 44589 protein includes at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or preferably, twelve transmembrane domains. As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15 amino acid residues in length that spans the plasma membrane. More preferably, a transmembrane domain includes about at least 15, 20, 23, 24, 25, 30 or 35 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an a-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N. et al, (1996) Annual Rev. Neuronsci. 19: 235-63, the contents of which are incorporated herein by reference. Amino acid residues 163-185, 199-215, 283-303, 310-333, 353-369, 396-416, 781-805, 842-863, 919-935, 942-958, 1030-1047, and 1052-1069 of SEQ ID NO: 34 comprise transmembrane domains in a 44589 protein.

[1865] In a preferred embodiment, a 44589 polypeptide or protein has at least one transmembrane domain or a region which includes at least 15, 20, 23, 24, 25, 30 or 35 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “transmembrane domain,” e.g., at least one transmembrane domain of human 44589 (e.g., residues 163-185, 199-215, 283-303, 310-333, 353-369, 396-416, 781-805, 842-863, 919-935, 942-958, 1030-1047, and 1052-1069 of SEQ ID NO: 34). Preferably, the transmembrane domain interacts with a molecule traversing the plasma membrane, e.g., an ion and/or a toxic substance such as a drug molecule.

[1866] In another embodiment, a 44589 protein include at least one extracellular loop. As defined herein, the term “loop” includes an amino acid sequence having a length of at least about 4, preferably about 5-10, more preferably about 10-20, and even more preferably about 20-30 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring 44589 or 44589-like molecule, and the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring 44589 or 44589-like molecule. As used herein, an “extracellular loop” includes an amino acid sequence located outside of a cell, or extracellularly. For example, an extracellular loop can be found at about amino acids 186-198, 304-309, 370-395, 806-841, 936-941, and 1048-1051 of SEQ ID NO: 34.

[1867] In a preferred embodiment, a 44589 polypeptide or protein has at least one extracellular loop or a region which includes at least about 4, preferably about 5-10, more preferably about 10-20, more preferably about 20-30, and most preferably about 30-40 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “extracellular loop,” e.g., at least one extracellular loop of human 44589 (e.g., residues 186-198, 304-309, 370-395, 806-841, 936-941, and 1048-1051 of SEQ ID NO: 34).

[1868] In another embodiment, a 44589 protein includes at least one cytoplasmic loop, also referred to herein as a cytoplasmic domain. As used herein, a “cytoplasmic loop” includes an amino acid sequence having a length of at least about 4, preferably about 5-10, more preferably about 10-20, more preferably about 20-30, and most preferably about 30-40 amino acid residues located within a cell or within the cytoplasm of a cell. For example, a cytoplasmic loop is found at about amino acids 1-162, 216-282, 334-352, 417-780, 864-918, 959-1029, and 1070-1360 of SEQ ID NO: 34.

[1869] In a preferred embodiment, a 44589 polypeptide or protein has at least one cytoplasmic loop or a region which includes at least about 4, preferably about 5-10, more preferably about 10-20, more preferably about 20-30, and most preferably about 30-40 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “cytoplasmic loop,” e.g., at least one cytoplasmic loop of human 44589 (e.g., residues 1-162, 216-282, 334-352, 417-780, 864-918, 959-1029, and 1070-1360 of SEQ ID NO: 34). Preferably, the cytoplasmic loop is capable of interacting with a nucleotide, e.g., ATP.

[1870] In another embodiment, a 44589 protein includes a “C-terminal cytoplasmic domain”, also referred to herein as a C-terminal cytoplasmic tail, in the sequence of the protein. As used herein, a “C-terminal cytoplasmic domain” includes an amino acid sequence having a length of at least about 50, preferably about 50-150, more preferably about 50-300 amino acid residues, and is located within a cell or within the cytoplasm of a cell. Accordingly, the N-terminal amino acid residue of a “C-terminal cytoplasmic domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally-occurring 44589 or 44589-like protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 1070-1360 of SEQ ID NO: 34.

[1871] In a preferred embodiment, a 44589 polypeptide or protein has a C-terminal cytoplasmic domain or a region which includes at least about 5, preferably about 50-150, more preferably about 50-300 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “C-terminal cytoplasmic domain,” e.g., the C-terminal cytoplasmic domain of human 44589 (e.g., residues 1070-1360 of SEQ ID NO: 34).

[1872] Accordingly, a 44589 family member can include at least one, and preferably six, or twelve, transmembrane domains and/or at least one cytoplasmic loop, and/or at least one extracellular loop. In another embodiment, the 44589 further includes an N-terminal cytoplasmic domain and/or a C-terminal cytoplasmic domain. In another embodiment, the 44589 can include twelve transmembrane domains, seven cytoplasmic loops, six extracellular loops and can further include an N-terminal cytoplasmic domain and/or a C-terminal cytoplasmic domain.

[1873] A 44589 family member can include: at least one and preferably two ABC transporter ATP cassette domains; and at least one and preferably two ABC transporter transmembrane regions.

[1874] A 44589 family member can further include at least one and preferably two ATP/GTP-binding site motifs A (P-loops).

[1875] A 44589 family member can further include: at least one, two, three, four, five, six, seven, and preferably eight N-glycosylation sites (PS00001); at least one glycosaminoglycan attachment site (PS00002); at least one, two, and preferably three cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, and preferably 19 Protein Kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, and preferably 16 Casein Kinase II phosphorylation sites (PS00006); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and preferably 20 N-myristoylation sites (PS00008); and at least one and preferably two ABC transporter family signatures (PS00185).

[1876] As the 44589 polypeptides of the invention may modulate 44589-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 44589-mediated or related disorders, as described below.

[1877] As used herein, a “44589 activity”, “biological activity of 44589” or “functional activity of 44589”, refers to an activity exerted by a 44589 protein, polypeptide or nucleic acid molecule. For example, a 44589 activity can be an activity exerted by 44589 in a physiological milieu on, e.g., a 44589-responsive cell or on a 44589 substrate, e.g., a protein substrate. A 44589 activity can be determined in vivo or in vitro. In one embodiment, a 44589 activity is a direct activity, such as an association with a 44589 target molecule. A “target molecule” or “binding partner” is a molecule with which a 44589 protein binds or interacts in nature. Exemplary embodiments of a 44589 target molecule include an ion, a toxic substance, and/or a nucleotide, e.g., ATP.

[1878] A 44589 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 44589 protein with an ion, a toxic substance, and/or a nucleotide. The features of the 44589 molecules of the present invention can provide similar biological activities as ABC Transporter family members. For example, the 44589 proteins of the present invention can have one or more of the following activities: (1) mediating the active transport of biological molecules, e.g., ions, across a cell membrane, e.g., a plasma membrane; (2) mediating the active efflux of cytotoxic substances, e.g., drug molecules such as chemotherapeutic agents, from a cell; (3) participating in ion channel formation; (4) participating in ion channel regulation; (5) binding to a nucleotide, e.g., ATP; (6) hydrolyzing a nucleotide, e.g., ATP; (7) using the energy of ATP hydrolysis to reduce intracellular drug accumulation; (8) contributing to the chemoresistance of a tumor cell; or (9) being subject to blockade by the sulfoyl urea class of agents.

[1879] The 44589 protein may act as a pump that removes toxic substances from a cell. For example, the 44589 protein is homologous to both the Pgp (MDR) and MRP family of proteins (see e.g., FIGS. 18A-18D). Innate or acquired expression of Pgp (MDR) and/or MRP proteins in a cancer cell aids the cell in resisting treatment with certain chemotherapeutic agents. Inhibiting the activity of 44589 is therefore an important strategy for, e.g., increasing the chemosensitivity of a cancer cell.

[1880] Based on the relatedness of the 44589 protein to the MRP5 and Sulfonylurea receptor (SUR) proteins, 44589 is predicted to have similar activities. Thus, the 44589 protein may function as an ion channel. MRP5 has been shown to function as an anion transporter, as well as a transporter of CdCl2 and potassium antimonyl tartrate (McAleer et al. (1999) J. Biol. Chem. 274:23541-23548). SURs are required subunits of ATP-sensitive potassium channels, which serve as a vital link between cellular metabolism and membrane electrical activity in excitable cells (e.g., pancreatic islets, cardiac muscle, smooth muscle, skeletal muscle, neurons, and epithelia). ATP-sensitive potassium channels are involved in processes such as the control of insulin secretion from pancreatic beta islet cells, the response of cardiac and cerebral cells to ischemia, regulation of vascular smooth muscle tone, and modulation of transmitter release at brain synapses. Specifically, SUR1 is a subunit of the pancreatic beta-cell ATP-sensitive potassium channel and plays a key role in the regulation of glucose-induced insulin secretion. Pharmacologically, ATP-sensitive potassium channels can be blocked by the sulfonylurea class of agents, e.g., glibenclamide. The ATP-sensitive potassium channel is a complex of two subunits, a SUR and an inward rectifier Kir6.2 subunit (Nasonkin et al. (1999) J. Biol. Chem. 274:29420-29425. Based upon sequence relatedness, 44589 may participate in the formation of ATP-sensitive potassium channels and may be sensitive to blockade by the sulfonylurea class of agents.

[1881] 44589 may be associated with diseases and/or syndromes associated with misfunction and/or misexpression of members of the ABC transporter family. Several diseases are associated with activity of members of the ABC transporter family. For example, expression of some members of the ABC transporter family, e.g., MDR1, MRP1, and MRP2, is upregulated in various tumor types and is believed to contribute to the resistance of some tumor cells to anticancer chemotherapeutic agents, e.g., cisplatin (Hinoshita et al. (2000) Clin. Cancer Res. 6:2401-2407). A mutation in MRP2 has been detected in Dubin-Johnson syndrome, a pathology characterized by a defect in hepatic multispecific organic ion transport (Kast and Gros (1997) J. Biol. Chem. 272:26479-26487). Stargardt disease, a macular dystrophy of childhood characterized by bilateral loss of central vision over a period of several months, has been attributed to inherited mutations in the retinal specific ATP binding transporter gene (ABCR) (Rozet et al. (1999) J. Med. Genet. 36:447-451).

[1882] The 44589 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders and/or liver disorders.

[1883] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[1884] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[1885] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[1886] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[1887] Examples of proliferative breast diseases include, but are not limited to, epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors including, but not limited to, stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, no special type, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms.

[1888] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[1889] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[1890] Disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and cirrhosis, such as cirrhosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and toxin-induced liver disease, such as alcoholic liver disease; inborn errors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, α1-antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease, such as secondary biliary cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and anomalies of the biliary tree; circulatory disorders, such as impaired blood flow into the liver, including hepatic artery compromise and portal vein obstruction and thrombosis, impaired blood flow through the liver, including passive congestion and centrilobular necrosis and peliosis hepatis, hepatic vein outflow obstruction, including hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease associated with pregnancy, such as preeclampsia and eclampsia, acute fatty liver of pregnancy, and intrehepatic cholestasis of pregnancy; hepatic complications of organ or bone marrow transplantation, such as drug toxicity after bone marrow transplantation, graft-versus-host disease and liver rejection, and nonimmunologic damage to liver allografts; tumors and tumorous conditions, such as nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.

[1891] The 44589 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 34 thereof are collectively referred to as “polypeptides or proteins of the invention” or “44589 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “44589 nucleic acids.” 44589 molecules refer to 44589 nucleic acids, polypeptides, and antibodies.

[1892] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[1893] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[1894] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2× SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[1895] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 33 or SEQ ID NO: 35, corresponds to a naturally-occurring nucleic acid molecule.

[1896] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include at least an open reading frame encoding a 44589 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 44589 protein or derivative thereof.

[1897] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 44589 protein is at least 10% pure. In a preferred embodiment, the preparation of 44589 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-44589 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-44589 chemicals. When the 44589 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[1898] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 44589 without abolishing or substantially altering a 44589 activity. Preferably the alteration does not substantially alter the 44589 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 44589, results in abolishing a 44589 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 44589 are predicted to be particularly unamenable to alteration.

[1899] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 44589 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 44589 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 44589 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 33 or SEQ ID NO: 35, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[1900] As used herein, a “biologically active portion” of a 44589 protein includes a fragment of a 44589 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 44589 molecule and a non-44589 molecule or between a first 44589 molecule and a second 44589 molecule (e.g., a dimerization interaction). Biologically active portions of a 44589 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 44589 protein, e.g., the amino acid sequence shown in SEQ ID NO: 34, which include less amino acids than the full length 44589 proteins, and exhibit at least one activity of a 44589 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 44589 protein, e.g., the ability to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP. A biologically active portion of a 44589 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 44589 protein can be used as targets for developing agents which modulate a 44589 mediated activity, e.g., the ability to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP.

[1901] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[1902] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[1903] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[1904] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[1905] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[1906] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 44589 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 44589 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[1907] Particularly preferred 44589 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 34. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 34 are termed substantially identical.

[1908] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 33 or 3 are termed substantially identical. “Misexpression or aberrant expression”, as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[1909] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[1910] A “purified preparation of cells”, as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[1911] Various aspects of the invention are described in further detail below.

[1912] Isolated 44589 Nucleic Acid Molecules

[1913] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 44589 polypeptide described herein, e.g., a full-length 44589 protein or a fragment thereof, e.g., a biologically active portion of 44589 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 44589 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[1914] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 33, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 44589 protein (i.e., “the coding region” of SEQ ID NO: 33, as shown in SEQ ID NO: 35), as well as 5′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 33 (e.g., SEQ ID NO: 35) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid 515 to 686 of SEQ ID NO: 34. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid 1146 to 1329 of SEQ ID NO: 34. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid 163 to 445 of SEQ ID NO: 34. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid 784 to 1073 of SEQ ID NO: 34.

[1915] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 33 or SEQ ID NO: 35, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 33 or SEQ ID NO: 35, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO: 33 or 35, thereby forming a stable duplex.

[1916] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 33 or SEQ ID NO: 35, or a portion, preferably of the same length, of any of these nucleotide sequences.

[1917] 44589 Nucleic Acid Fragments

[1918] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 33 or 35. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 44589 protein, e.g., an immunogenic or biologically active portion of a 44589 protein. A fragment can comprise those nucleotides of SEQ ID NO: 33, which encode an ABC transporter ATP cassette domain and/or the ABC transporter transmembrane region of human 44589. The nucleotide sequence determined from the cloning of the 44589 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 44589 family members, or fragments thereof, as well as 44589 homologues, or fragments thereof, from other species.

[1919] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[1920] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 44589 nucleic acid fragment can include a sequence corresponding to an ABC transporter ATP cassette domain and/or the ABC transporter transmembrane region.

[1921] 44589 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 33 or SEQ ID NO: 35, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 33 or SEQ ID NO: 35. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.

[1922] In one embodiment, the probe or primer is attached to a solid support, e.g., a solid support described herein.

[1923] One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g., the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 34. The reverse primer can anneal to the ultimate codon, e.g., the codon immediately before the stop codon, e.g., the codon encoding amino acid residue 1360 of SEQ ID NO: 34. In a preferred embodiment, the annealing temperatures of the forward and reverse primers differ by no more than 5, 4, 3, or 2° C.

[1924] In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1925] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: an ABC transporter ATP cassette domain which extends from about amino acids 515-686 of SEQ ID NO: 34; an ABC transporter ATP cassette domain which extends from about amino acids 1146-1329 of SEQ ID NO: 34; an ABC transporter transmembrane region which extends from about amino acids 163-445 of SEQ ID NO: 34; an ABC transporter transmembrane region which extends from about amino acids 784-1073 of SEQ ID NO: 34; an ATP/GTP-binding site motif A (P-loop) which extends from about amino acids 522-529 of SEQ ID NO: 34; an ATP/GTP-binding site motif A (P-loop) which extends from about amino acids 1153-1160 of SEQ ID NO: 34; an ABC transporter family signature which extends from about amino acids 612-626 of SEQ ID NO: 34; an ABC transporter family signature which extends from about amino acids 1256-1270 of SEQ ID NO: 34; an N-terminal cytoplasmic domain which extends from about amino acids 1-162 of SEQ ID NO: 34; one or more of the six extracellular loops which extend from about amino acids 186-198, 304-309, 370-395, 806-841, 936-941, and 1048-1051 of SEQ ID NO: 34; one or more of the five cytoplasmic loops which extend from about amino acids 216-282, 334-352, 417-780, 864-918, and 959-1029 of SEQ ID NO: 34; or a C-terminal cytoplasmic domain which extend from about amino acids 1070-1360 of SEQ ID NO: 34.

[1926] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 44589 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: an ABC transporter ATP cassette domain; an ABC transporter transmembrane region; a cytoplasmic domain; any or all of the extracellular loops and/or any or all of the cytoplasmic loops as defined above relative to SEQ ID NO: 34.

[1927] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[1928] A nucleic acid fragment encoding a “biologically active portion of a 44589 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 33 or 35, which encodes a polypeptide having a 44589 biological activity (e.g., the biological activities of the 44589 proteins are described herein), expressing the encoded portion of the 44589 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 44589 protein. For example, a nucleic acid fragment encoding a biologically active portion of 44589 includes an ABC transporter ATP cassette domain and/or the ABC transporter transmembrane region, e.g., amino acid residues about 515 to 686, 1146-1329, 163-445, or 784-1073 of SEQ ID NO: 34. A nucleic acid fragment encoding a biologically active portion of a 44589 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

[1929] In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 33, or SEQ ID NO: 35.

[1930] In preferred embodiments, the fragment includes at least one, and preferably at least 5, 10, 15, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 nucleotides from nucleotides 1-1379 or 459-1379 of SEQ ID NO: 33.

[1931] In preferred embodiments, the fragment includes the nucleotide sequence of SEQ ID NO: 35 and at least one, and preferably at least 5, 10, 15, 25, 50, 75, 100, 200, 300, or 500 consecutive nucleotides of SEQ ID NO: 33.

[1932] In preferred embodiments, the fragment includes at least one, and preferably at least 5, 10, 15, 25, 50, 75, 100, 200, 300, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides encoding a protein including at least 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 consecutive amino acids of SEQ ID NO: 34. In one embodiment, the encoded protein includes at least 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 375 consecutive amino acids from residues 1-393 of SEQ ID NO: 34

[1933] In preferred embodiments, the nucleic acid fragment includes a nucleotide sequence that is other than a sequence described in WO 01/32706, BE089591, AI401832, AI676121, AW372862, or AW372855.

[1934] In preferred embodiments, the fragment comprises the coding region of 44589, e.g., the nucleotide sequence of SEQ ID NO: 35.

[1935] 44589 Nucleic Acid Variants

[1936] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 33 or SEQ ID NO: 35. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 44589 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 34. If alignment is needed for this comparison the sequences should be aligned for maximum homology. The encoded protein can differ by no more than 5, 4, 3, 2, or 1 amino acid. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1937] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. Coli, yeast, human, insect, or CHO cells.

[1938] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[1939] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 33 or 35, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid. The nucleic acid can differ by no more than 5, 4, 3, 2, or 1 nucleotide. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[1940] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 34 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO: 34 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 44589 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 44589 gene.

[1941] Preferred variants include those that are correlated with the ability to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP.

[1942] Allelic variants of 44589, e.g., human 44589, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 44589 protein within a population that maintain the ability to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 34, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 44589, e.g., human 44589, protein within a population that do not have the ability to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 34, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[1943] Moreover, nucleic acid molecules encoding other 44589 family members and, thus, which have a nucleotide sequence which differs from the 44589 sequences of SEQ ID NO: 33 or SEQ ID NO: 35 are intended to be within the scope of the invention.

[1944] Antisense Nucleic Acid Molecules, Ribozymes and Modified 44589 Nucleic Acid Molecules

[1945] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 44589. An “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 44589 coding strand, or to only a portion thereof (e.g., the coding region of human 44589 corresponding to SEQ ID NO: 35). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 44589 (e.g., the 5′ and 3′ untranslated regions).

[1946] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 44589 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 44589 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 44589 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[1947] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[1948] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 44589 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[1949] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[1950] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 44589-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 44589 cDNA disclosed herein (i.e., SEQ ID NO: 33 or SEQ ID NO: 35), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 44589-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 44589 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[1951] 44589 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 44589 (e.g., the 44589 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 44589 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N. Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[1952] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

[1953] A 44589 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulmé (2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.

[1954] For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[1955] PNAs of 44589 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 44589 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[1956] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[1957] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 44589 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 44589 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

[1958] Isolated 44589 Polypeptides

[1959] In another aspect, the invention features, an isolated 44589 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-44589 antibodies. 44589 protein can be isolated from cells or tissue sources using standard protein purification techniques. 44589 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[1960] Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[1961] In a preferred embodiment, a 44589 polypeptide has one or more of the following characteristics:

[1962] (i) it has the ability to mediate the cellular transport of ions and/or toxic substances;

[1963] (ii) it has the ability to bind a nucleotide, e.g., ATP;

[1964] (iii) it has the ability to hydrolyze a nucleotide, e.g., ATP;

[1965] (iv) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition, or other physical characteristic of a 44589 polypeptide, e.g., a polypeptide of SEQ ID NO: 34;

[1966] (v) it has an overall sequence similarity of at least 60%, more preferably at least 70, 80, 90, or 95%, with a polypeptide of SEQ ID NO: 34;

[1967] (vi) it can be found inserted in the cell membrane;

[1968] (vii) it has an ABC transporter ATP cassette domain which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues about 515-686 and/or 1146-1329 of SEQ ID NO: 34;

[1969] (viii) it has an ABC transporter transmembrane region which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues about 163-445 and/or 784-1073 of SEQ ID NO: 34;

[1970] (ix) it can colocalize with a subunit of an ATP-dependent ion channel;

[1971] (x) it has the ability to promote the chemoresistance of cells in which it is expressed; or

[1972] (xi) it has at least 60% preferably 70%, and most preferably 90% of the cysteines found amino acid sequence of the native protein.

[1973] In a preferred embodiment the 44589 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID: 2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 34 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 34. (If this comparison requires aliginnent the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the ABC transporter ATP cassette domains and/or the ABC transporter transmembrane regions. In another preferred embodiment one or more differences are in the ABC transporter ATP cassette domain and/or the ABC transporter transmembrane region.

[1974] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 44589 proteins differ in amino acid sequence from SEQ ID NO: 34, yet retain biological activity.

[1975] In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 34.

[1976] A 44589 protein or fragment is provided which varies from the sequence of SEQ ID NO: 34 in regions defined by amino acids about 1-163 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 34 in regions defined by amino acids about 515-686, 1146-1329, 163-445, and/or 784-1073. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[1977] In one embodiment, a biologically active portion of a 44589 protein includes an ABC transporter ATP cassette domain and/or an ABC transporter transmembrane region. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 44589 protein.

[1978] In a preferred embodiment, the 44589 protein has an amino acid sequence shown in SEQ ID NO: 34, or a fragment thereof (e.g., 515 to 686, 1146-1329, 163-445, or 784-1073 of SEQ ID NO: 34). In other embodiments, the 44589 protein is substantially identical to SEQ ID NO: 34, or a fragment thereof (e.g., 515 to 686, 1146-1329, 163-445, or 784-1073 of SEQ ID NO: 34). In yet another embodiment, the 44589 protein is substantially identical to SEQ ID NO: 34 and retains the functional activity of the protein of SEQ ID NO: 34, as described in detail in the subsections above. In other embodiments, the 44589 protein includes a fragment of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 980, 990 consecutive amino acids of SEQ ID NO: 34 and includes at least 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 375, 380, or 390 consecutive amino acids from residues 1-393 of SEQ ID NO: 34. In other embodiments, the 44589 protein includes a fragment of about 989 or more amino acids of SEQ ID NO: 34.

[1979] 44589 Chimeric or Fusion Proteins

[1980] In another aspect, the invention provides 44589 chimeric or fusion proteins. As used herein, a 44589 “chimeric protein” or “fusion protein” includes a 44589 polypeptide linked to a non-44589 polypeptide. A “non-44589 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 44589 protein, e.g., a protein which is different from the 44589 protein and which is derived from the same or a different organism. The 44589 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a 44589 amino acid sequence. In a preferred embodiment, a 44589 fusion protein includes at least one (or two) biologically active portion of a 44589 protein. The non-44589 polypeptide can be fused to the N-terminus or C-terminus of the 44589 polypeptide.

[1981] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-44589 fusion protein in which the 44589 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 44589. Alternatively, the fusion protein can be a 44589 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 44589 can be increased through use of a heterologous signal sequence.

[1982] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[1983] The 44589 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 44589 fusion proteins can be used to affect the bioavailability of a 44589 substrate. 44589 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 44589 protein; (ii) mis-regulation of the 44589 gene; and (iii) aberrant post-translational modification of a 44589 protein.

[1984] Moreover, the 44589-fusion proteins of the invention can be used as immunogens to produce anti-44589 antibodies in a subject, to purify 44589 ligands and in screening assays to identify molecules which inhibit the interaction of 44589 with a 44589 substrate.

[1985] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 44589-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 44589 protein.

[1986] Variants of 44589 Proteins

[1987] In another aspect, the invention also features a variant of a 44589 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 44589 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 44589 protein. An agonist of the 44589 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a 44589 protein. An antagonist of a 44589 protein can inhibit one or more of the activities of the naturally occurring form of the 44589 protein by, for example, competitively modulating a 44589-mediated activity of a 44589 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 44589 protein.

[1988] Variants of a 44589 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 44589 protein for agonist or antagonist activity.

[1989] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 44589 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 44589 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[1990] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 44589 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 44589 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[1991] Cell based assays can be exploited to analyze a variegated 44589 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 44589 in a substrate-dependent manner. The transfected cells are then contacted with 44589 and the effect of the expression of the mutant on signaling by the 44589 substrate can be detected, e.g., the ability to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 44589 substrate, and the individual clones further characterized.

[1992] In another aspect, the invention features a method of making a 44589 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 44589 polypeptide, e.g., a naturally occurring 44589 polypeptide. The method includes: altering the sequence of a 44589 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[1993] In another aspect, the invention features a method of making a fragment or analog of a 44589 polypeptide a biological activity of a naturally occurring 44589 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 44589 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[1994] Anti-44589 Antibodies

[1995] In another aspect, the invention provides an anti-44589 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[1996] The anti-44589 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[1997] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[1998] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 44589 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-44589 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[1999] The anti-44589 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[2000] Phage display and combinatorial methods for generating anti-44589 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[2001] In one embodiment, the anti-44589 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art.

[2002] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[2003] An anti-44589 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[2004] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[2005] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a 44589 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[2006] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[2007] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 44589 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[2008] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[2009] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[2010] In preferred embodiments an antibody can be made by immunizing with purified 44589 antigen, or a fragment thereof, e.g., a fragment described herein, membrane associated antigen, tissue, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g., membrane fractions.

[2011] A full-length 44589 protein or, antigenic peptide fragment of 44589 can be used as an immunogen or can be used to identify anti-44589 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 44589 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 34 and encompasses an epitope of 44589. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[2012] Fragments of 44589 which include residues about 45-65 or 485-510 of SEQ ID NO: 34 can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophilic regions of the 44589 protein. Similarly, fragments of 44589 which include residues about 300-340 or 920-960 of SEQ ID NO: 34 can be used to make an antibody against a hydrophobic region of the 44589 protein; fragments of 44589 which include residues about 186-198, 304-309, 370-395, 806-841, 936-941, or 1048-1051 of SEQ ID NO: 34 can be used to make an antibody against an extracellular region of the 44589 protein; fragments of 44589 which include residues about 1-162, 216-282, 334-352, 417-780, 864-918, 959-1029, or 1070-1360 of SEQ ID NO: 34 can be used to make an antibody against an intracellular region of the 44589 protein; a fragment of 44589 which include residues about 515-686 or 1146-1329 of SEQ ID NO: 34 can be used to make an antibody against the ABC transporter ATP cassette domain of the 44589 protein; and a fragment of 44589 which include residues about 163-445 or 784-1073 of SEQ ID NO: 34 can be used to make an antibody against the ABC transporter transmembrane region of the 44589 protein.

[2013] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[2014] Antibodies which bind only native 44589 protein, only denatured or otherwise non-native 44589 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies that bind to native, but not denatured 44589 protein.

[2015] Preferred epitopes encompassed by the antigenic peptide are regions of 44589 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 44589 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 44589 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[2016] In a preferred embodiment the antibody can bind to the extracellular portion of the 44589 protein, e.g., it can bind to a whole cell which expresses the 44589 protein. In another embodiment, the antibody binds an intracellular portion of the 44589 protein.

[2017] In preferred embodiments antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g., tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g., membrane fractions.

[2018] The anti-44589 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 44589 protein.

[2019] In a preferred embodiment the antibody has effector function and/or can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement.

[2020] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[2021] In a preferred embodiment, an anti-44589 antibody alters (e.g., increases or decreases) the ability of a 44589 polypeptide to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP. For example, the antibody can bind at or in proximity to the active site, e.g., to an epitope that includes a residue located from about 522-529, 1153-1160, 616-626, or 1256-1270 of SEQ ID NO: 34.

[2022] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[2023] An anti-44589 antibody (e.g., monoclonal antibody) can be used to isolate 44589 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-44589 antibody can be used to detect 44589 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-44589 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[2024] The invention also includes a nucleic acid which encodes an anti-44589 antibody, e.g., an anti-44589 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[2025] The invention also includes cell lines, e.g., hybridomas, which make an anti-44589 antibody, e.g., and antibody described herein, and method of using said cells to make a 44589 antibody.

[2026] 44589 Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[2027] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[2028] A vector can include a 44589 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 44589 proteins, mutant forms of 44589 proteins, fusion proteins, and the like).

[2029] The recombinant expression vectors of the invention can be designed for expression of 44589 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[2030] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[2031] Purified fusion proteins can be used in 44589 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 44589 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[2032] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[2033] The 44589 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[2034] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[2035] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

[2036] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[2037] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[2038] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 44589 nucleic acid molecule within a recombinant expression vector or a 44589 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[2039] A host cell can be any prokaryotic or eukaryotic cell. For example, a 44589 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) CellI23:175-182)). Other suitable host cells are known to those skilled in the art.

[2040] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[2041] A host cell of the invention can be used to produce (i.e., express) a 44589 protein. Accordingly, the invention further provides methods for producing a 44589 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a 44589 protein has been introduced) in a suitable medium such that a 44589 protein is produced. In another embodiment, the method further includes isolating a 44589 protein from the medium or the host cell.

[2042] In another aspect, the invention features, a cell or purified preparation of cells which include a 44589 transgene, or which otherwise misexpress 44589. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a 44589 transgene, e.g., a heterologous form of a 44589, e.g., a gene derived from humans (in the case of a non-human cell). The 44589 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 44589, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 44589 alleles or for use in drug screening.

[2043] In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, a breast cell, or a liver cell, transformed with nucleic acid which encodes a subject 44589 polypeptide.

[2044] Also provided are cells, preferably human cells, e.g., a hematopoietic stem cell, a breast cell, or a liver cell, or a fibroblast cell, in which an endogenous 44589 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 44589 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 44589 gene. For example, an endogenous 44589 gene which is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[2045] In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 44589 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Production of 44589 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 44589 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

[2046] 44589 Transgenic Animals

[2047] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 44589 protein and for identifying and/or evaluating modulators of 44589 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the-expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 44589 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[2048] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 44589 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 44589 transgene in its genome and/or expression of 44589 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 44589 protein can further be bred to other transgenic animals carrying other transgenes.

[2049] 44589 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[2050] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[2051] Uses of 44589

[2052] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[2053] The isolated nucleic acid molecules of the invention can be used, for example, to express a 44589 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a 44589 mRNA (e.g., in a biological sample) or a genetic alteration in a 44589 gene, and to modulate 44589 activity, as described further below. The 44589 proteins can be used to treat disorders characterized by insufficient or excessive production of a 44589 substrate or production of 44589 inhibitors. In addition, the 44589 proteins can be used to screen for naturally occurring 44589 substrates, to screen for drugs or compounds which modulate 44589 activity, as well as to treat disorders characterized by insufficient or excessive production of 44589 protein or production of 44589 protein forms which have decreased, aberrant or unwanted activity compared to 44589 wild type protein (e.g., cancer). Moreover, the anti-44589 antibodies of the invention can be used to detect and isolate 44589 proteins, regulate the bioavailability of 44589 proteins, and modulate 44589 activity.

[2054] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 44589 polypeptide is provided. The method includes: contacting the compound with the subject 44589 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 44589 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 44589 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 44589 polypeptide. Screening methods are discussed in more detail below.

[2055] 44589 Screening Assays

[2056] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 44589 proteins, have a stimulatory or inhibitory effect on, for example, 44589 expression or 44589 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 44589 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 44589 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[2057] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 44589 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 44589 protein or polypeptide or a biologically active portion thereof.

[2058] In one embodiment, the ability of a 44589 protein bind to and/or hydrolyze ATP can be assayed, as follows. Methods of detecting the hydrolysis of ATP by a protein containing a nucleotide binding domain are described in, for example, Li et al. (1996) J. Biol. Chem. 271:28463-28468 and Gadsby et al. (1999) Physiol. Rev. 79:S77-S107.

[2059] A purified protein containing an nucleotide binding domain of 44589 can be evaluated for its ability to mediate ATPase activity in vitro. The assay can be performed in the presence of a test compound to determine the ability of the test compound to modulate the ATPase activity of the purified protein. In addition, or alternatively, the purified protein used in an ATPase activity assay can be a variant or a fragment of 44589, and the assay can be performed to determine the ATPase activity of the fragment or variant.

[2060] ATPase activity can measured as the production of [α32-P]ADP from [α32-P]ATP, using polyethyleneimine-cellulose chromatography for separation of the nucleotides. The assay can be carried out in a 15 μl reaction mixture containing 50 mM Tris, 50 mM NaCl, pH 7.5, 2 mM MgCl2, 10% glycerol, 0.5 mM CHAPS, and 8 μCi of [α32-P]ATP. Reaction mixtures are incubated at 30° C. and are stopped by the addition of 5 μl of 10% SDS. One μl samples are spotted on a polyethyleneimine-cellulose plate and developed in 1 M formic acid, 0.5 M LiCl. The location and quantitation of the radiolabeled ATP and ADP can determined with a Molecular Dynamics PhosphorImager. Data can be analyzed using the ImageQuant software package (Molecular Dynamics). See, e.g., Li et al. (1996) J. Biol. Chem. 271:28463-28468 for additional details on methods detecting ATPase activity by nucleotide binding domain-containing proteins and variants thereof.

[2061] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[2062] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[2063] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[2064] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 44589 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 44589 activity is determined. Determining the ability of the test compound to modulate 44589 activity can be accomplished by monitoring, for example, the ability to mediate the cellular transport of ions and/or toxic substances and/or the ability to bind ATP. The cell, for example, can be of mammalian origin, e.g., human.

[2065] The ability of the test compound to modulate 44589 binding to a compound, e.g., a 44589 substrate, or to bind to 44589 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 44589 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 44589 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 44589 binding to a 44589 substrate in a complex. For example, compounds (e.g., 44589 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[2066] The ability of a compound (e.g., a 44589 substrate) to interact with 44589 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 44589 without the labeling of either the compound or the 44589. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 44589.

[2067] In yet another embodiment, a cell-free assay is provided in which a 44589 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 44589 protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the 44589 proteins to be used in assays of the present invention include fragments which participate in interactions with non-44589 molecules, e.g., fragments with high surface probability scores.

[2068] Soluble and/or membrane-bound forms of isolated proteins (e.g., 44589 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[2069] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[2070] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[2071] In another embodiment, determining the ability of the 44589 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[2072] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[2073] It may be desirable to immobilize either 44589, an anti-44589 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 44589 protein, or interaction of a 44589 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/44589 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigmna Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 44589 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 44589 binding or activity determined using standard techniques.

[2074] Other techniques for immobilizing either a 44589 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 44589 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[2075] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[2076] In one embodiment, this assay is performed utilizing antibodies reactive with 44589 protein or target molecules but which do not interfere with binding of the 44589 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 44589 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 44589 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 44589 protein or target molecule.

[2077] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[2078] In a preferred embodiment, the assay includes contacting the 44589 protein or biologically active portion thereof with a known compound which binds 44589 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 44589 protein, wherein determining the ability of the test compound to interact with a 44589 protein includes determining the ability of the test compound to preferentially bind to 44589 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[2079] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 44589 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a 44589 protein through modulation of the activity of a downstream effector of a 44589 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[2080] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[2081] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[2082] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[2083] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[2084] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[2085] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[2086] In yet another aspect, the 44589 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 44589 (“44589-binding proteins” or “44589-bp”) and are involved in 44589 activity. Such 44589-bps can be activators or inhibitors of signals by the 44589 proteins or 44589 targets as, for example, downstream elements of a 44589-mediated signaling pathway.

[2087] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 44589 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 44589 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 44589-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 44589 protein.

[2088] In another embodiment, modulators of 44589 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 44589 mRNA or protein evaluated relative to the level of expression of 44589 mRNA or protein in the absence of the candidate compound. When expression of 44589 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 44589 mRNA or protein expression. Alternatively, when expression of 44589 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 44589 mRNA or protein expression. The level of 44589 mRNA or protein expression can be determined by methods described herein for detecting 44589 mRNA or protein.

[2089] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a 44589 protein can be confirmed in vivo, e.g., in an animal such as an animal model for cancer.

[2090] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 44589 modulating agent, an antisense 44589 nucleic acid molecule, a 44589-specific antibody, or a 44589-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[2091] 44589 Detection Assays

[2092] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 44589 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[2093] 44589 Chromosome Mapping

[2094] The 44589 nucleotide sequences or portions thereof can be used to map the location of the 44589 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 44589 sequences with genes associated with disease.

[2095] Briefly, 44589 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 44589 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 44589 sequences will yield an amplified fragment.

[2096] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[2097] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 44589 to a chromosomal location.

[2098] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[2099] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[2100] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[2101] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 44589 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the, chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[2102] 44589 Tissue Typing

[2103] 44589 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[2104] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 44589 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[2105] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 33 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 35 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[2106] If a panel of reagents from 44589 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[2107] Use of Partial 44589 Sequences in Forensic Biology

[2108] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[2109] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 33 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 33 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[2110] The 44589 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 44589 probes can be used to identify tissue by species and/or by organ type.

[2111] In a similar fashion, these reagents, e.g., 44589 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[2112] Predictive Medicine of 44589

[2113] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[2114] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 44589.

[2115] Such disorders include, e.g., a disorder associated with the misexpression of 44589 gene, e.g., cancer; a disorder of the hepatic system.

[2116] The method includes one or more of the following:

[2117] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 44589 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[2118] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 44589 gene;

[2119] detecting, in a tissue of the subject, the misexpression of the 44589 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[2120] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 44589 polypeptide.

[2121] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 44589 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[2122] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 33, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 44589 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[2123] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 44589 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 44589.

[2124] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[2125] In preferred embodiments the method includes determining the structure of a 44589 gene, an abnormal structure being indicative of risk for the disorder.

[2126] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 44589 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[2127] Diagnostic and Prognostic Assays of 44589

[2128] Diagnostic and prognostic assays of the invention include method for assessing the expression level of 44589 molecules and for identifying variations and mutations in the sequence of 44589 molecules.

[2129] Expression Monitoring and Profiling:

[2130] The presence, level, or absence of 44589 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 44589 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 44589 protein such that the presence of 44589 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 44589 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 44589 genes; measuring the amount of protein encoded by the 44589 genes; or measuring the activity of the protein encoded by the 44589 genes.

[2131] The level of mRNA corresponding to the 44589 gene in a cell can be determined both by in situ and by in vitro formats.

[2132] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 44589 nucleic acid, such as the nucleic acid of SEQ ID NO: 33, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100; 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 44589 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[2133] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 44589 genes.

[2134] The level of mRNA in a sample that is encoded by one of 44589 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[2135] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 44589 gene being analyzed.

[2136] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 44589 mRNA, or genomic DNA, and comparing the presence of 44589 mRNA or genomic DNA in the control sample with the presence of 44589 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 44589 transcript levels.

[2137] A variety of methods can be used to determine the level of protein encoded by 44589. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[2138] The detection methods can be used to detect 44589 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 44589 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 44589 protein include introducing into a subject a labeled anti-44589 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-44589 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[2139] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 44589 protein, and comparing the presence of 44589 protein in the control sample with the presence of 44589 protein in the test sample.

[2140] The invention also includes kits for detecting the presence of 44589 in a biological sample. For example, the kit can include a compound or agent capable of detecting 44589 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 44589 protein or nucleic acid.

[2141] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[2142] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[2143] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 44589 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as cancer or deregulated cell proliferation.

[2144] In one embodiment, a disease or disorder associated with aberrant or unwanted 44589 expression or activity is identified. A test sample is obtained from a subject and 44589 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 44589 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 44589 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[2145] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 44589 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a cell proliferation related disorder, e.g., cancer.

[2146] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 44589 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 44589 (e.g., other genes associated with a 44589-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[2147] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 44589 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose a cell proliferation related disorder, e.g., cancer, in a subject wherein an increase in 44589 expression is an indication that the subject has or is disposed to having a cell proliferation related disorder, e.g., cancer. Increased expression of 44589 can also be used as an indicator of drug resistance, e.g., resistance of a cancer cell to chemotherapeutic agents, in an individual diagnosed as having cancer. The method can be used to monitor a treatment for cell proliferation related disorder, e.g., cancer in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[2148] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 44589 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[2149] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 44589 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[2150] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[2151] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 44589 expression.

[2152] 44589 Arrays and uses thereof

[2153] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a 44589 molecule (e.g., a 44589 nucleic acid or a 44589 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm2 , and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[2154] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a 44589 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 44589. Each address of the subset can include a capture probe that hybridizes to a different region of a 44589 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for a 44589 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 44589 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 44589 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[2155] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[2156] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a 44589 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 44589 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-44589 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[2157] In another aspect, the invention features a method of analyzing the expression of 44589. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 44589-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[2158] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 44589. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 44589. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[2159] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 44589 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[2160] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[2161] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 44589-associated disease or disorder; and processes, such as a cellular transformation associated with a 44589-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 44589-associated disease or disorder

[2162] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 44589) that could serve as a molecular target for diagnosis or therapeutic intervention.

[2163] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon a 44589 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99% identical to a 44589 polypeptide or fragment thereof. For example, multiple variants of a 44589 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[2164] The polypeptide array can be used to detect a 44589 binding compound, e.g., an antibody in a sample from a subject with specificity for a 44589 polypeptide or the presence of a 44589-binding protein or ligand.

[2165] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 44589 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[2166] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 44589 or from a cell or subject in which a 44589 mediated response has been elicited, e.g., by contact of the cell with 44589 nucleic acid or protein, or administration to the cell or subject 44589 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 44589 (or does not express as highly as in the case of the 44589 positive plurality of capture probes) or from a cell or subject which in which a 44589 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 44589 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[2167] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 44589 or from a cell or subject in which a 44589-mediated response has been elicited, e.g., by contact of the cell with 44589 nucleic acid or protein, or administration to the cell or subject 44589 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 44589 (or does not express as highly as in the case of the 44589 positive plurality of capture probes) or from a cell or subject which in which a 44589 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[2168] In another aspect, the invention features a method of analyzing 44589, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 44589 nucleic acid or amino acid sequence; comparing the 44589 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 44589.

[2169] Detection of 44589 Variations or Mutations

[2170] The methods of the invention can also be used to detect genetic alterations in a 44589 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 44589 protein activity or nucleic acid expression, such as a cell proliferation related disorder, e.g., cancer. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 44589-protein, or the mis-expression of the 44589 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 44589 gene; 2) an addition of one or more nucleotides to a 44589 gene; 3) a substitution of one or more nucleotides of a 44589 gene, 4) a chromosomal rearrangement of a 44589 gene; 5) an alteration in the level of a messenger RNA transcript of a 44589 gene, 6) aberrant modification of a 44589 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a 44589 gene, 8) a non-wild type level of a 44589-protein, 9) allelic loss of a 44589 gene, and 10) inappropriate post-translational modification of a 44589-protein.

[2171] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 44589-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 44589 gene under conditions such that hybridization and amplification of the 44589-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[2172] In another embodiment, mutations in a 44589 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[2173] In other embodiments, genetic mutations in 44589 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 44589 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 44589 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 44589 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[2174] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 44589 gene and detect mutations by comparing the sequence of the sample 44589 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[2175] Other methods for detecting mutations in the 44589 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[2176] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 44589 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[2177] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 44589 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 44589 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[2178] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[2179] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[2180] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[2181] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 44589 nucleic acid.

[2182] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 33 or the complement of SEQ ID NO: 33. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[2183] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 44589. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[2184] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the Tm of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[2185] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 44589 nucleic acid.

[2186] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 44589 gene.

[2187] Use of 44589 Molecules as Surrogate Markers

[2188] The 44589 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 44589 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 44589 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[2189] The 44589 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 44589 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-44589 antibodies may be employed in an immune-based detection system for a 44589 protein marker, or 44589-specific radiolabeled probes may be used to detect a 44589 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[2190] The 44589 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 44589 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 44589 DNA may correlate 44589 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[2191] Pharmaceutical Compositions of 44589

[2192] The nucleic acid and polypeptides, fragments thereof, as well as anti-44589 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[2193] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[2194] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[2195] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[2196] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[2197] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[2198] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[2199] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[2200] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[2201] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[2202] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[2203] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[2204] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[2205] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[2206] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[2207] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[2208] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactive ions include, but are not limited to iodine, yttrium and praseodymium.

[2209] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[2210] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[2211] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[2212] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[2213] Methods of Treatment for 44589

[2214] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 44589 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[2215] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 44589 molecules of the present invention or 44589 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[2216] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 44589 expression or activity, by administering to the subject a 44589 or an agent which modulates 44589 expression or at least one 44589 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 44589 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 44589 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 44589 aberrance, for example, a 44589, 44589 agonist or 44589 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[2217] It is possible that some 44589 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[2218] The 44589 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of disorders associated with bone metabolism, immune disorders, cardiovascular disorders, viral diseases, pain or metabolic disorders.

[2219] Aberrant expression and/or activity of 44589 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 44589 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 44589 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 44589 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[2220] The 44589 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders. Examples of immune disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

[2221] Examples of disorders involving the heart or “cardiovascular disorder” include, but are not limited to, a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, congestive heart failure, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

[2222] Additionally, 44589 molecules may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 44589 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 44589 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

[2223] Additionally, 44589 may play an important role in the regulation of metabolism or pain disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[2224] As discussed, successful treatment of 44589 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 44589 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[2225] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[2226] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[2227] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 44589 expression is through the use of aptamer molecules specific for 44589 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 44589 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[2228] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 44589 disorders. For a description of antibodies, see the Antibody section above.

[2229] In circumstances wherein injection of an animal or a human subject with a 44589 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 44589 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 44589 protein. Vaccines directed to a disease characterized by 44589 expression may also be generated in this fashion.

[2230] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[2231] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 44589 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[2232] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[2233] Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 44589 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 44589 can be readily monitored and used in calculations of IC50.

[2234] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[2235] Another aspect of the invention pertains to methods of modulating 44589 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 44589 or agent that modulates one or more of the activities of 44589 protein activity associated with the cell. An agent that modulates 44589 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a 44589 protein (e.g., a 44589 substrate or receptor), a 44589 antibody, a 44589 agonist or antagonist, a peptidomimetic of a 44589 agonist or antagonist, or other small molecule.

[2236] In one embodiment, the agent stimulates one or 44589 activities. Examples of such stimulatory agents include active 44589 protein and a nucleic acid molecule encoding 44589. In another embodiment, the agent inhibits one or more 44589 activities. Examples of such inhibitory agents include antisense 44589 nucleic acid molecules, anti-44589 antibodies, and 44589 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 44589 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 44589 expression or activity. In another embodiment, the method involves administering a 44589 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 44589 expression or activity.

[2237] Stimulation of 44589 activity is desirable in situations in which 44589 is abnormally downregulated and/or in which increased 44589 activity is likely to have a beneficial effect. For example, stimulation of 44589 activity is desirable in situations in which a 44589 is downregulated and/or in which increased 44589 activity is likely to have a beneficial effect. Likewise, inhibition of 44589 activity is desirable in situations in which 44589 is abnormally upregulated and/or in which decreased 44589 activity is likely to have a beneficial effect.

[2238] 44589 Pharmacogenomics

[2239] The 44589 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 44589 activity (e.g., 44589 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 44589 associated disorders, e.g., a cell proliferation related disorder (e.g., cancer). In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 44589 molecule or 44589 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 44589 molecule or 44589 modulator.

[2240] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[2241] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[2242] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 44589 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[2243] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 44589 molecule or 44589 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[2244] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 44589 molecule or 44589 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[2245] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 44589 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 44589 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[2246] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a 44589 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 44589 gene expression, protein levels, or upregulate 44589 activity, can be monitored in clinical trials of subjects exhibiting decreased 44589 gene expression, protein levels, or downregulated 44589 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 44589 gene expression, protein levels, or downregulate 44589 activity, can be monitored in clinical trials of subjects exhibiting increased 44589 gene expression, protein levels, or upregulated 44589 activity. In such clinical trials, the expression or activity of a 44589 gene, and preferably, other genes that have been implicated in, for example, a 44589-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[2247] 44589 Informatics

[2248] The sequence of a 44589 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 44589. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 44589 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[2249] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[2250] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[2251] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[2252] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[2253] Thus, in one aspect, the invention features a method of analyzing 44589, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 44589 nucleic acid or amino acid sequence; comparing the 44589 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 44589. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[2254] The method can include evaluating the sequence identity between a 44589 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[2255] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[2256] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[2257] Thus, the invention features a method of making a computer readable record of a sequence of a 44589 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[2258] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 44589 sequence, or record, in machine-readable form; comparing a second sequence to the 44589 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 44589 sequence includes a sequence being compared. In a preferred embodiment the 44589 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 44589 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[2259] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 44589-associated disease or disorder or a pre-disposition to a 44589-associated disease or disorder, wherein the method comprises the steps of determining 44589 sequence information associated with the subject and based on the 44589 sequence information, determining whether the subject has a 44589-associated disease or disorder or a pre-disposition to a 44589-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[2260] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 44589-associated disease or disorder or a pre-disposition to a disease associated with a 44589 wherein the method comprises the steps of determining 44589 sequence information associated with the subject, and based on the 44589 sequence information, determining whether the subject has a 44589-associated disease or disorder or a pre-disposition to a 44589-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 44589 sequence of the subject to the 44589 sequences in the database to thereby determine whether the subject as a 44589-associated disease or disorder, or a pre-disposition for such.

[2261] The present invention also provides in a network, a method for determining whether a subject has a 44589 associated disease or disorder or a pre-disposition to a 44589-associated disease or disorder associated with 44589, said method comprising the steps of receiving 44589 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 44589 and/or corresponding to a 44589-associated disease or disorder (e.g., a cell proliferation related disorder, e.g., cancer), and based on one or more of the phenotypic information, the 44589 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 44589-associated disease or disorder or a pre-disposition to a 44589-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[2262] The present invention also provides a method for determining whether a subject has a 44589-associated disease or disorder or a pre-disposition to a 44589-associated disease or disorder, said method comprising the steps of receiving information related to 44589 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 44589 and/or related to a 44589-associated disease or disorder, and based on one or more of the phenotypic information, the 44589 information, and the acquired information, determining whether the subject has a 44589-associated disease or disorder or a pre-disposition to a 44589-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[2263] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

[2264] Background of the 84226 Invention

[2265] The concentration of metallic ions such as cadmium, zinc, and cobalt is maintained within a narrow range in mammalian cells. Members of cation transporter protein family are integral membrane proteins, which are found to increase tolerance to cation ions such as cadmium, zinc, or cobalt. A diverse family of cation transporter proteins, the “cation diffusion facilitator” family, contributes to the maintenance of cellular metallic ion homeostasis (Paulsen et al. (1997) J. Membr. Biol. 156: 99-103).

[2266] Zinc transporter proteins are one exemplary subclass of this family of cation transporter proteins. Zinc is an essential component of many metalloenzymes, transcription factors, and other proteins, but can be toxic to mammalian cells at high concentrations. Various homeostatic mechanisms are thought to be used by cells to regulate intracellular zinc: regulation of zinc influx across the plasma membrane; regulation of zinc efflux across the plasma membrane; sequestration of zinc within subcellular compartments; and synthesis of molecules, e.g., metallothioneins, that bind tightly to zinc (Palmiter et al. (1996) EMBO J. 15: 1784-1791; Palmiter et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14934-14939).

[2267] The genes encoding several zinc transporters have been cloned. Each of the proteins encoded by these genes appears to contribute to cellular resistance to zinc toxicity. Zinc transporter-1 (ZnT-1) encodes a plasma membrane protein that stimulates zinc efflux. ZnT-1 appears to be activated by excess cellular zinc concentrations (Palmiter et al. (1995) EMBO J. 14: 639-649). Zinc transporter-2 (ZnT-2) encodes a vesicular protein that promotes the vesicular sequestration of zinc. Thus, ZnT-2 appears to help protect cells from zinc toxicity by facilitating zinc transport into an endosomal/lysosomal compartment (Palmiter et al. (1996) EMBO J. 15: 1784-1791). Zinc transporter-3 (ZnT-3) encodes a putative transporter of zinc into synaptic vesicles. ZnT-3, which is expressed in the brain and testis, is proposed to be a component of the complex that sequesters zinc in synaptic vesicles, thereby serving as a neuromodulator (Palmiter et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14934-14939). ZnT-1, ZnT-2, and ZnT-3 share a common topology characterized by six membrane-spanning domains, a histidine-rich cytoplasmic loop between membrane spanning regions four and five, and a long C-terminal tail.

[2268] Summary of the 84226 Invention

[2269] The present invention is based, in part, on the discovery of a novel cation transporter family member, referred to herein as “84226”. The nucleotide sequence of a cDNA encoding 84226 is shown in SEQ ID NO: 39, and the amino acid sequence of an 84226 polypeptide is shown in SEQ ID NO: 40. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 41.

[2270] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes an 84226 protein or polypeptide, e.g., a biologically active portion of the 84226 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 40. In other embodiments, the invention provides isolated 84226 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 39, SEQ ID NO: 41, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 39, SEQ ID NO: 41, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 39, SEQ ID NO: 41, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 84226 protein or an active fragment thereof.

[2271] In a related aspect, the invention further provides nucleic acid constructs that include an 84226 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 84226 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 84226 nucleic acid molecules and polypeptides.

[2272] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 84226-encoding nucleic acids.

[2273] In still another related aspect, isolated nucleic acid molecules that are antisense to an 84226 encoding nucleic acid molecule are provided.

[2274] In another aspect, the invention features, 84226 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 84226-mediated or -related disorders. In another embodiment, the invention provides 84226 polypeptides having an 84226 activity. Preferred polypeptides are 84226 proteins including at least one cation efflux domain, or a transmembrane domain, and, preferably, having an 84226 activity, e.g., an 84226 activity as described herein.

[2275] In other embodiments, the invention provides 84226 polypeptides, e.g., an 84226 polypeptide having the amino acid sequence shown in SEQ ID NO: 40 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 40 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number ______; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 39, SEQ ID NO: 41, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number ______, wherein the nucleic acid encodes a full length 84226 protein or an active fragment thereof.

[2276] In a related aspect, the invention further provides nucleic acid constructs which include an 84226 nucleic acid molecule described herein.

[2277] In a related aspect, the invention provides 84226 polypeptides or fragments operatively linked to non-84226 polypeptides to form fusion proteins.

[2278] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 84226 polypeptides or fragments thereof, e.g., a cation efflux domain.

[2279] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 84226 polypeptides or nucleic acids.

[2280] In still another aspect, the invention provides a process for modulating 84226 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 84226 polypeptides or nucleic acids, such as conditions involving metal transport-related disorders, e.g., disorders associated with cellular toxicity resulting from aberrant or deficient cation diffusion, pancreatic disorders, e.g., pancreatic cancer, metabolic disorder, and aberrant or deficient cellular proliferation or differentiation.

[2281] The invention also provides assays for determining the activity of or the presence or absence of 84226 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[2282] In yet another aspect, the invention provides methods for inhibiting the proliferation or inducing the killing, of an 84226-expressing cell, e.g., a hyper-proliferative 84226-expressing cell. The method includes contacting the cell with a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 84226 polypeptide or nucleic acid. In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol. In a preferred embodiment, the cell is a pancreas cell.

[2283] In a preferred embodiment, the compound is an inhibitor of an 84226 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety selected from a cytotoxin, a cytotoxic agent and a radioactive metal ion). In another preferred embodiment, the compound is an inhibitor of an 84226 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.

[2284] In a preferred embodiment, the compound is administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation.

[2285] In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant cellular proliferation or differentiation of an 84226-expressing cell, in a subject. Preferably, the method includes administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 84226 polypeptide or nucleic acid. In a preferred embodiment, the disorder is a cancerous or pre-cancerous condition, e.g., pancreatic cancer. The disorder is a metal transport-related disorder, e.g., a disorder associated with cellular toxicity resulting from aberrant or deficient cation diffusion, or a metabolic disorder.

[2286] In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., proliferative disorder such as pancreatic cancer or a metal transport-related disorder such as a disorder associated with cellular toxicity resulting from aberrant or deficient cation diffusion disorder or a metabolic disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with one or more of: chemotherapy, radiation, and/or a compound identified using the methods described herein); and evaluating the expression of an 84226 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of an 84226 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 84226 nucleic acid or polypeptide expression can be detected by any method described herein.

[2287] In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of an 84226 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.

[2288] In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent or an anti-pancreatic cancer agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein, a cytotoxic agent) and, evaluating the expression of 84226 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 84226 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 84226 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a cancerous tissue or a pancreas tissue.

[2289] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in an 84226 polypeptide or nucleic acid molecule, including for disease diagnosis.

[2290] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes an 84226 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to an 84226 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 84226 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[2291] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[2292] Detailed Description of 84226

[2293] The human 84226 sequence (see SEQ ID NO: 39, as recited in Example 26), which is approximately 1630 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1119 nucleotides, including the termination codon. The coding sequence encodes a 372 amino acid protein (see SEQ ID NO: 40, as recited in Example 26).

[2294] Human 84226 contains the following regions or other structural features:

[2295] one predicted cation efflux domain (PFAM Accession Number PF01545) located at about amino acid residues 74 to 361 of SEQ ID NO: 40;

[2296] six predicted transmembrane domains, located at about amino acids 74 to 95, 107 to 123, 141 to 163, 178 to 196, 219 to 243, and 253 to 277 of SEQ ID NO: 40;

[2297] four predicted cytoplasmic domains, located at about amino acids 1 (amino terminus) to 73, 124 to 140, 197 to 218, and 278 to 373 (carboxy terminus) of SEQ ID NO: 40;

[2298] three predicted non-cytoplasmic (e.g., lumenal or extracellular) loops, located at about amino acids 96 to 106, 164 to 177, and 244 to 252 of SEQ ID NO: 40;

[2299] one predicted glycosaminoglycan attachment site (PS00002) located at about amino acids 199 to 202 of SEQ ID NO: 40;

[2300] four predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 124 to 126, 216 to 218, 281 to 283, and 338 to 340 of SEQ ID NO: 40;

[2301] one predicted Casein Kinase II phosphorylation site (PS00006) located at about amino acids 61 to 64 of SEQ ID NO: 40; and

[2302] six predicted N-myristylation sites (PS00008) located at about amino acids 91 to 96, 143 to 148, 183 to 188, 233 to 238, 264 to 269, and 280 to 285 of SEQ ID NO: 40.

[2303] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[2304] A plasmid containing the nucleotide sequence encoding human 84226 (clone “Fbh84226FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[2305] The 84226 protein contains a significant number of structural characteristics in common with members of the cation transporter family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[2306] Members of the cation transporter family of proteins are membrane proteins that increase cellular tolerance to divalent metal ions such as zinc, cadmium, and cobalt by mediating cation diffusion across membranes. Some cation transporter proteins are efflux pumps that remove divalent metal ions from cells. Other cation transporter proteins function to increase cellular tolerance to metal ions by mediating the sequestration of ions in subcellular compartments. Some cation transporter proteins are characterized by a topology of six membrane spanning domains, a histidine-rich loop between the fourth and fifth membrane spanning domains, and a long C-terminal tail. Examples of cation transporter proteins include ZnT-1, ZnT-2, and ZnT-3. ZnT-1, a plasma membrane protein, functions as a zinc transporter, mediating the cellular efflux of zinc. ZnT-2 is located in vesicles within a cell and mediates the vesicular sequestration of zinc. ZnT-3 can participate in the accumulation of zinc in synaptic vesicles. As the 84226 protein has the structural features of cation transporter proteins, it is likely to mediate tolerance to divalent metal ions, e.g., zinc, in the cells in which it is expressed. The 84226 protein, like other members of the cation transporter protein family, is a transmembrane protein that can include six membrane spanning domains, a histidine-rich loop between the fourth and fifth membrane spanning domains, and a long C-terminal tail.

[2307] An 84226 polypeptide can include at least one “cation efflux domain” or regions homologous with a “cation efflux domain.”

[2308] As used herein, the term “cation efflux domain” includes an amino acid sequence of about 100 to 500 amino acid residues in length and having a bit score for the alignment of the sequence to the cation transporter domain (PFAM Accession Number PF01545) of at least 150. Preferably, a cation efflux domain includes at least about 200 to 400 amino acids, more preferably about 250 to 300 amino acid residues, and has a bit score for the alignment of the sequence to the cation transporter domain (PFAM Accession Number PF01545) of at least 200, 250, 300, 320 or greater. The cation transporter domain (HMM) has been assigned the PFAM Accession Number PF01545 (http://genome.wustl.edu/Pfam/.html). An alignment of the cation transporter domain (amino acids 74 to 361 of SEQ ID NO: 40) of human 84226 with a consensus amino acid sequence (SEQ ID NO: 42) derived from a hidden Markov model is depicted in FIG. 20.

[2309] In a preferred embodiment 84226 polypeptide or protein has a “cation efflux domain” or a region which includes at least about 100 to 500 more preferably about 200 to 400, or 250 to 300 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “cation efflux domain,” e.g., the cation transporter protein domain of human 84226 (e.g., residues 74 to 361 of SEQ ID NO: 40).

[2310] To identify the presence of a “cation transporter” domain in an 84226 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3): 405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183: 146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84: 4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2: 305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a “cation transporter” domain in the amino acid sequence of human 84226 at about residues 74 to 361 of SEQ ID NO: 40 (see FIG. 20).

[2311] An 84226 polypeptide can include a “transmembrane domain” or regions homologous with a “transmembrane domain.”

[2312] As used herein, the term “transmembrane domain” includes an amino acid sequence of at least about 15 amino acid residues in length which spans a phospholipid bilayer. More preferably, a transmembrane domain includes about at least 10, 15, or 20 amino acid residues and spans a phospholipid bilayer. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucine, isoleucine, valine, alanine, glycine, tyrosine, phenylalanine, or tryptophan. Transmembrane domains are described in, for example, Zagotta W. N. et al. (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. An 84226 protein has at least one, preferably two, three, four, five, most preferably six transmembrane domains. Amino acid residues at about 74 to 95, 107 to 123, 141 to 163, 178 to 196, 219 to 243, and 253 to 277 of the 84226 protein (SEQ ID NO: 40) are predicted to comprise six transmembrane domains. Accordingly, 84226 proteins having at least 50% to 60% homology, preferably about 60% to 70%, more preferably about 70% to 80%, or about 80% to 90% homology with a transmembrane domain of human 84226 are within the scope of the invention.

[2313] In one embodiment, an 84226 protein includes at least one cytoplasmic domain. When located at the N-terminal domain the cytoplasmic domain is referred to herein as an “N-terminal cytoplasmic domain.” As used herein, a “N-terminal cytoplasmic domain” includes an amino acid sequence having about 1 to 300, preferably about 1 to 250, 1 to 200, more preferably about 1 to 150, 1 to 100, or even more preferably about 1 to 80 amino acid residues in length and is located inside of a cell or intracellularly. The C-terminal amino acid residue of a “N-terminal cytoplasmic domain” is adjacent to a N-terminal amino acid residue of a transmembrane domain in an 84226 protein. For example, a N-terminal cytoplasmic domain is located at about amino acid residues 1 to 73 of SEQ ID NO: 40.

[2314] In a preferred embodiment, an 84226 polypeptide or protein has at least one cytoplasmic domain or a region which includes at least about 5, preferably about 10 to 200, and more preferably about 15 to 110 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “cytoplasmic domain,” e.g., at least one cytoplasmic domain of human 84226 protein (e.g., residues 1 to 73, 124 to 140, 197 to 218, and 278 to 373 of SEQ ID NO: 40).

[2315] In another embodiment, an 84226 protein includes at least one non-cytoplasmic loop. As used herein, the term “loop” includes an amino acid sequence that resides outside of a phospholipid membrane, having a length of at least about 4, preferably about 5-80, and more preferably about 5 to 50 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Non-cytoplasmic loops include extracellular domains (i.e., outside of the cell) and intracellular domains (i.e., within the cell). When referring to membrane-bound proteins found in intracellular organelles (e.g., mitochondria, endoplasmic reticulum, peroxisomes microsomes, vesicles, endosomes, and lysosomes), non-cytoplasmic loops include those domains of the protein that reside in the lumen of the organelle or the matrix or the intermembrane space. Accordingly, the N-terminal amino acid of a non-cytoplasmic loop is adjacent to a C-terminal amino acid of a transmembrane domain in an 84226 protein, and the C-terminal amino acid of a non-cytoplasmic loop is adjacent to an N-terminal amino acid of a transmembrane domain in an 84226 protein. As used herein, a “non-cytoplasmic loop” includes an amino acid sequence located outside of a cell or within an intracellular organelle. For example, a “non-cytoplasmic loop” can be found at about amino acids 96 to 106, 164 to 177, and 244 to 252 of SEQ ID NO: 40.

[2316] In a preferred embodiment, an 84226 polypeptide or protein has at least one non-cytoplasmic loop or a region which includes at least about 4, preferably about 5-10, and more preferably about 5-20 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “non-cytoplasmic loop,” e.g., at least one non-cytoplasmic loop of human 84226 (e.g., residues 96 to 106, 164 to 177, and 244 to 252 of SEQ ID NO: 40).

[2317] In another embodiment, an 84226 protein includes a “C-terminal cytoplasmic domain,” also referred to herein as a C-terminal cytoplasmic tail, in the sequence of the protein. As used herein, a “C-terminal cytoplasmic domain” includes an amino acid sequence having a length of at least about 30, preferably about 50 to 300, preferably about 60 to 200, more preferably about 80 to 130 amino acid residues and is located within a cell or within the cytoplasm of a cell. Accordingly, the N-terminal amino acid residue of a “C-terminal cytoplasmic domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in an 84226 protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 278 to 373 of SEQ ID NO: 40.

[2318] Histidine residues in cation transporter proteins play important roles in binding to divalent metal ions such as zinc. Histidine residues located in the cytoplasmic domain between the fourth and fifth transmembrane domains, e.g., at about amino acids 197 to 218 of SEQ ID NO: 40, as well as those located in the C-terminal cytoplasmic domain, e.g., at about amino acids 278 to 373 of SEQ ID NO: 40 can be of particular importance.

[2319] An 84226 protein can have four histidine residues in the cytoplasmic domain between the fourth and fifth transmembrane domain at about amino acids 197, 201, 203, and 205. An 84226 protein can also have five histidine residues in the C-terminal cytoplasmic domain at about amino acids 304, 307, 321, 346, and 348. A preferred 84226 polypeptide has at least one, preferably two, three, or four histidine residues between the fourth and fifth transmembrane domains, and has at least one, preferably two, three, four, or five histidine residues in a C-terminal cytoplasmic domain.

[2320] An 84226 polypeptide can optionally include at least one glycosaminoglycan attachment site (PS00002); at least one, two, three, or preferably four protein kinase C phosphorylation sites (PS00005); at least one casein kinase II phosphorylation site (PS00006); and at least one, two, three, four, five, or preferably six N-myristoylation sites (PS00008).

[2321] As the 84226 polypeptides of the invention may modulate 84226-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 84226-mediated or related disorders, as described below.

[2322] As used herein, an “84226 activity,” “biological activity of 84226” or “functional activity of 84226,” refers to an activity exerted by an 84226 protein, polypeptide or nucleic acid molecule. For example, an 84226 activity can be an activity exerted by 84226 in a physiological milieu on, e.g., an 84226-responsive cell or on an 84226 substrate, e.g., a protein substrate. An 84226 activity can be determined in vivo or in vitro. In one embodiment, an 84226 activity is a direct activity, such as an association with an 84226 target molecule. A “target molecule” or “binding partner” is a molecule with which an 84226 protein binds or interacts in nature.

[2323] An 84226 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 84226 protein with an 84226 receptor. The features of the 84226 molecules of the present invention can provide similar biological activities as cation transporter family members. For example, the 84226 proteins of the present invention can have one or more of the following activities: (1) modulating cellular tolerance and/or resistance to a metal ion, e.g., zinc; (2) facilitating cation diffusion; (3) modulating cellular efflux of a metal ion, e.g., zinc; (4) modulating vesicular sequestration of a metal ion, e.g., zinc; (5) modulating sequestration of a metal ion, e.g., zinc, in synaptic vesicles; (6) binding to a metal ion, e.g., zinc; (7) modulating (e.g., stimulating) cell differentiation, e.g., differentiation of pancreatic cells; (8) modulating cell proliferation, e.g., proliferation of pancreatic cells; or (9) modulating (increasing or decreasing) apoptosis, e.g., apoptosis of a cancer cell, e.g., a pancreatic cancer cell.

[2324] Based upon the above-described sequence similarities and the detetced expression patterns of 84226 described in Table 8 of Example 27 (e.g., pancreas cells), the 84226 molecules of the present invention are predicted to have similar biological activities as cation transporter family members. Thus, the 84226 molecule can act as novel diagnostic targets and therapeutic agents for controlling metal transport-related disorders, e.g., disorders associated with cellular toxicity resulting from aberrant or deficient cation diffusion. Furthermore, an 84226 molecule can be used for metal detoxification, e.g., to treat cells or individuals containing excessive or unwanted amounts of metal ions.

[2325] Additionally, 84226 mRNA is highly expressed in human pancreas, and slightly expressed in human heart, kidney, skeletal muscle, and small intestine (Table 8 of Examiner 2). Thus, the 84226 molecule can act as novel diagnostic targets and therapeutic agents for pancreatic disorders, and metabolic disorders.

[2326] Examples of pancreatic disorders include, but are not limited to, pancreatitis (an inflammation of the pancreas), hypoglycemia (over utilization of glucose) resulting from hyperinsulinism, and pancreatic cancer. Hyperinsulinism can be due to an insulinoma, which include single solid tumors, microadenomatosis and islet cell hyperplasia (nesidioblastosis). In addition, inherited pancreatic disorders include cystic fibrosis, Shwachman diamond syndrome, Johansson Blizzard syndrome, Pearson's bone marrow syndrome and hereditary pancreatitis.

[2327] 84226 may also play an important role in the regulation of metabolism or pain disorders, e.g., disorders associated with cellular toxicity resulting from aberrant or deficient cation diffusion. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain. An “angiogenic disorder” refers to a disorder characterized by aberrant, unregulated, or unwanted vascularization. Angiogenic disorders include, but are not limited to, hemangiomas, Kaposi's sarcoma, von Hippel-Lindau disease; psoriasis; diabetic retinopathy; endometriosis; Grave's disease; chronic inflammatory diseases (e.g., rheumatoid arthritis); aberrant or excess angiogenesis in diseases such as a Castleman's disease or fibrodysplasia ossificans progressiva; aberrant or deficient angiogenesis associated with aging, complications of healing certain wounds and complications of diseases such as diabetes and rheumatoid arthritis; or aberrant or deficient angiogenesis associated with hereditary hemorrhagic telangiectasia, autosomal dominant polycystic kidney disease, myelodysplastic syndrome or Klippel-Trenaunay-Weber syndrome.

[2328] In addition to the diseases described above, the 84226 molecules can act as novel diagnostic targets and therapeutic agents for controlling disorders associated with heart or kidney disorders.

[2329] Examples of disorders involving the heart or “cardiovascular disorder” include, but are not limited to, a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, congestive heart failure, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

[2330] Examples of disorders involving the kidney include, but are not limited to, amyloidosis, e.g., primary amyloidosis or dialysis-related amyloidosis, analgesic nephropathy, anemia in kidney, childhood nephrotic syndrome, cystoscopy and ureteroscopy, diabetes insipidus, hemodialysis, glomerular diseases including Glomerulonephritis and Glomerulosclerosis, goodpasture syndrome, hemolytic uremic syndrome, IgA nephropathy, polycystic kidney disease, proteinuria, renal tubular acidosis, renal osteodystrophy, and vesicoureteral reflux.

[2331] The 84226 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 40 thereof are collectively referred to as “polypeptides or proteins of the invention” or “84226 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “84226 nucleic acids.” 84226 molecules refer to 84226 nucleic acids, polypeptides, and antibodies.

[2332] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[2333] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[2334] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[2335] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 39 or SEQ ID NO: 41, corresponds to a naturally-occurring nucleic acid molecule.

[2336] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein.

[2337] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include at least an open reading frame encoding an 84226 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 84226 protein or derivative thereof.

[2338] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 84226 protein is at least 10% pure. In a preferred embodiment, the preparation of 84226 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-84226 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-84226 chemicals. When the 84226 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[2339] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 84226 without abolishing or substantially altering an 84226 activity. Preferably the alteration does not substantially alter the 84226 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 84226, results in abolishing an 84226 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 84226 are predicted to be particularly unamenable to alteration.

[2340] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an 84226 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an 84226 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 84226 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 39 or SEQ ID NO: 41, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[2341] As used herein, a “biologically active portion” of an 84226 protein includes a fragment of an 84226 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between an 84226 molecule and a non-84226 molecule or between a first 84226 molecule and a second 84226 molecule (e.g., a dimerization interaction). Biologically active portions of an 84226 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 84226 protein, e.g., the amino acid sequence shown in SEQ ID NO: 40, which include less amino acids than the full length 84226 proteins, and exhibit at least one activity of an 84226 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 84226 protein, e.g., (1) modulating cellular tolerance and/or resistance to a metal ion, e.g., zinc; (2) facilitating cation diffusion; (3) modulating cellular efflux of a metal ion, e.g., zinc; (4) modulating vesicular sequestration of a metal ion, e.g., zinc; (5) modulating sequestration of a metal ion, e.g., zinc, in synaptic vesicles; (6) binding to a metal ion, e.g., zinc; (7) modulating (e.g., stimulating) cell differentiation, e.g., differentiation of pancreatic cells; (8) modulating cell proliferation, e.g., proliferation of pancreatic cells; or (9) modulating (increasing or decreasing) apoptosis, e.g., apoptosis of a cancer cell, e.g., a pancreatic cancer cell. A biologically active portion of an 84226 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of an 84226 protein can be used as targets for developing agents which modulate an 84226 mediated activity, e.g., (1) modulating cellular tolerance and/or resistance to a metal ion, e.g., zinc; (2) facilitating cation diffusion; (3) modulating cellular efflux of a metal ion, e.g., zinc; (4) modulating vesicular sequestration of a metal ion, e.g., zinc; (5) modulating sequestration of a metal ion, e.g., zinc, in synaptic vesicles; (6) binding to a metal ion, e.g., zinc; (7) modulating (e.g., stimulating) cell differentiation, e.g., differentiation of pancreatic cells; (8) modulating cell proliferation, e.g., proliferation of pancreatic cells; or (9) modulating (increasing or decreasing) apoptosis, e.g., apoptosis of a cancer cell, e.g., a pancreatic cancer cell.

[2342] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[2343] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[2344] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[2345] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[2346] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[2347] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 84226 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 84226 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[2348] Particularly preferred 84226 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 40. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 40 are termed substantially identical.

[2349] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 39 or 41 are termed substantially identical. “Misexpression or aberrant expression,” as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[2350] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[2351] A “purified preparation of cells,” as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[2352] Various aspects of the invention are described in further detail below.

[2353] Isolated 84226 Nucleic Acid Molecules

[2354] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes an 84226 polypeptide described herein, e.g., a full-length 84226 protein or a fragment thereof, e.g., a biologically active portion of 84226 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 84226 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[2355] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 39, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 84226 protein (i.e., “the coding region” of SEQ ID NO: 39, as shown in SEQ ID NO: 41), as well as 5′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 39 (e.g., SEQ ID NO: 41) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid 74 to 361.

[2356] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 39 or SEQ ID NO: 41, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 39 or SEQ ID NO: 41, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO: 39 or 41, thereby forming a stable duplex.

[2357] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 39 or SEQ ID NO: 41, or a portion, preferably of the same length, of any of these nucleotide sequences.

[2358] 84226 Nucleic Acid Fragments

[2359] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 39 or 41. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of an 84226 protein, e.g., an immunogenic or biologically active portion of an 84226 protein. A fragment can comprise those nucleotides of SEQ ID NO: 39, which encode a cation efflux domain of human 84226. The nucleotide sequence determined from the cloning of the 84226 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 84226 family members, or fragments thereof, as well as 84226 homologues, or fragments thereof, from other species.

[2360] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[2361] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, an 84226 nucleic acid fragment can include a sequence corresponding to a cation efflux domain.

[2362] 84226 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 39 or SEQ ID NO: 41, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 39 or SEQ ID NO: 41. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.

[2363] In one embodiment, the probe or primer is attached to a solid support, e.g., a solid support described herein.

[2364] One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g., the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 40. The reverse primer can anneal to the ultimate codon, e.g., the codon immediately before the stop codon, e.g., the codon encoding amino acid residue 372 of SEQ ID NO: 40. In a preferred embodiment, the annealing temperatures of the forward and reverse primers differ by no more than 5, 4, 3, or 2° C.

[2365] In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[2366] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a cation efflux domain located at residues of 74 to 361 of SEQ ID NO: 40.

[2367] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of an 84226 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: a cation efflux domain from about amino acid 74 to 361 of SEQ ID NO: 40.

[2368] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[2369] A nucleic acid fragment encoding a “biologically active portion of an 84226 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 39 or 41, which encodes a polypeptide having an 84226 biological activity (e.g., the biological activities of the 84226 proteins are described herein), expressing the encoded portion of the 84226 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 84226 protein. For example, a nucleic acid fragment encoding a biologically active portion of 84226 includes a cation efflux domain, e.g., amino acid residues about 74 to 361 of SEQ ID NO: 40. A nucleic acid fragment encoding a biologically active portion of an 84226 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

[2370] In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 39, or SEQ ID NO: 41.

[2371] In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank™ accession numbers H16506, H07440, H16516, H07460, AK023491, and AK023504. Differ can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 39 or SEQ ID NO: 41 outside the region of nucleotides 74 to 361; not include all of the nucleotides Genbank™ accession numbers H16506, H07440, H16516, H07460, AK023491, and AK023504, e.g., can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank™ accession numbers H16506, H07440, H16516, H07460, AK023491, and AK023504; or can differ by one or more nucleotides in the region of overlap.

[2372] 84226 Nucleic Acid Variants

[2373] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 39 or SEQ ID NO: 41. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 84226 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 40. If alignment is needed for this comparison the sequences should be aligned for maximum homology. The encoded protein can differ by no more than 5, 4, 3, 2, or 1 amino acid. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[2374] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[2375] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[2376] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 39 or 41, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid. The nucleic acid can differ by no more than 5, 4, 3, 2, or I nucleotide. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[2377] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 40 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO: 40 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 84226 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 84226 gene.

[2378] Preferred variants include those that are correlated with (1) modulating cellular tolerance and/or resistance to a metal ion, e.g., zinc; (2) facilitating cation diffusion; (3) modulating cellular efflux of a metal ion, e.g., zinc; (4) modulating vesicular sequestration of a metal ion, e.g., zinc; (5) modulating sequestration of a metal ion, e.g., zinc, in synaptic vesicles; (6) binding to a metal ion, e.g., zinc; (7) modulating (e.g., stimulating) cell differentiation, e.g., differentiation of pancreatic cells; (8) modulating cell proliferation, e.g., proliferation of pancreatic cells; or (9) modulating (increasing or decreasing) apoptosis, e.g., apoptosis of a cancer cell, e.g., a pancreatic cancer cell. Allelic variants of 84226, e.g., human 84226, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 84226 protein within a population that maintain the ability to bind zinc ions. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 40, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 84226, e.g., human 84226, protein within a population that do not have the ability to (1) modulate cellular tolerance and/or resistance to a metal ion, e.g., zinc; (2) facilitate cation diffusion; (3) modulate cellular efflux of a metal ion, e.g., zinc; (4) modulate vesicular sequestration of a metal ion, e.g., zinc; (5) modulate sequestration of a metal ion, e.g., zinc, in synaptic vesicles; (6) bind to a metal ion, e.g., zinc; (7) modulate (e.g., stimulate) cell differentiation, e.g., differentiation of pancreatic cells; (8) modulate cell proliferation, e.g., proliferation of pancreatic cells; or (9) modulate (increase or decrease) apoptosis, e.g., apoptosis of a cancer cell, e.g., a pancreatic cancer cell. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 40, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[2379] Moreover, nucleic acid molecules encoding other 84226 family members and, thus, which have a nucleotide sequence which differs from the 84226 sequences of SEQ ID NO: 39 or SEQ ID NO: 41 are intended to be within the scope of the invention.

[2380] Antisense Nucleic Acid Molecules, Ribozymes and Modified 84226 Nucleic Acid Molecules

[2381] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 84226. An “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 84226 coding strand, or to only a portion thereof (e.g., the coding region of human 84226 corresponding to SEQ ID NO: 41). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 84226 (e.g., the 5′ and 3′ untranslated regions).

[2382] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 84226 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 84226 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 84226 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[2383] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[2384] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an 84226 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[2385] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215: 327-330).

[2386] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 84226-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of an 84226 cDNA disclosed herein (i.e., SEQ ID NO: 39 or SEQ ID NO: 41), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334: 585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 84226-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 84226 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.

[2387] 84226 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 84226 (e.g., the 84226 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 84226 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6: 569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660: 27-36; and Maher, L. J. (1992) Bioassays 14: 807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[2388] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

[2389] An 84226 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulmé (2001) Nature Biotech. 19: 17 and Faria et al. (2001) Nature Biotech. 19: 40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.

[2390] For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[2391] PNAs of 84226 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 84226 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[2392] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6: 958-976) or intercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[2393] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to an 84226 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 84226 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

[2394] Isolated 84226 Polypeptides

[2395] In another aspect, the invention features, an isolated 84226 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-84226 antibodies. 84226 protein can be isolated from cells or tissue sources using standard protein purification techniques. 84226 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[2396] Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[2397] In a preferred embodiment, an 84226 polypeptide has one or more of the following characteristics:

[2398] (i) it has the ability to modulate cellular tolerance and/or resistance to a metal ion, e.g., zinc;

[2399] (ii) it has the ability to facilitate cation diffusion;

[2400] (iii) it has the ability to modulate cellular efflux of a metal ion, e.g., zinc;

[2401] (iv) it has the ability to modulate vesicular sequestration of a metal ion, e.g., zinc;

[2402] (v) it has the ability to modulate sequestration of a metal ion, e.g., zinc, in synaptic vesicles;

[2403] (vi) it has the ability to bind to a metal ion, e.g., zinc;

[2404] (vii) it has the ability to modulate (e.g., stimulate) cell differentiation, e.g., differentiation of pancreatic cells;

[2405] (viii) it has the ability to modulate cell proliferation, e.g., proliferation of pancreatic cells;

[2406] (ix) it has the ability to modulate (increase or decrease) apoptosis, e.g., apoptosis of a cancer cell, e.g., a pancreatic cancer cell;

[2407] (x) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of SEQ ID NO: 40;

[2408] (xi) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a polypeptide a of SEQ ID NO: 40;

[2409] (xii) it has a cation efflux domain which is preferably about 70%, 80%, 90% or 95% with amino acid residues about 74 to 361 of SEQ ID NO: 40; and

[2410] (xiii) it has at least four histidine residues in the cytoplasmic domain and five histidine residues in the C-terminal cytoplasmic.

[2411] In a preferred embodiment the 84226 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID: 2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 40 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 40. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the cation efflux domain at residues 74 to 361 of SEQ ID NO: 40. In another preferred embodiment one or more differences are in the cation efflux domain at residues 74 to 361 of SEQ ID NO: 40.

[2412] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 84226 proteins differ in amino acid sequence from SEQ ID NO: 40, yet retain biological activity.

[2413] In one embodiment, the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 40.

[2414] An 84226 protein or fragment is provided which varies from the sequence of SEQ ID NO: 40 in regions defined by amino acids about 1 to 73 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 40 in regions defined by amino acids about 74 to 361. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[2415] In one embodiment, a biologically active portion of an 84226 protein includes a cation efflux domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 84226 protein.

[2416] In a preferred embodiment, the 84226 protein has an amino acid sequence shown in SEQ ID NO: 40. In other embodiments, the 84226 protein is substantially identical to SEQ ID NO: 40. In yet another embodiment, the 84226 protein is substantially identical to SEQ ID NO: 40 and retains the functional activity of the protein of SEQ ID NO: 40, as described in detail in the subsections above.

[2417] In a preferred embodiment, a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues from a sequence in Genbank™ accession numbers H16506, H07440, H16516, H07460, AK023491, and AK023504. Differ can include differing in length or sequence identity. E.g., a fragment can: include one or more amino acid residues from SEQ ID NO: 40 outside the region encoded by nucleotides 74-361; not include all of the amino acid residues of a sequence in Genbank™ accession numbers H16506, H07440, H16516, H07460, AK023491, and AK023504, e.g., can be one or more amino acid residues shorter (at one or both ends) than a sequence in Genbank™ accession numbers H16506, H07440, H16516, H07460, AK023491, and AK023504; or can differ by one or more amino acid residues in the region of overlap.

[2418] 84226 Chimeric or Fusion Proteins

[2419] In another aspect, the invention provides 84226 chimeric or fusion proteins. As used herein, an 84226 “chimeric protein” or “fusion protein” includes an 84226 polypeptide linked to a non-84226 polypeptide. A “non-84226 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 84226 protein, e.g., a protein which is different from the 84226 protein and which is derived from the same or a different organism. The 84226 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of an 84226 amino acid sequence. In a preferred embodiment, an 84226 fusion protein includes at least one (or two) biologically active portion of an 84226 protein. The non-84226 polypeptide can be fused to the N-terminus or C-terminus of the 84226 polypeptide.

[2420] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-84226 fusion protein in which the 84226 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 84226. Alternatively, the fusion protein can be an 84226 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 84226 can be increased through use of a heterologous signal sequence.

[2421] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[2422] The 84226 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 84226 fusion proteins can be used to affect the bioavailability of an 84226 substrate. 84226 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding an 84226 protein; (ii) mis-regulation of the 84226 gene; and (iii) aberrant post-translational modification of an 84226 protein.

[2423] Moreover, the 84226-fusion proteins of the invention can be used as immunogens to produce anti-84226 antibodies in a subject, to purify 84226 ligands and in screening assays to identify molecules which inhibit the interaction of 84226 with an 84226 substrate.

[2424] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An 84226-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 84226 protein.

[2425] Variants of 84226 Proteins

[2426] In another aspect, the invention also features a variant of an 84226 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 84226 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of an 84226 protein. An agonist of the 84226 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of an 84226 protein. An antagonist of an 84226 protein can inhibit one or more of the activities of the naturally occurring form of the 84226 protein by, for example, competitively modulating an 84226-mediated activity of an 84226 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 84226 protein.

[2427] Variants of an 84226 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an 84226 protein for agonist or antagonist activity.

[2428] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of an 84226 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of an 84226 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[2429] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 84226 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 84226 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[2430] Cell based assays can be exploited to analyze a variegated 84226 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 84226 in a substrate-dependent manner. The transfected cells are then contacted with 84226 and the effect of the expression of the mutant on signaling by the 84226 substrate can be detected, e.g., by measuring the binding to zinc ions. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 84226 substrate, and the individual clones further characterized.

[2431] In another aspect, the invention features a method of making an 84226 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 84226 polypeptide, e.g., a naturally occurring 84226 polypeptide. The method includes: altering the sequence of an 84226 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[2432] In another aspect, the invention features a method of making a fragment or analog of an 84226 polypeptide a biological activity of a naturally occurring 84226 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of an 84226 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[2433] Anti-84226 Antibodies

[2434] In another aspect, the invention provides an anti-84226 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[2435] The anti-84226 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[2436] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[2437] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 84226 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-84226 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[2438] The anti-84226 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[2439] Phage display and combinatorial methods for generating anti-84226 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio/Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al. (1991) PNAS 88: 7978-7982, the contents of all of which are incorporated by reference herein).

[2440] In one embodiment, the anti-84226 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art.

[2441] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. (1994) Nature 368: 856-859; Green, L. L. et al. (1994) Nature Genet. 7: 13-21; Morrison, S. L. et al. (1994) Proc. Natl. Acad. Sci. USA 81: 6851-6855; Bruggeman et al. (1993) Year Immunol 7: 33-40; Tuaillon et al. (1993) PNAS 90: 3720-3724; Bruggeman et al. (1991) Eur J Immunol 21: 1323-1326).

[2442] An anti-84226 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[2443] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al. (1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988)J. Natl Cancer Inst. 80: 1553-1559).

[2444] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to an 84226 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[2445] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[2446] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L. (1985) Science 229: 1202-1207, by Oi et al. (1986) BioTechniques 4: 214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against an 84226 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[2447] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science 239:1534; Beidler et al. (1988) J. Immunol. 141: 4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[2448] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[2449] In preferred embodiments an antibody can be made by immunizing with purified 84226 antigen, or a fragment thereof, e.g., a fragment described herein, membrane associated antigen, tissue, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g., membrane fractions.

[2450] A full-length 84226 protein or, antigenic peptide fragment of 84226 can be used as an immunogen or can be used to identify anti-84226 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 84226 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 40 and encompasses an epitope of 84226. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[2451] Fragments of 84226 which include residues from about 45 to 75, from about 200 to 218, and from about 248 to 258 of SEQ ID NO: 40 can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophilic regions of the 84226 protein. Similarly, fragments of 84226 which include residues from about 80 to 92, from about 140 to 152, from about 232 to 248, and from about 312 to 328 of SEQ ID NO: 40 can be used to make an antibody against a hydrophobic region of the 84226 protein; fragments of 84226 which include residues 96 to 106, 164 to 177, and 244 to 252 of SEQ ID NO: 40 can be used to make an antibody against an extracellular region of the 84226 protein; fragments of 84226 which include residues 1 (amino terminus) to 73, 124 to 140, 197 to 218, and 278 to 373 (carboxy terminus) of SEQ ID NO: 40 can be used to make an antibody against an intracellular region of the 84226 protein; a fragment of 84226 which include residues about 74 to 361 of SEQ ID NO: 40 can be used to make an antibody against the cation transporter region of the 84226 protein.

[2452] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[2453] Antibodies which bind only native 84226 protein, only denatured or otherwise non-native 84226 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 84226 protein.

[2454] Preferred epitopes encompassed by the antigenic peptide are regions of 84226 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 84226 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 84226 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[2455] In a preferred embodiment the antibody can bind to the extracellular portion of the 84226 protein, e.g., it can bind to a whole cell which expresses the 84226 protein. In another embodiment, the antibody binds an intracellular portion of the 84226 protein. In preferred embodiments antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g., tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g., membrane fractions, e.g., residues 74 to 95, 107 to 123, 141 to 163, 178 to 196, 219 to 243, and 253 to 277 of SEQ ID NO: 40.

[2456] The anti-84226 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann NY Acad Sci 880: 263-80; and Reiter, Y. (1996) Clin Cancer Res 2: 245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 84226 protein.

[2457] In a preferred embodiment the antibody has effector function and/or can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement.

[2458] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[2459] In a preferred embodiment, an anti-84226 antibody alters (e.g., increases or decreases) the zinc binding activity of an 84226 polypeptide.

[2460] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[2461] An anti-84226 antibody (e.g., monoclonal antibody) can be used to isolate 84226 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-84226 antibody can be used to detect 84226 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-84226 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidinibiotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I,35S or 3H.

[2462] The invention also includes a nucleic acid which encodes an anti-84226 antibody, e.g., an anti-84226 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[2463] The invention also includes cell lines, e.g., hybridomas, which make an anti-84226 antibody, e.g., and antibody described herein, and method of using said cells to make an 84226 antibody.

[2464] 84226 Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[2465] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[2466] A vector can include an 84226 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 84226 proteins, mutant forms of 84226 proteins, fusion proteins, and the like).

[2467] The recombinant expression vectors of the invention can be designed for expression of 84226 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[2468] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[2469] Purified fusion proteins can be used in 84226 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 84226 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[2470] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[2471] The 84226 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[2472] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[2473] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

[2474] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banedji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the (α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[2475] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[2476] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., an 84226 nucleic acid molecule within a recombinant expression vector or an 84226 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[2477] A host cell can be any prokaryotic or eukaryotic cell. For example, an 84226 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Cell 23:175-182)). Other suitable host cells are known to those skilled in the art.

[2478] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[2479] A host cell of the invention can be used to produce (i.e., express) an 84226 protein. Accordingly, the invention further provides methods for producing an 84226 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding an 84226 protein has been introduced) in a suitable medium such that an 84226 protein is produced. In another embodiment, the method further includes isolating an 84226 protein from the medium or the host cell.

[2480] In another aspect, the invention features, a cell or purified preparation of cells which include an 84226 transgene, or which otherwise misexpress 84226. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include an 84226 transgene, e.g., a heterologous form of a 84226, e.g., a gene derived from humans (in the case of a non-human cell). The 84226 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 84226, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 84226 alleles or for use in drug screening.

[2481] In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid which encodes a subject 84226 polypeptide.

[2482] Also provided are cells, preferably human cells, e.g., human hematopoietic or fibroblast cells, in which an endogenous 84226 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 84226 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 84226 gene. For example, an endogenous 84226 gene which is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[2483] In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding an 84226 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Production of 84226 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for an 84226 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

[2484] 84226 Transgenic Animals

[2485] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of an 84226 protein and for identifying and/or evaluating modulators of 84226 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 84226 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[2486] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of an 84226 protein to particular cells. A transgenic founder animal can be identified based upon the presence of an 84226 transgene in its genome and/or expression of 84226 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding an 84226 protein can further be bred to other transgenic animals carrying other transgenes.

[2487] 84226 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[2488] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[2489] Uses of 84226

[2490] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[2491] The isolated nucleic acid molecules of the invention can be used, for example, to express an 84226 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect an 84226 mRNA (e.g., in a biological sample) or a genetic alteration in an 84226 gene, and to modulate 84226 activity, as described further below. The 84226 proteins can be used to treat disorders characterized by insufficient or excessive production of an 84226 substrate or production of 84226 inhibitors. In addition, the 84226 proteins can be used to screen for naturally occurring 84226 substrates, to screen for drugs or compounds which modulate 84226 activity, as well as to treat disorders characterized by insufficient or excessive production of 84226 protein or production of 84226 protein forms which have decreased, aberrant or unwanted activity compared to 84226 wild type protein (e.g., pancreatic disorders such as pancreatic cancer). Moreover, the anti-84226 antibodies of the invention can be used to detect and isolate 84226 proteins, regulate the bioavailability of 84226 proteins, and modulate 84226 activity.

[2492] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 84226 polypeptide is provided. The method includes: contacting the compound with the subject 84226 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 84226 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 84226 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 84226 polypeptide. Screening methods are discussed in more detail below.

[2493] 84226 Screening Assays

[2494] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 84226 proteins, have a stimulatory or inhibitory effect on, for example, 84226 expression or 84226 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of an 84226 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 84226 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[2495] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of an 84226 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of an 84226 protein or polypeptide or a biologically active portion thereof.

[2496] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[2497] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[2498] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[2499] In one embodiment, an assay is a cell-based assay in which a cell which expresses an 84226 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 84226 activity is determined. Determining the ability of the test compound to modulate 84226 activity can be accomplished by monitoring, for example, zinc binding activity. The cell, for example, can be of mammalian origin, e.g., human.

[2500] The ability of the test compound to modulate 84226 binding to a compound, e.g., an 84226 substrate, or to bind to 84226 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 84226 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 84226 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 84226 binding to an 84226 substrate in a complex. For example, compounds (e.g., 84226 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[2501] The ability of a compound (e.g., an 84226 substrate) to interact with 84226 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 84226 without the labeling of either the compound or the 84226. McConnell, H. M. et al. (1992) Science 257: 1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 84226.

[2502] In yet another embodiment, a cell-free assay is provided in which an 84226 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 84226 protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the 84226 proteins to be used in assays of the present invention include fragments which participate in interactions with non-84226 molecules, e.g., fragments with high surface probability scores.

[2503] Soluble and/or membrane-bound forms of isolated proteins (e.g., 84226 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton(® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[2504] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[2505] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[2506] In another embodiment, determining the ability of the 84226 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[2507] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[2508] It may be desirable to immobilize either 84226, an anti-84226 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to an 84226 protein, or interaction of an 84226 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/84226 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 84226 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 84226 binding or activity determined using standard techniques.

[2509] Other techniques for immobilizing either an 84226 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 84226 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[2510] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways.

[2511] Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[2512] In one embodiment, this assay is performed utilizing antibodies reactive with 84226 protein or target molecules but which do not interfere with binding of the 84226 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 84226 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 84226 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 84226 protein or target molecule.

[2513] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[2514] In a preferred embodiment, the assay includes contacting the 84226 protein or biologically active portion thereof with a known compound which binds 84226 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an 84226 protein, wherein determining the ability of the test compound to interact with an 84226 protein includes determining the ability of the test compound to preferentially bind to 84226 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[2515] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 84226 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of an 84226 protein through modulation of the activity of a downstream effector of an 84226 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[2516] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[2517] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[2518] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[2519] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[2520] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[2521] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[2522] In yet another aspect, the 84226 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 84226 (“84226-binding proteins” or “84226-bp”) and are involved in 84226 activity. Such 84226-bps can be activators or inhibitors of signals by the 84226 proteins or 84226 targets as, for example, downstream elements of a 84226-mediated signaling pathway.

[2523] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an 84226 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 84226 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 84226-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 84226 protein.

[2524] In another embodiment, modulators of 84226 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 84226 mRNA or protein evaluated relative to the level of expression of 84226 mRNA or protein in the absence of the candidate compound. When expression of 84226 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 84226 mRNA or protein expression. Alternatively, when expression of 84226 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 84226 mRNA or protein expression. The level of 84226 mRNA or protein expression can be determined by methods described herein for detecting 84226 mRNA or protein.

[2525] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of an 84226 protein can be confirmed in vivo, e.g., in an animal such as an animal model for pancreatic disorders.

[2526] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., an 84226 modulating agent, an antisense 84226 nucleic acid molecule, a 84226-specific antibody, or a 84226-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[2527] 84226 Detection Assays

[2528] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 84226 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[2529] 84226 Chromosome Mapping

[2530] The 84226 nucleotide sequences or portions thereof can be used to map the location of the 84226 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 84226 sequences with genes associated with disease.

[2531] Briefly, 84226 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 84226 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 84226 sequences will yield an amplified fragment.

[2532] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[2533] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 84226 to a chromosomal location.

[2534] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[2535] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[2536] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325: 783-787.

[2537] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 84226 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[2538] 84226 Tissue Typing

[2539] 84226 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[2540] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 84226 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[2541] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 39 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 41 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[2542] If a panel of reagents from 84226 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[2543] Use of Partial 84226 Sequences in Forensic Biology

[2544] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[2545] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 39 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 39 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[2546] The 84226 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 84226 probes can be used to identify tissue by species and/or by organ type.

[2547] In a similar fashion, these reagents, e.g., 84226 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[2548] Predictive Medicine of 84226

[2549] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[2550] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 84226.

[2551] Such disorders include, e.g., a disorder associated with the misexpression of 84226 gene; a disorder of the pancreas.

[2552] The method includes one or more of the following:

[2553] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 84226 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[2554] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 84226 gene;

[2555] detecting, in a tissue of the subject, the misexpression of the 84226 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[2556] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of an 84226 polypeptide.

[2557] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 84226 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[2558] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 39, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 84226 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[2559] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 84226 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 84226.

[2560] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[2561] In preferred embodiments the method includes determining the structure of an 84226 gene, an abnormal structure being indicative of risk for the disorder.

[2562] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 84226 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[2563] Diagnostic and Prognostic Assays of 84226

[2564] Diagnostic and prognostic assays of the invention include method for assessing the expression level of 84226 molecules and for identifying variations and mutations in the sequence of 84226 molecules.

[2565] Expression Monitoring and Profiling:

[2566] The presence, level, or absence of 84226 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 84226 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 84226 protein such that the presence of 84226 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 84226 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 84226 genes; measuring the amount of protein encoded by the 84226 genes; or measuring the activity of the protein encoded by the 84226 genes.

[2567] The level of mRNA corresponding to the 84226 gene in a cell can be determined both by in situ and by in vitro formats.

[2568] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 84226 nucleic acid, such as the nucleic acid of SEQ ID NO: 39, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 84226 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[2569] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 84226 genes.

[2570] The level of mRNA in a sample that is encoded by one of 84226 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[2571] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 84226 gene being analyzed.

[2572] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 84226 mRNA, or genomic DNA, and comparing the presence of 84226 mRNA or genomic DNA in the control sample with the presence of 84226 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 84226 transcript levels.

[2573] A variety of methods can be used to determine the level of protein encoded by 84226. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[2574] The detection methods can be used to detect 84226 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 84226 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 84226 protein include introducing into a subject a labeled anti-84226 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-84226 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[2575] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 84226 protein, and comparing the presence of 84226 protein in the control sample with the presence of 84226 protein in the test sample.

[2576] The invention also includes kits for detecting the presence of 84226 in a biological sample. For example, the kit can include a compound or agent capable of detecting 84226 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 84226 protein or nucleic acid.

[2577] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[2578] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[2579] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 84226 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as pancreatic disorders or deregulated cell proliferation.

[2580] In one embodiment, a disease or disorder associated with aberrant or unwanted 84226 expression or activity is identified. A test sample is obtained from a subject and 84226 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 84226 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 84226 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[2581] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 84226 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a pancreas cell with a pancreatic disorder.

[2582] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 84226 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 84226 (e.g., other genes associated with a 84226-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[2583] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 84226 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose a disorder in a subject wherein an increase in 84226 expression is an indication that the subject has or is disposed to having a pancreatic disorder. The method can be used to monitor a treatment for pancreatic disorders in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[2584] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 84226 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[2585] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 84226 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[2586] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[2587] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 84226 expression.

[2588] 84226 Arrays and Uses Thereof

[2589] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to an 84226 molecule (e.g., an 84226 nucleic acid or an 84226 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm2, and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[2590] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to an 84226 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 84226. Each address of the subset can include a capture probe that hybridizes to a different region of an 84226 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for an 84226 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 84226 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 84226 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[2591] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[2592] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to an 84226 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 84226 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-84226 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[2593] In another aspect, the invention features a method of analyzing the expression of 84226. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 84226-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[2594] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 84226. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 84226. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[2595] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 84226 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[2596] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[2597] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 84226-associated disease or disorder; and processes, such as a cellular transformation associated with a 84226-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 84226-associated disease or disorder.

[2598] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 84226) that could serve as a molecular target for diagnosis or therapeutic intervention.

[2599] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon an 84226 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1 999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99% identical to an 84226 polypeptide or fragment thereof For example, multiple variants of an 84226 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[2600] The polypeptide array can be used to detect an 84226 binding compound, e.g., an antibody in a sample from a subject with specificity for an 84226 polypeptide or the presence of a 84226-binding protein or ligand.

[2601] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 84226 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[2602] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 84226 or from a cell or subject in which an 84226 mediated response has been elicited, e.g., by contact of the cell with 84226 nucleic acid or protein, or administration to the cell or subject 84226 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 84226 (or does not express as highly as in the case of the 84226 positive plurality of capture probes) or from a cell or subject which in which an 84226 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than an 84226 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[2603] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 84226 or from a cell or subject in which a 84226-mediated response has been elicited, e.g., by contact of the cell with 84226 nucleic acid or protein, or administration to the cell or subject 84226 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 84226 (or does not express as highly as in the case of the 84226 positive plurality of capture probes) or from a cell or subject which in which an 84226 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[2604] In another aspect, the invention features a method of analyzing 84226, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing an 84226 nucleic acid or amino acid sequence; comparing the 84226 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 84226.

[2605] Detection of 84226 Variations or Mutations

[2606] The methods of the invention can also be used to detect genetic alterations in an 84226 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 84226 protein activity or nucleic acid expression, such as a pancreatic disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 84226-protein, or the mis-expression of the 84226 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an 84226 gene; 2) an addition of one or more nucleotides to an 84226 gene; 3) a substitution of one or more nucleotides of an 84226 gene, 4) a chromosomal rearrangement of an 84226 gene; 5) an alteration in the level of a messenger RNA transcript of an 84226 gene, 6) aberrant modification of an 84226 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an 84226 gene, 8) a non-wild type level of a 84226-protein, 9) allelic loss of an 84226 gene, and 10) inappropriate post-translational modification of a 84226-protein.

[2607] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 84226-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an 84226 gene under conditions such that hybridization and amplification of the 84226-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[2608] In another embodiment, mutations in an 84226 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[2609] In other embodiments, genetic mutations in 84226 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of an 84226 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of an 84226 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7:244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations in 84226 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[2610] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 84226 gene and detect mutations by comparing the sequence of the sample 84226 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[2611] Other methods for detecting mutations in the 84226 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[2612] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 84226 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[2613] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 84226 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 84226 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[2614] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[2615] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[2616] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[2617] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to an 84226 nucleic acid.

[2618] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 39 or the complement of SEQ ID NO: 39. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[2619] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 84226. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[2620] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the Tm of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[2621] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, an 84226 nucleic acid.

[2622] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an 84226 gene.

[2623] Use of 84226 Molecules as Surrogate Markers

[2624] The 84226 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 84226 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 84226 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35:258-264; and James (1994) AIDS Treatment News Archive 209.

[2625] The 84226 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., an 84226 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-84226 antibodies may be employed in an immune-based detection system for an 84226 protein marker, or 84226-specific radiolabeled probes may be used to detect an 84226 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[2626] The 84226 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 84226 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 84226 DNA may correlate 84226 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[2627] Pharmaceutical Compositions of 84226

[2628] The nucleic acid and polypeptides, fragments thereof, as well as anti-84226 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[2629] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[2630] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[2631] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[2632] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.

[2633] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[2634] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[2635] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[2636] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[2637] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[2638] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[2639] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[2640] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[2641] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[2642] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[2643] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[2644] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[2645] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactive ions include, but are not limited to iodine, yttrium and praseodymium.

[2646] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[2647] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[2648] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[2649] Methods of Treatment for 84226

[2650] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 84226 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[2651] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 84226 molecules of the present invention or 84226 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[2652] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 84226 expression or activity, by administering to the subject an 84226 or an agent which modulates 84226 expression or at least one 84226 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 84226 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 84226 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 84226 aberrance, for example, a 84226, 84226 agonist or 84226 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[2653] It is possible that some 84226 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[2654] The 84226 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, cardiovascular disorders, liver disorders, viral diseases, pain or metabolic disorders.

[2655] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[2656] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[2657] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[2658] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[2659] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[2660] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.

[2661] Aberrant expression and/or activity of 84226 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 84226 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 84226 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 84226 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[2662] The 84226 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders. Examples of immune disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

[2663] Examples of disorders involving the heart or “cardiovascular disorder” include, but are not limited to, a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, congestive heart failure, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

[2664] Disorders which may be treated or diagnosed by methods described herein include, but are not limited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, A1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

[2665] Additionally, 84226 molecules may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 84226 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 84226 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

[2666] Additionally, 84226 may play an important role in the regulation of metabolism or pain disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[2667] As discussed, successful treatment of 84226 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 84226 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[2668] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[2669] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[2670] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 84226 expression is through the use of aptamer molecules specific for 84226 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1:5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 84226 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[2671] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 84226 disorders. For a description of antibodies, see the Antibody section above.

[2672] In circumstances wherein injection of an animal or a human subject with an 84226 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 84226 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 84226 protein. Vaccines directed to a disease characterized by 84226 expression may also be generated in this fashion.

[2673] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[2674] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 84226 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[2675] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[2676] Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 84226 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 84226 can be readily monitored and used in calculations of IC50.

[2677] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[2678] Another aspect of the invention pertains to methods of modulating 84226 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with an 84226 or agent that modulates one or more of the activities of 84226 protein activity associated with the cell. An agent that modulates 84226 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an 84226 protein (e.g., an 84226 substrate or receptor), an 84226 antibody, an 84226 agonist or antagonist, a peptidomimetic of an 84226 agonist or antagonist, or other small molecule.

[2679] In one embodiment, the agent stimulates one or 84226 activities. Examples of such stimulatory agents include active 84226 protein and a nucleic acid molecule encoding 84226. In another embodiment, the agent inhibits one or more 84226 activities. Examples of such inhibitory agents include antisense 84226 nucleic acid molecules, anti-84226 antibodies, and 84226 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of an 84226 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 84226 expression or activity. In another embodiment, the method involves administering an 84226 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 84226 expression or activity.

[2680] Stimulation of 84226 activity is desirable in situations in which 84226 is abnormally downregulated and/or in which increased 84226 activity is likely to have a beneficial effect. For example, stimulation of 84226 activity is desirable in situations in which an 84226 is downregulated and/or in which increased 84226 activity is likely to have a beneficial effect. Likewise, inhibition of 84226 activity is desirable in situations in which 84226 is abnormally upregulated and/or in which decreased 84226 activity is likely to have a beneficial effect.

[2681] 84226 Pharmacogenomics

[2682] The 84226 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 84226 activity (e.g., 84226 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 84226 associated disorders (e.g., pancreatic disorders) associated with aberrant or unwanted 84226 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an 84226 molecule or 84226 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an 84226 molecule or 84226 modulator.

[2683] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[2684] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[2685] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., an 84226 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[2686] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., an 84226 molecule or 84226 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[2687] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an 84226 molecule or 84226 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[2688] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 84226 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 84226 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[2689] Monitoring the influence of agents (e.g., drugs) on the expression or activity of an 84226 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 84226 gene expression, protein levels, or upregulate 84226 activity, can be monitored in clinical trials of subjects exhibiting decreased 84226 gene expression, protein levels, or downregulated 84226 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 84226 gene expression, protein levels, or downregulate 84226 activity, can be monitored in clinical trials of subjects exhibiting increased 84226 gene expression, protein levels, or upregulated 84226 activity. In such clinical trials, the expression or activity of an 84226 gene, and preferably, other genes that have been implicated in, for example, a 84226-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[2690] 84226 Informatics

[2691] The sequence of an 84226 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 84226. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 84226 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[2692] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[2693] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[2694] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[2695] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[2696] Thus, in one aspect, the invention features a method of analyzing 84226, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing an 84226 nucleic acid or amino acid sequence; comparing the 84226 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 84226. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[2697] The method can include evaluating the sequence identity between an 84226 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[2698] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[2699] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[2700] Thus, the invention features a method of making a computer readable record of a sequence of an 84226 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[2701] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing an 84226 sequence, or record, in machine-readable form; comparing a second sequence to the 84226 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 84226 sequence includes a sequence being compared. In a preferred embodiment the 84226 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 84226 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof, the 5′ end of the translated region.

[2702] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 84226-associated disease or disorder or a pre-disposition to a 84226-associated disease or disorder, wherein the method comprises the steps of determining 84226 sequence information associated with the subject and based on the 84226 sequence information, determining whether the subject has a 84226-associated disease or disorder or a pre-disposition to a 84226-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[2703] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 84226-associated disease or disorder or a pre-disposition to a disease associated with an 84226 wherein the method comprises the steps of determining 84226 sequence information associated with the subject, and based on the 84226 sequence information, determining whether the subject has a 84226-associated disease or disorder or a pre-disposition to a 84226-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 84226 sequence of the subject to the 84226 sequences in the database to thereby determine whether the subject as a 84226-associated disease or disorder, or a pre-disposition for such.

[2704] The present invention also provides in a network, a method for determining whether a subject has an 84226 associated disease or disorder or a pre-disposition to a 84226-associated disease or disorder associated with 84226, said method comprising the steps of receiving 84226 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 84226 and/or corresponding to a 84226-associated disease or disorder (e.g., a pancreatic disorder), and based on one or more of the phenotypic information, the 84226 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 84226-associated disease or disorder or a pre-disposition to a 84226-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[2705] The present invention also provides a method for determining whether a subject has an 84226-associated disease or disorder or a pre-disposition to a 84226-associated disease or disorder, said method comprising the steps of receiving information related to 84226 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 84226 and/or related to a 84226-associated disease or disorder, and based on one or more of the phenotypic information, the 84226 information, and the acquired information, determining whether the subject has a 84226-associated disease or disorder or a pre-disposition to a 84226-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[2706] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

[2707] Background of the 8105 Invention

[2708] Cellular membranes differentiate the contents of a cell from the surrounding environment. Membranes also serve as effective barriers against the unregulated influx of hazardous or unwanted compounds, and the unregulated efflux of desirable compounds. However, the cell does need a supply of desired compounds and removal of waste products. Transport proteins that are embedded (singly or in complexes) in the cellular membrane (reviewed by Oh and Amidon (1999) in Membrane Transporters as Drug Targets, ed. Amidon and Sadee, Kluwer Academic/Plenum Publishers, New York, Chapter 1) are major providers of these functions. There are two general classes of membrane transport proteins: channels or pores, and transporters (also known as carriers or permeases). Channels and transporters differ in their translocation mechanisms. Channels are hydrophilic group-lined protein tunnels whose opening by a regulatory event allow free, rapid passage of their charge-, size-, and geometry-selected small ions down their concentration gradients. Transporters specifically and selectively bind the molecules they move, some with and some against their concentration gradients, across membranes. The binding mechanism causes the action of transporters to be slow and saturable.

[2709] Transport molecules are specific for a particular target solute or class of solutes, and are also present in one or more specific membranes. Transport molecules localized to the plasma membrane permit an exchange of solutes with the surrounding environment, while transport molecules localized to intracellular membranes (e.g., membranes of the mitochondrion, peroxisome, lysosome, endoplasmic reticulum, nucleus, or vacuole) permit import and export of molecules from organelle to organelle or to the cytoplasm. For example, in the case of the mitochondrion, transporters in the inner and outer mitochondrial membranes permit the import of sugar molecules, calcium ions, and water (among other molecules) into the organelle and the export of newly synthesized ATP to the cytosol.

[2710] Transporters can move molecules by two types of processes. In one process, “facilitated diffusion,” transporters move molecules with their concentration gradients. In the other process, “active transport,” transporters move molecules against their concentration gradients. Active transport to move a molecule against its gradient requires energy, in contrast to facilitated diffusion, which does not require energy.

[2711] Transporters play important roles in the ability of the cell to regulate homeostasis, to grow and divide, and to communicate with other cells, e.g., to transport metabolic compounds (e.g., sugars, e.g., glucose) or metabolic intermediates, signaling molecules, such as hormones, reactive oxygen species, ions, neurotransmitters, or vitamins. A wide variety of human diseases and disorders are associated with defects in transporter or other membrane transport molecules, including certain types of liver disorders (e.g., due to defects in transport of long-chain fatty acids (Al Odaib et al. (1998) New Eng. J. Med. 339:1752-1757), hyperlysinemia (mitochondrial lysine transport defect (Oyanagi et al. (1986) Inherit. Metab. Dis. 9:313-316)), and cataract (Wintour (1997) Clin. Exp. Pharmacol. Physiol. 24(1):1-9). In addition, some sugar transporters are known to be involved in the regulation of cellular metabolism and, thus, can play a role in body weight disorders such as obesity, as well as related disorders like diabetes, hyperphagia, hypertension, and abnormalities associated with arterial and venous thrombosis, growth, sexual development, fertility (in both men and women), and sense of taste.

[2712] Many sugar transporters act by a facilitated diffusion mechanism to transport various monosaccharides across the cell membrane (Walmsley et al. (1998) Trends in Biochem. Sci. 23:476-481; Barrett et al. (1999) Curr. Op. Cell Biol. 11:496-502). Thus, they can be included in the major facilitator superfamily of transporters. In humans, there are over 30 families of transporters, also known as solute carriers or SLC (reviewed by Berger, et al. (2000) in The Kidney: Physiology and Pathophysiology, eds. Seldin D W and Giebisch G., Lippincott, Williams & Wilkins, Philadelphia 1:107-138; see also www.gene.ucl.ac.uk/nomenclature for names of human SLC genes). The SLC families are classified according to the molecules they transport across the membrane. The major facilitator or facilitated diffusion human sugar transporters are in the SLC2 family and transport glucose or fructose or participate in glucose homeostasis.

[2713] Summary of the 8105 Invention

[2714] The present invention is based, in part, on the discovery of a novel sugar transporter family member, referred to herein as “8105”. The nucleotide sequence of a cDNA encoding 8105 is shown in SEQ ID NO: 43, and the amino acid sequence of a 8105 polypeptide is shown in SEQ ID NO: 44. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO: 45.

[2715] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 8105 protein or polypeptide, e.g., a biologically active portion of the 8105 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 44. In other embodiments, the invention provides isolated 8105 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 43, SEQ ID NO: 45, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number as described herein. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 43, SEQ ID NO: 45, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number as described herein. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 43, SEQ ID NO: 45, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number as described herein, wherein the nucleic acid encodes a full length 8105 protein or an active fragment thereof.

[2716] In a related aspect, the invention further provides nucleic acid constructs that include a 8105 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 8105 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 8105 nucleic acid molecules and polypeptides.

[2717] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 8105-encoding nucleic acids.

[2718] In still another related aspect, isolated nucleic acid molecules that are antisense to a 8105 encoding nucleic acid molecule are provided.

[2719] In another aspect, the invention features, 8105 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 8105-mediated or -related disorders, e.g., obesity and related disorders, e.g., diabetes, hormonal disorders, hypertension, hyperphagia, and/or cardiovascular disorders. In another embodiment, the invention provides 8105 polypeptides having a 8105 activity. Preferred polypeptides are 8105 proteins including at least one sugar transporter domain, and, preferably, having a 8105 activity, e.g., a 8105 activity as described herein.

[2720] In other embodiments, the invention provides 8105 polypeptides, e.g., a 8105 polypeptide having the amino acid sequence shown in SEQ ID NO: 44 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number as described herein; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 44 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number as described herein; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 43, SEQ ID NO: 45, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number as described herein, wherein the nucleic acid encodes a full length 8105 protein or an active fragment thereof, e.g., a fragment of at least 540 amino acid residues of SEQ ID NO: 44.

[2721] In a related aspect, the invention further provides nucleic acid constructs which include a 8105 nucleic acid molecule described herein.

[2722] In a related aspect, the invention provides 8105 polypeptides or fragments operatively linked to non-8105 polypeptides to form fusion proteins.

[2723] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 8105 polypeptides or fragments thereof, e.g., an extracellular domain of an 8105 polypeptide.

[2724] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 8105 polypeptides or nucleic acids.

[2725] In still another aspect, the invention provides a process for modulating 8105 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 8105 polypeptides or nucleic acids, such as conditions involving obesity and related disorders, e.g., diabetes, hormonal disorders, hypertension, hyperphagia, and cardiovascular disorders.

[2726] The invention also provides assays for determining the activity of or the presence or absence of 8105 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[2727] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 8105 polypeptide or nucleic acid molecule, including for disease diagnosis.

[2728] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 8105 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 8105 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 8105 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[2729] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[2730] Detailed Description of 8105

[2731] The human 8105 sequence (see SEQ ID NO: 43, as recited in Example 31), which is approximately 4385 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1689 nucleotides, including the termination codon. The coding sequence encodes a 562 amino acid protein (see SEQ ID NO: 44, as recited in Example 31).

[2732] Human 8105 contains the following regions or other structural features:

[2733] a sugar transporter domain (PFAM Accession Number PF00083) located at about amino acid residues 31 to 533 of SEQ ID NO: 44;

[2734] two sugar transport signature 1 sites (Prosite PS00216) located at about amino acid residues 86 to 102, and 308 to 324 of SEQ ID NO: 44;

[2735] twelve predicted transmembrane domains (predicted by MEMSAT, Jones et al. (1994) Biochemistry 33:3038-3049) located at about amino acid residues 17 to 41, 70 to 90, 98 to 121, 128 to 144, 154 to 174, 188 to 210, 255 to 279, 290 to 312, 319 to 342, 433 to 456, 468 to 488, and 496 to 518 of SEQ ID NO: 44;

[2736] two predicted protein kinase C phosphorylation sites (Prosite PS00005) located at about amino acid residues 215 to 217, and 391 to 393 of SEQ ID NO: 44;

[2737] eight predicted casein kinase II phosphorylation sites (Prosite PS00006) located at about amino acid residues 64 to 67, 158 to 161, 215 to 218, 238 to 241, 380 to 383, 486 to 489, 525 to 528, and 554 to 557 of SEQ ID NO: 44;

[2738] one predicted cAMP/cGMP-dependent protein kinase phosphorylation site (Prosite PS00004) located at about amino acid residues 536 to 539 of SEQ ID NO: 44;

[2739] two predicted N-glycosylation sites (Prosite PS00001) from about amino acid residues 355 to 358, and 547 to 550 of SEQ ID NO: 44;

[2740] one predicted glycosaminoglycan attachment site (Prosite PS00002) located at about amino acid residues 333 to 336 of SEQ ID NO: 44;

[2741] two predicted amidation sites (Prosite PS00009) located at about amino acid residues 93 to 96, and 315 to 318 of SEQ ID NO: 44; and

[2742] eleven predicted N-myristoylation sites (Prosite PS00008) located at about amino acid residues 40 to 45, 78 to 83, 114 to 119, 154 to 159, 163 to 168, 180 to 185, 209 to 214, 286 to 291, 495 to 500, 523 to 528, and 550 to 555 of SEQ ID NO: 44.

[2743] In addition, three predicted arginine methylation sites are located at about amino acid residues 153-154, 250-251, and 467-466 of SEQ ID NO: 44; two predicted SH2 domain binding sites are located at about amino acid residues 237-240 and 553 to 556 of SEQ ID NO: 44; one predicted SH3 domain binding site is located at about amino acid residues 232 to 235 of SEQ ID NO: 44; and one LAMMER kinase phosphorylation site is located at about amino acid residues 545 to 548 of SEQ ID NO: 44 (these predictions were made using the BinderFinder algorithm).

[2744] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

[2745] A plasmid containing the nucleotide sequence encoding human 8105 (clone “Fbh8105FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[2746] The 8105 protein contains a significant number of structural characteristics in common with members of the sugar transporter family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[2747] As used herein, the term “sugar transporter” or “SLC2 family member” includes a protein or polypeptide which is capable of mediating facilitated diffusion, e.g., driven by the substrate concentration gradient, of a molecule, e.g. a monosaccharide (e.g. glucose, fructose, galactose or myo-inositol) across a membrane, e.g. a cell (e.g, a nerve cell, pancreatic cell, endothelial cell, smooth muscle cell, or liver cell) or organelle (e.g, a mitochondrion) membrane. Sugar transporters play a role in or function in a variety of cellular processes, e.g., maintenance of sugar homeostasis and, typically, have sugar substrate specificity. Examples of sugar transporters include glucose transporters, fructose transporters, galactose transporters and yeast myo-inositol transporters.

[2748] The sugar transporter, or SLC2 family of proteins are characterized by twelve amphipathic (i.e. having hydrophilic or charged residue(s) along one face of an otherwise hydrophobic helix) transmembrane domains included within a sugar transporter domain, intracellular N- and C-termini, a large non-cytoplasmic hydrophilic loop between transmembrane domains one and two, and again between transmembrane domains nine and ten, a large cytoplasmic hydrophilic loop between transmembrane domains six and seven, and an oscillating pore mechanism of function (Barrett et al., supra). Typically, the transmembrane domains anchor the transporter within a membrane and through coordinated allosteric movements, effect the transport function along their hydrophilic faces across the membrane, while contributing to the sugar type selectivity. The hydrophilic non-transmembrane loops between and beyond the transmembrane domains of the transporter determine the ion binding specificity and provide the ion binding sites, the trigger for the transport conformational change, and release activity for the transporter.

[2749] An 8105 polypeptide can include a “sugar transporter domain” or regions homologous with a “sugar transporter domain”. A 8105 polypeptide can further include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, and preferably twelve “transmembrane domains” or regions homologous with a “transmembrane domain.”

[2750] As used herein, the term “sugar transporter domain” or “sugar (and other) transporter domain” includes an amino acid sequence of about 400 to 650 amino acid residues in length and having a bit score for the alignment of the sequence to the sugar transporter domain (HMM) of at least 150. Preferably a sugar transporter domain mediates facilitated diffusion of molecules, e.g. monosaccharides (e.g. glucose, fructose, galactose or myo-inositol) across a membrane. Preferably, a sugar transporter domain includes at least about 450 to 600 amino acids, more preferably about 470 to 550 amino acid residues, or about 490 to 510 amino acids and has a bit score for the alignment of the sequence to the sugar transporter domain (HMM) of at least 200, 210, 220 or greater.

[2751] Sugar transporter domains can include two Prosite signature sequences for sugar transport proteins (PS00216, or sequences homologous thereto). The first sugar transport protein signature sequence (GGFLIDCYGRKQAILGS, SEQ ID NO: 47) is located roughly between the second and third transmembrane domains of human 8105 polypeptide and corresponds to about amino acid residues 86 to 102 of SEQ ID NO: 44. In the above conserved motif, and other motifs described herein, the standard IUPAC one-letter code for the amino acids is used. The second sugar transport signature sequence (amino acids AMGLVDRAGRRALLLAG, SEQ ID NO: 48) is located roughly between the eighth and ninth transmembrane domains of human 8105 polypeptide and corresponds to about amino acids 308 to 324 of SEQ ID NO: 44. These signature sequences are involved in the conformational change required for transport. The sugar transporter domain (HMM) has been assigned the PFAM Accession Number PF00083. An alignment of the sugar transporter domain (amino acids 31 to 533 of SEQ ID NO: 44) of human 8105 with a consensus amino acid sequence (SEQ ID NO: 46) derived from a hidden Markov model is depicted in FIGS. 22A-22B.

[2752] In a preferred embodiment, a 8105 polypeptide or protein has a “sugar transporter domain” or a region which includes at least about 400 to 650 amino acids, 450 to 600 amino acids, more preferably about 470 to 550 amino acid residues, or about 490 to 510 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “sugar transporter domain,” e.g., the sugar transporter domain of human 8105 (e.g., residues 31 to 533 of SEQ ID NO: 44).

[2753] To identify the presence of a “sugar transporter” domain in a 8105 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28:405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a “sugar (and other) transporter domain” domain in the amino acid sequence of human 8105 at about residues 31 to 533 of SEQ ID NO: 44 (see FIGS. 22A-22B).

[2754] An 8105 polypeptide can include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, and preferably twelve “transmembrane domains” or regions homologous with a “transmembrane domain.” As used herein, the term “transmembrane domain” includes an amino acid sequence of about 10 to 40 amino acid residues in length and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains typically have alpha-helical structures and are described in, for example, Zagotta, W. N. et al., (1996) Annual Rev. Neurosci. 19:235-263, the contents of which are incorporated herein by reference.

[2755] In a preferred embodiment, an 8105 polypeptide or protein has at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, and preferably twelve “transmembrane domains” or regions which include at least about 12 to 35 more preferably about 15 to 30 or 16 to 25 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “transmembrane domain,” e.g., the transmembrane domains of human 8105 (e.g., residues 17 to 41, 70 to 90, 98 to 121, 128 to 144, 154 to 174, 188 to 210, 255 to 279, 290 to 312, 319 to 342, 433 to 456, 468 to 488, and 496 to 518 of SEQ ID NO: 44). The transmembrane domain of human 8105 is visualized in the hydropathy plot (FIG. 21) as regions of about 17 to 25 amino acids where the hydropathy trace is mostly above the horizontal line.

[2756] To identify the presence of a “transmembrane” domain in a 8105 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be analyzed by a transmembrane prediction method that predicts the secondary structure and topology of integral membrane proteins based on the recognition of topological models (MEMSAT, Jones et al., (1994) Biochemistry 33:3038-3049).

[2757] An 8105 polypeptide can include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, preferably thirteen “non-transmembrane regions.” As used herein, the term “non-transmembrane region” includes an amino acid sequence not identified as a transmembrane domain. The non-transmembrane regions in 8105 are located at about amino acid residues 1 to 16, 42 to 69, 91 to 97, 122 to 127, 145 to 153, 175 to 187, 211 to 254, 280 to 289, 313 to 318, 343 to 432, 457 to 467, 489 to 495, and 519 to 562 of SEQ ID NO: 44.

[2758] The non-transmembrane regions of 8105 include at least one, two, three, four, five, six, preferably seven cytoplasmic regions. When located at the N-terminus, the cytoplasmic region is referred to herein as the “N-terminal cytoplasmic domain.” As used herein, an “N-terminal cytoplasmic domain” includes an amino acid sequence having about 1 to 90, preferably about 1 to 40, more preferably about 1 to 30, or even more preferably about 1 to 20 amino acid residues in length and is located inside of a cell or within the cytoplasm of a cell. The C-terminal amino acid residue of an “N-terminal cytoplasmic domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a 8105 protein. For example, an N-terminal cytoplasmic domain is located at about amino acid residues 1 to 16 of SEQ ID NO: 44.

[2759] In a preferred embodiment, a polypeptide or protein has an N-terminal cytoplasmic domain or a region which includes at least about 5, preferably about 1 to 40, and more preferably about 1 to 20 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with an “N-terminal cytoplasmic domain,” e.g., the N-terminal cytoplasmic domain of human 8105 (e.g., residues 1 to 16 of SEQ ID NO: 44).

[2760] In another embodiment, a cytoplasmic region of an 8105 protein can include the C-terminus and can be a “C-terminal cytoplasmic domain,” also referred to herein as a “C-terminal cytoplasmic tail.” As used herein, a “C-terminal cytoplasmic domain” includes an amino acid sequence having a length of at least about about 20 to 90, more preferably about 40 to 50 amino acid residues and is located inside of a cell or within the cytoplasm of a cell. The N-terminal amino acid residue of a “C-terminal cytoplasmic domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a 8105 protein. For example, a C-terminal cytoplasmic domain is located at about amino acid residues 519 to 562 of SEQ ID NO: 44.

[2761] In a preferred embodiment, a 8105 polypeptide or protein has a C-terminal cytoplasmic domain or a region which includes at least about 20 to 90, and more preferably about 40 to 50 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a C-terminal cytoplasmic domain,“e.g., the C-terminal cytoplasmic domain of human 8105 (e.g., residues 519 to 562 of SEQ ID NO: 44).

[2762] In another embodiment, an 8105 protein includes at least one, two, three, four, preferably five cytoplasmic loops. As used herein, the term “loop” includes an amino acid sequence that resides outside of a phospholipid membrane, having a length of at least about 4, preferably about 5 to 80, more preferably about 5 to 45 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in an 8105 molecule, and the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a 8105 molecule. As used herein, a “cytoplasmic loop” includes a loop located inside of a cell or within the cytoplasm of a cell. For example, a “cytoplasmic loop” can be found at about amino acid residues 91 to 97, 145 to 153, 211 to 254, 313 to 318, and 457 to 467 of SEQ ID NO: 44.

[2763] In a preferred embodiment, a 8105 polypeptide or protein has a cytoplasmic loop or a region which includes at least about 4, preferably about 5 to 80, more preferably about 5 to 45 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a cytoplasmic loop,“e.g., a cytoplasmic loop of human 8105 (e.g., residues 91 to 97, 145 to 153, 211 to 254, 313 to 318, and 457 to 467 of SEQ ID NO: 44).

[2764] In another embodiment, a 8105 protein includes at least one, two, three, four, five, preferably six non-cytoplasmic loops. As used herein, a “non-cytoplasmic loop” includes an amino acid sequence located outside of a cell or within an intracellular organelle. Non-cytoplasmic loops include extracellular domains (i.e., outside of the cell) and intracellular domains (i.e., within the cell). When referring to membrane-bound proteins found in intracellular organelles (e.g., mitochondria, endoplasmic reticulum, peroxisomes microsomes, vesicles, endosomes, and lysosomes), non-cytoplasmic loops include those domains of the protein that reside in the lumen of the organelle or the matrix or the intermembrane space. For example, a “non-cytoplasmic loop” can be found at about amino acid residues 42 to 69, 122 to 127, 175 to 187, 280 to 289, 343 to 432, and 489 to 495 of SEQ ID NO: 44.

[2765] In a preferred embodiment, a 8105 polypeptide or protein has at least one non-cytoplasmic loop or a region which includes at least about 4, preferably about 5 to 100, more preferably about 5 to 90 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “non-cytoplasmic loop,” e.g., at least one non-cytoplasmic loop of human 8105 (e.g., residues 42 to 69, 122 to 127, 175 to 187, 280 to 289, 343 to 432, and 489 to 495 of SEQ ID NO: 44).

[2766] An 8105 family member can include at least one sugar transporter domain; at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, and preferably twelve transmembrane domains; at least one, two, three, four, five, six, preferably seven cytoplasmic regions, including N- and C-terminal cytoplasmic domains and at least one, two, three, four, preferably five cytoplasmic loops; and at least one, two, three, four, five, preferably six non-cytoplasmic loops. A 8105 family member also can include at least one, preferably two sugar transporter signature 1 sequences (PS00216). Furthermore, a 8105 family member can include at least one, preferably two predicted protein kinase C phosphorylation sites (Prosite PS00005); at least one, two, three, four, five, six, seven, preferably eight predicted casein kinase II phosphorylation sites (Prosite PS00006); at least one predicted cAMP/cGMP protein kinase phosphorylation site (Prosite PS00004); at least one, preferably two predicted N-glycosylation sites (Prosite PS00001); at least one predicted glycosaminoglycan attachment site (Prosite PS00002); at least one, preferably two predicted amidation sites (Prosite PS00009); at least one, two, three, four, five, six, seven, eight, nine, ten, and preferably eleven predicted N-myristoylation sites (Prosite PS00008); at least one, two, preferably three predicted arginine methylation sites; at least one, preferably two predicted SH2 domain binding sites; at least one predicted SH3 domain binding site; and at least one LAMMER kinase phosphorylation site.

[2767] As the 8105 polypeptides of the invention can modulate 8105-mediated activities, they can be useful for developing novel diagnostic and therapeutic agents for sugar transporter-associated or other 8105-associated disorders, as described below. As used herein, a “sugar transporter-mediated activity” includes an activity which involves transport of a molecule, e.g. a monosaccharide (e.g. glucose, fructose, galactose or myo-inositol) across a membrane, e.g. a cell (e.g, a nerve cell, fat cell, muscle cell, or blood cell, such as an erythrocyte) or organelle (e.g, a mitochondrion) membrane. Sugar transporters of the SLC2 family play important roles in sugar homeostasis, i.e., in making monosaccharides available to cells to use as an energy source. Besides a general role in metabolism, this role of sugar transporters is evident especially in the neurological and cardiovascular systems, with specific or high energy demands. As a result, glucose transporters are being investigated in relation to infantile seizures (Klepper et al. (1999) Neurochem. Res. 24:587-94) and coronary artery disease (Young et al. (1999) Am. J. Cardiol. 83:25H-30H).

[2768] As used herein, a “8105 activity”, “biological activity of 8105” or “functional activity of 8105”, refers to an activity exerted by a 8105 protein, polypeptide or nucleic acid molecule e.g., a 8105-responsive cell or on a 8105 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, a 8105 activity is a direct activity, such as an association with a 8105 target molecule. A “target molecule” or “binding partner” is a molecule with which a 8105 protein binds or interacts in nature. In an exemplary embodiment, 8105 is a transporter, e.g., sugar transporter, e.g., an SLC2 family member, and thus binds to or interacts in nature with a molecule, e.g., a monosaccharide (e.g., glucose, fructose, galactose or myo-inositol).

[2769] An 8105 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 8105 protein with a 8105 receptor.

[2770] Based on the above-described sequence structures and similarities to molecules of known function, the 8105 molecules of the present invention have similar biological activities as sugar transporter family members. For example, the 8105 proteins of the present invention can have one or more of the following activities: (1) the ability to reside within a membrane, e.g., a cell membrane (e.g., a nerve cell membrane, pancreatic cell membrane, endothelial cell membrane, smooth muscle cell membrane, and/or liver cell membrane) or organelle (e.g., mitochondrion) membrane; (2) the ability to interact with a substrate or target molecule, e.g., a monosaccharide, (e.g., glucose, fructose, galactose or myo-inositol); (3) the ability to transport the substrate or target molecule across the membrane; (4) the ability to interact with and/or modulate a second non-transporter protein; (5) the ability to modulate sugar homeostasis in a cell; (6) the ability to modulate insulin and/or glucagon secretion; or (7) the ability to modulate metabolism.

[2771] The expression pattern of 8105 (as described in Examples 2 and 3), particularly the expression in the brain, hypothalamus, pancreas, and vasculature, supports a role for the 8105 molecules of the invention in the regulation of metabolic processes. Without wanting to be bound by theory, it is possible that the 8105 molecules present in the brain and hypothalamus are part of a signaling network that monitors the levels of sugars (e.g., glucose) and leptins in the blood and integrates the information before sending out signals to other tissues in the body concerning hunger and metabolism. For example, glucose is known to influence the activity of a Na+/K+ pump in pancreatic cells (see Elmi et al. (2000), Int. J. Exp. Diabetes Res. 1(2):155-64, the contents of which are incorporated herein by reference), and leptins (the products of the obese gene) are known to activate an ATP-sensitive potassium channel in hypothalamic neurons (see Spanswick et al. (1997), Nature 390(6659):521-5, the contents of which are incorporated herein by reference). Consequently, since leptins are active in hypothalamic cells, where 8105 molecules are expressed, they could both be acting to modify the Na+ and K+ concentrations within the neurons, thereby altering the signaling properties of the neurons, as well as the signals that the neurons are sending to other cells in the body. 8105 molecules in other tissues, e.g., other neurons or pancreatic cells, could be functioning similarly, either in conjunction with leptins or with other molecules involved in the control of metabolism, e.g., hormones like NPY, MC4-R, and AGRP.

[2772] Thus, the 8105 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more sugar transporter-associated disorders. As used herein, a “human sugar transporter-associated disorder” includes a disorder, disease, or condition which is caused by, characterized by, or associated with a misregulation, e.g., an aberrant or deficient (e.g., downregulation or upregulation) of a sugar transporter mediated activity. Sugar transporter-associated disorders typically result in, e.g., upregulated or downregulated, sugar levels in a cell. Examples of sugar transporter-associated disorders include disorders associated with sugar homeostasis, such as obesity, anorexia, type-1 diabetes, type-2 diabetes, hypoglycemia, glycogen storage disease (Von Gierke disease), type I glycogenosis, bipolar disorder, seasonal affective disorder, and cluster B personality disorders.

[2773] Human sugar transporter-associated disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, and cellular regulation of homeostasis, e.g., glucose homeostasis; inter- or intra-cellular communication, e.g., involving neurons; tissue function, such as cardiovascular function (e.g., thrombosis and hypertension) or musculoskeletal function; systemic responses in an organism, such as nervous system responses, hormonal responses (e.g., regulation of metabolism and reproduction), or immune responses; and protection of cells from toxic compounds (e.g., carcinogens, toxins, mutagens, and toxic byproducts of metabolic activity, e.g., reactive oxygen species). Accordingly, the 8105 molecules of the invention, as human sugar transporters, can mediate various human sugar transporter-associated disorders, including, but not limited to, metabolic disorders, hormonal disorders, neurological disorders, pancreatic disorders, liver disorders kidney disorders, cardiovascular disorders, blood vessel disorders, pain disorders, disorders of bone metabolism, and cellular proliferative and/or differentiative disorders.

[2774] The 8105 molecules of the invention can play an important role in the regulation of metabolic disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, hyperphagia, anorexia nervosa, cachexia, and lipid disorders, and disorders in the regulation of blood sugar levels, e.g., diabetes type I and type II, and hypoglycemia. Metabolic disorders such as obesity can be associated with secondary disorders such as hormonal disorders (see below), hypertension, cardiovascular disorders (e.g., propensity for arterial and venous thrombosis), and sensory disorders (e.g., altered sense of taste), all of which could be influenced by the acitivity of 8105 molecules.

[2775] Human sugar transporter-associated disorders can include hormonal disorders, such as conditions or diseases in which the production and/or regulation of hormones in an organism is aberrant. Examples of such disorders and diseases include type I and type II diabetes mellitus, pituitary disorders (e.g., growth disorders), thyroid disorders (e.g., hypothyroidism or hyperthyroidism), and reproductive or fertility disorders (e.g., disorders which affect the organs of the reproductive system, e.g., the prostate gland, the uterus, or the vagina; disorders which involve an imbalance in the levels of a reproductive hormone in a subject; disorders affecting the ability of a subject to reproduce; and disorders affecting secondary sex characteristic development, e.g., adrenal hyperplasia).

[2776] Disorders involving the pancreas include those of the exocrine pancreas such as congenital anomalies, including but not limited to, ectopic pancreas; pancreatitis, including but not limited to, acute pancreatitis; cysts, including but not limited to, pseudocysts; tumors, including but not limited to, cystic tumors and carcinoma of the pancreas; and disorders of the endocrine pancreas such as, diabetes mellitus; islet cell tumors, including but not limited to, insulinomas, gastrinomas, and other rare islet cell tumors.

[2777] Disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and cirrhosis, such as cirrhosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and toxin-induced liver disease, such as alcoholic liver disease; inborn errors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, a1-antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease, such as secondary biliary cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and anomalies of the biliary tree; circulatory disorders, such as impaired blood flow into the liver, including hepatic artery compromise and portal vein obstruction and thrombosis, impaired blood flow through the liver, including passive congestion and centrilobular necrosis and peliosis hepatis, hepatic vein outflow obstruction, including hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease associated with pregnancy, such as preeclampsia and eclampsia, acute fatty liver of pregnancy, and intrehepatic cholestasis of pregnancy; hepatic complications of organ or bone marrow transplantation, such as drug toxicity after bone marrow transplantation, graft-versus-host disease and liver rejection, and nonimmunologic damage to liver allografts; tumors and tumorous conditions, such as nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.

[2778] Disorders involving the kidney include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases of the kidney, that include but are not limited to, cystic renal dysplasia, polycystic kidney diseases, and cystic diseases of renal medulla; glomerular diseases including pathologies of glomerular injury; glomerular lesions associated with systemic disease, and thrombotic microangiopathies.

[2779] Disorders of the CNS or neurological disorders such as cognitive and neurodegenerative disorders, include, but are not limited to, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, Korsakoff's psychosis, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Such neurological disorders include, for example, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicella-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer's disease and Pick's disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson's disease (paralysis agitans) and other Lewy diffuse body diseases, progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington's disease, senile dementia, Gilles de la Tourette's syndrome, epilepsy, and Jakob-Creutzfieldt disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin B1) deficiency and vitamin B12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease. Further CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

[2780] As used herein, disorders involving the heart, or “cardiovascular disease” or a “cardiovascular disorder” includes a disease or disorder which affects the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. A cardiovascular disorder includes, but is not limited to disorders such as arteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, valvular disease, including but not limited to, valvular degeneration caused by calcification, rheumatic heart disease, endocarditis, or complications of artificial valves; atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, pericardial disease, including but not limited to, pericardial effusion and pericarditis; cardiomyopathies, e.g., dilated cardiomyopathy or idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, ischemic disease, arrhythmia, sudden cardiac death, and cardiovascular developmental disorders (e.g., arteriovenous malformations, arteriovenous fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome, causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrial septal defects, atrioventricular canal, coarctation of the aorta, ebsteins anomaly, hypoplastic left heart syndrome, interruption of the aortic arch, mitral valve prolapse, ductus arteriosus, patent foramen ovale, partial anomalous pulmonary venous return, pulmonary atresia with ventricular septal defect, pulmonary atresia without ventricular septal defect, persistance of the fetal circulation, pulmonary valve stenosis, single ventricle, total anomalous pulmonary venous return, transposition of the great vessels, tricuspid atresia, truncus arteriosus, ventricular septal defects). A cardiovascular disease or disorder also can include an endothelial cell disorder.

[2781] As used herein, an “endothelial cell disorder” includes a disorder characterized by aberrant, unregulated, or unwanted endothelial cell activity, e.g., proliferation, migration, angiogenesis, or vascularization; or aberrant expression of cell surface adhesion molecules or genes associated with angiogenesis, e.g., TIE-2, FLT and FLK. Endothelial cell disorders include tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy, endometriosis, Grave's disease, ischemic disease (e.g., atherosclerosis), and chronic inflammatory diseases (e.g., rheumatoid arthritis).

[2782] Disorders involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease, e.g., an arteritis condition; Raynaud disease; aneurysms and dissection; disorders of veins and lymphatics, such as varicose veins, thrombophlebitis and phlebothrombosis, or other obstructions, lymphangitis and lymphedema; tumors, including benign tumors and tumor-like conditions, such as hemangioma, vascular ectasias, and bacillary angiomatosis, and intermediate-grade (borderline low-grade malignant) tumors, such as Kaposi's sarcoma and hemangloendothelioma, and malignant tumors, such as angiosarcoma and hemangiopericytoma; and pathology of therapeutic interventions in vascular disease, such as balloon angioplasty and related techniques and vascular replacement, such as coronary artery bypass graft surgery.

[2783] Aberrant expression and/or activity of 8105 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 8105 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 8105 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 8105 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[2784] Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[2785] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[2786] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[2787] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[2788] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[2789] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[2790] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myclogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.

[2791] The 8105 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 44 thereof are collectively referred to as “polypeptides or proteins of the invention” or “8105 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “8105 nucleic acids.” 8105 molecules refer to 8105 nucleic acids, polypeptides, and antibodies.

[2792] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[2793] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[2794] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2× SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[2795] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 43 or SEQ ID NO: 45, corresponds to a naturally-occurring nucleic acid molecule.

[2796] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include at least an open reading frame encoding a 8105 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 8105 protein or derivative thereof.

[2797] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 8105 protein is at least 10% pure. In a preferred embodiment, the preparation of 8105 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-8105 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-8105 chemicals. When the 8105 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[2798] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 8105 without abolishing or substantially altering a 8105 activity. Preferably the alteration does not substantially alter the 8105 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 8105, results in abolishing a 8105 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 8105 are predicted to be particularly unamenable to alteration.

[2799] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 8105 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 8105 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 8105 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 43 or SEQ ID NO: 45, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[2800] As used herein, a “biologically active portion” of a 8105 protein includes a fragment of a 8105 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 8105 molecule and a non-8105 molecule or between a first 8105 molecule and a second 8105 molecule (e.g., a dimerization interaction). Biologically active portions of a 8105 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 8105 protein, e.g., the amino acid sequence shown in SEQ ID NO: 44, which include less amino acids than the full length 8105 proteins, and exhibit at least one activity of a 8105 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 8105 protein, e.g., the transport of sugar molecules, e.g., glucose, accross cell membranes, e.g., the plasma membrane. A biologically active portion of a 8105 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 8105 protein can be used as targets for developing agents which modulate a 8105 mediated activity, e.g., the transport of sugar molecules, e.g., glucose, accross cell membranes, e.g., the plasma membrane.

[2801] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[2802] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[2803] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[2804] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[2805] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[2806] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 8105 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 8105 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[2807] Particularly preferred 8105 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 44. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 44 are termed substantially identical.

[2808] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43 or 45 are termed substantially identical.

[2809] “Misexpression or aberrant expression”, as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[2810] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[2811] A “purified preparation of cells”, as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[2812] Various aspects of the invention are described in further detail below.

[2813] Isolated 8105 Nucleic Acid Molecules

[2814] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 8105 polypeptide described herein, e.g., a full-length 8105 protein or a fragment thereof, e.g., a biologically active portion of 8105 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 8105 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[2815] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 43, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 8105 protein (i.e., “the coding region” of SEQ ID NO: 43, as shown in SEQ ID NO: 45), as well as 5′ untranslated sequences (nucleotides 1 to 173 of SEQ ID NO: 43), 3′ untranslated sequences (nucleotides 1860 to 4385 of SEQ ID NO: 43), or both 5′ and 3′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 43 (e.g., SEQ ID NO: 45) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from about amino acid residues 23 to 562, or 31 to 533 of SEQ ID NO: 44.

[2816] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 43 or SEQ ID NO: 45, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 43 or SEQ ID NO: 45, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO: 43 or 45, thereby forming a stable duplex.

[2817] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about: 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 43 or SEQ ID NO: 45, or a portion, preferably of the same length, of any of these nucleotide sequences.

[2818] 8105 Nucleic Acid Fragments

[2819] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 43 or 45. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 8105 protein, e.g., an immunogenic or biologically active portion of a 8105 protein. A fragment can comprise those nucleotides of SEQ ID NO: 43 which encode a sugar transporter domain of human 8105. The nucleotide sequence determined from the cloning of the 8105 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 8105 family members, or fragments thereof, as well as 8105 homologues, or fragments thereof, from other species.

[2820] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 520, 540, 545, 550, 555, 560, or more amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[2821] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 8105 nucleic acid fragment can include a sequence corresponding to a sugar transporter domain, e.g., about amino acid residues 23 to 562, or about 31 to 533 of SEQ ID NO: 44; or a transmembrane domain from about amino acid residues 17 to 41, 70 to 90, 98 to 121, 128 to 144, 154 to 174, 188 to 210, 255 to 279, 290 to 312, 319 to 342, 433 to 456, 468 to 488, and 496 to 518 of SEQ ID NO: 44.

[2822] 8105 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 43 or SEQ ID NO: 45, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 43 or SEQ ID NO: 45. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.

[2823] In one embodiment, the probe or primer is attached to a solid support, e.g., a solid support described herein.

[2824] One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g., the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 44. The reverse primer can anneal to the ultimate codon, e.g., the codon immediately before the stop codon, e.g., the codon encoding amino acid residue 562 of SEQ ID NO: 44. In a preferred embodiment, the annealing temperatures of the forward and reverse primers differ by no more than 5, 4, 3, or 2° C.

[2825] In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[2826] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a sugar transporter domain from about amino acid residues 23 to 562, or 31 to 533 of SEQ ID NO: 44; or a transmembrane domain from about amino acid residues 17 to 41, 70 to 90, 98 to 121, 128 to 144, 154 to 174, 188 to 210, 255 to 279, 290 to 312, 319 to 342, 433 to 456, 468 to 488, and 496 to 518 of SEQ ID NO: 44.

[2827] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 8105 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: a sugar transporter domain from about amino acid residues 23 to 562, or 31 to 533 of SEQ ID NO: 44; or a transmembrane domain from about amino acid residues 17 to 41, 70 to 90, 98 to 121, 128 to 144, 154 to 174, 188 to 210, 255 to 279, 290 to 312, 319 to 342, 433 to 456, 468 to 488, and 496 to 518 of SEQ ID NO: 44.

[2828] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[2829] A nucleic acid fragment encoding a “biologically active portion of a 8105 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 43 or 45, which encodes a polypeptide having a 8105 biological activity (e.g., the biological activities of the 8105 proteins are described herein), expressing the encoded portion of the 8105 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 8105 protein. For example, a nucleic acid fragment encoding a biologically active portion of 8105 includes a sugar transporter domain, e.g., amino acid residues about 23 to 562, or 31 to 533 of SEQ ID NO: 44. A nucleic acid fragment encoding a biologically active portion of a 8105 polypeptide, may comprise a nucleotide sequence which is greater than 300, 550, 691, 820, 882, 960, 1100, 1284, 1641, 1781, 2000, 2092 or more nucleotides in length.

[2830] In preferred embodiments, a nucleic acid fragment includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400 or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: 43 or SEQ ID NO: 45. In a preferred embodiment, the nucleic acid fragment includes at least one contiguous nucleotide from a region of about nucleotides 1-240, 200-1000, 800-2000, 1600-2400, 2200-2500, 2400-3200, 3000-3800, 3600-4400, 4000-4200, or 4100-4385.

[2831] In preferred embodiments, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AL137188, AF321240, AF248053, AK055548, or AL031055, or SEQ ID NO: 26 of WO 02/04520, or SEQ ID NO: 2 of WO 02/02586 or WO 02/18621. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 43 or SEQ ID NO: 45 located outside the region of nucleotides 241 to 2332 or 2333 to 4113; not include all of the nucleotides of AL137188, AF321240, AF248053, AK055548, or AL031055, or SEQ ID NO: 26 of WO 02/04520, or SEQ ID NO: 2 of WO 02/02586 or WO 02/18621, e.g., can be one or more nucleotides shorter (at one or both ends) than the sequence of AL137188, AF321240, AF248053, AK055548, or AL031055, or SEQ ID NO: 26 of WO 02/04520, or SEQ ID NO: 2 of WO 02/02586 or WO 02/18621; or can differ by one or more nucleotides in the region of overlap.

[2832] 8105 Nucleic Acid Variants

[2833] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 43 or SEQ ID NO: 45. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 8105 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 44. If alignment is needed for this comparison the sequences should be aligned for maximum homology. The encoded protein can differ by no more than 5, 4, 3, 2, or 1 amino acid. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[2834] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[2835] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[2836] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 43 or 45, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid. The nucleic acid can differ by no more than 5, 4, 3, 2, or 1 nucleotide. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[2837] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 44 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO: 44 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 8105 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 8105 gene.

[2838] Preferred variants include those that are correlated with the transport of sugar molecules, e.g., glucose, accross cell membranes, e.g., the plasma membranes.

[2839] Allelic variants of 8105, e.g., human 8105, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 8105 protein within a population that maintain the ability to bind and transport sugar molecules, e.g., glucose molecules. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 44, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 8105, e.g., human 8105, protein within a population that do not have the ability to bind and/or transport sugar molecules, e.g., glucose molecules. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 44, or a substitution, insertion, or deletion in critical residues or critical regions of the protein, e.g., in a sugar transport protein signature sequence, e.g., about amino acid residues 86 to 102 and 308 to 324 of SEQ ID NO: 44.

[2840] Moreover, nucleic acid molecules encoding other 8105 family members and, thus, which have a nucleotide sequence which differs from the 8105 sequences of SEQ ID NO: 43 or SEQ ID NO: 45 are intended to be within the scope of the invention.

[2841] Antisense Nucleic Acid Molecules, Ribozymes and Modified 8105 Nucleic Acid Molecules

[2842] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 8105. An “antisense” nucleic acid can include a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 8105 coding strand, or to only a portion thereof (e.g., the coding region of human 8105 corresponding to SEQ ID NO: 45). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 8105 (e.g., the 5′ and 3′ untranslated regions).

[2843] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 8105 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of 8105 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 8105 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[2844] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[2845] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 8105 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[2846] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[2847] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 8105-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 8105 cDNA disclosed herein (i.e., SEQ ID NO: 43 or SEQ ID NO: 45), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 8105-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 8105 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[2848] 8105 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 8105 (e.g., the 8105 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 8105 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N.Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[2849] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.

[2850] A 8105 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulmè (2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.

[2851] For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4:5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93:14670-675.

[2852] PNAs of 8105 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 8105 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[2853] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[2854] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 8105 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 8105 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

[2855] Isolated 8105 Polypeptides

[2856] In another aspect, the invention features, an isolated 8105 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-8105 antibodies. 8105 protein can be isolated from cells or tissue sources using standard protein purification techniques. 8105 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[2857] Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[2858] In a preferred embodiment, a 8105 polypeptide has one or more of the following characteristics:

[2859] it has the ability to the ability to reside within a membrane, e.g., a cell membrane (e.g., a nerve cell membrane, pancreatic cell membrane, endothelial cell membrane, smooth muscle cell membrane, and/or liver cell membrane) or organelle (e.g., mitochondrion) membrane;

[2860] it has the ability to interact with a substrate or target molecule, e.g., a monosaccharide, (e.g., glucose, fructose, galactose or myo-inositol);

[2861] it has the ability to transport the substrate or target molecule across the membrane;

[2862] it has the ability to modulate sugar homeostasis in a cell;

[2863] it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of a 8105 polypeptide, e.g., a polypeptide of SEQ ID NO: 44;

[2864] it has an overall sequence similarity of at least 90%, preferably 95%, more preferably 96%, 97%, 98%, 99%, or more with a polypeptide of SEQ ID NO: 44;

[2865] it has a sugar transporter domain which is preferably about 90%, preferably 95%, more preferably 96%, 97%, 98%, 99%, or more identical to amino acid residues about 31 to 533 of SEQ ID NO: 44;

[2866] it has at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, preferably twelve transmembrane domains which are preferably about 70%, 80%, 90%, 95%, 98%, 99%, or even 100% identical to amino acid residues about 17 to 41, 70 to 90, 98 to 121, 128 to 144, 154 to 174, 188 to 210, 255 to 279, 290 to 312, 319 to 342, 433 to 456, 468 to 488, and 496 to 518 of SEQ ID NO: 44;

[2867] it has two sugar transport signature 1 sites (Prosite PS00216);

[2868] it has one, preferably two predicted protein kinase C phosphorylation sites (Prosite PS00005);

[2869] it has one, two, three, four, five, six, seven, preferably eight predicted casein kinase II phosphorylation sites (Prosite PS00006);

[2870] it has one predicted cAMP/cGMP-dependent protein kinase phosphorylation site (Prosite PS00004);

[2871] it has one, preferably two predicted N-glycosylation sites (Prosite PS00001);

[2872] it has one predicted glycosaminoglycan attachment site (Prosite PS00002);

[2873] it has one, preferably two predicted amidation sites (Prosite PS00009);

[2874] it has one, two, three, four, five, six, seven, eight, nine, ten, preferably eleven predicted N-myristoylation sites (Prosite PS00008);

[2875] it has one, two, preferably three predicted arginine methylation sites it has one, preferably two predicted SH2 domain binding sites;

[2876] it has one predicted SH3 domain binding site; or

[2877] it has one LAMMER kinase phosphorylation site.

[2878] In a preferred embodiment the 8105 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID: 2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 44 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 44. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In a preferred embodiment the differences are not in the sugar transporter domain at about amino acid residues 31 to 533 of SEQ ID NO: 44. In another embodiment one or more differences are in the sugar transporter domain at about amino acid residues 31 to 533 of SEQ ID NO: 44.

[2879] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 8105 proteins differ in amino acid sequence from SEQ ID NO: 44, yet retain biological activity.

[2880] In one embodiment, the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 44.

[2881] A 8105 protein or fragment is provided which varies from the sequence of SEQ ID NO: 44 in regions defined by amino acids about 1 to 16 or 534 to 562 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 44 in regions defined by amino acids about 31 to 533. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[2882] In one embodiment, a biologically active portion of a 8105 protein includes a sugar transporter domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 8105 protein.

[2883] In a preferred embodiment, the 8105 protein has an amino acid sequence shown in SEQ ID NO: 44. In other embodiments, the 8105 protein is substantially identical to SEQ ID NO: 44. In yet another embodiment, the 8105 protein is substantially identical to SEQ ID NO: 44 and retains the functional activity of the protein of SEQ ID NO: 44, as described in detail in the subsections above.

[2884] In a preferred embodiment, a 8105 fragment is at least 300, 350, 400, 450, 500, 520, 540, 545, 550, 555, 560, or more amino acid residues in length and differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence in AL137188, AF321240, AF248053, AK055548, or AL031055, or SEQ ID NO: 26 of WO 02/04520, or SEQ ID NO: 2 of WO 02/02586 or WO 02/18621. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO: 44 outside the region encoded by nucleotides 24 to 562 of SEQ ID NO: 44; not include all of the amino acid residues of a sequence encoded by a sequence in AL137188, AF321240, AF248053, AK055548, or AL031055, or SEQ ID NO: 26 of WO 02/04520, or SEQ ID NO: 2 of WO 02/02586 or WO 02/186215, e.g., can be one or more amino acid residues shorter (at one or both ends) than such a sequence; or can differ by one or more amino acid residues in the region of overlap.

[2885] 8105 Chimeric or Fusion Proteins

[2886] In another aspect, the invention provides 8105 chimeric or fusion proteins. As used herein, a 8105 “chimeric protein” or “fusion protein” includes a 8105 polypeptide linked to a non-8105 polypeptide. A “non-8105 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 8105 protein, e.g., a protein which is different from the 8105 protein and which is derived from the same or a different organism. The 8105 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a 8105 amino acid sequence. In a preferred embodiment, a 8105 fusion protein includes at least one (or two) biologically active portion of a 8105 protein. The non-8105 polypeptide can be fused to the N-terminus or C-terminus of the 8105 polypeptide.

[2887] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-8105 fusion protein in which the 8105 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 8105. Alternatively, the fusion protein can be a 8105 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 8105 can be increased through use of a heterologous signal sequence.

[2888] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[2889] The 8105 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 8105 fusion proteins can be used to affect the bioavailability of a 8105 substrate. 8105 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 8105 protein; (ii) mis-regulation of the 8105 gene; and (iii) aberrant post-translational modification of a 8105 protein.

[2890] Moreover, the 8105-fusion proteins of the invention can be used as immunogens to produce anti-8105 antibodies in a subject, to purify 8105 ligands and in screening assays to identify molecules which inhibit the interaction of 8105 with a 8105 substrate.

[2891] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 8105-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 8105 protein.

[2892] Variants of 8105 Proteins

[2893] In another aspect, the invention also features a variant of a 8105 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 8105 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 8105 protein. An agonist of the 8105 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a 8105 protein. An antagonist of a 8105 protein can inhibit one or more of the activities of the naturally occurring form of the 8105 protein by, for example, competitively modulating a 8105-mediated activity of a 8105 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 8105 protein.

[2894] Variants of a 8105 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 8105 protein for agonist or antagonist activity.

[2895] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 8105 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 8105 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[2896] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 8105 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 8105 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[2897] Cell based assays can be exploited to analyze a variegated 8105 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 8105 in a substrate-dependent manner. The transfected cells are then contacted with 8105 and the effect of the expression of the mutant on signaling by the 8105 substrate can be detected, e.g., by measuring transport of a radiolabeled sugar, e.g., glucose, across the cell membrane. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 8105 substrate, and the individual clones further characterized.

[2898] In another aspect, the invention features a method of making a 8105 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 8105 polypeptide, e.g., a naturally occurring 8105 polypeptide. The method includes: altering the sequence of a 8105 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[2899] In another aspect, the invention features a method of making a fragment or analog of a 8105 polypeptide a biological activity of a naturally occurring 8105 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 8105 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[2900] Anti-8105 Antibodies

[2901] In another aspect, the invention provides an anti-8105 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[2902] The anti-8105 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[2903] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[2904] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 8105 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-8105 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH 1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[2905] The anti-8105 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[2906] Phage display and combinatorial methods for generating anti-8105 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 2:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[2907] In one embodiment, the anti-8105 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art.

[2908] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[2909] An anti-8105 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[2910] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fe constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988,J. Natl Cancer Inst. 80:1553-1559).

[2911] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a 8105 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[2912] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[2913] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 8105 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[2914] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[2915] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[2916] In preferred embodiments an antibody can be made by immunizing with purified 8105 antigen, or a fragment thereof, e.g., a fragment described herein, membrane associated antigen, tissue, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g., membrane fractions.

[2917] A full-length 8105 protein or, antigenic peptide fragment of 8105 can be used as an immunogen or can be used to identify anti-8105 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 8105 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 44 and encompasses an epitope of 8105. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[2918] Fragments of 8105 which include residues about 145 to 153, from about 223 to 240, from about 243 to 252, and from about 392 to 407 of SEQ ID NO: 44 can be used to make antibodies against hydrophilic regions of the 8105 protein (see FIG. 21). Similarly, fragments of 8105 which include residues about 70 to 90, from about 98 to 121, from about 319 to 342, and from about 496 to 518 of SEQ ID NO: 44 can be used to make antibodies against a hydrophobic region of the 8105 protein; fragments of 8105 which include residues about 42 to 49, about 122 to 127, about 175 to 187, about 280 to 289, about 343 to 432, about 489 to 495 or a subset thereof, e.g. about residues 370 to 385, or about residues 390 to 410, of SEQ ID NO: 44 can be used to make an antibody against a non-cytoplasmic region (e.g., an extracellular region) of the 8105 protein; fragments of 8105 which include residues about 1 to 16, about 91 to 97, about 145 to 153, about 211 to 254, about 313 to 318, about 457 to 467, or about 519 to 562 of SEQ ID NO: 44 can be used to make an antibody against an intracellular or cytoplasmic region of the 8105 protein; fragments of 8105 which include residues about 31 to 150, about 155 to 225, or about 300 to 400 of SEQ ID NO: 44 can be used to make an antibody against the sugar transporter region of the 8105 protein.

[2919] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[2920] Antibodies which bind only native 8105 protein, only denatured or otherwise non-native 8105 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 8105 protein.

[2921] Preferred epitopes encompassed by the antigenic peptide are regions of 8105 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 8105 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 8105 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[2922] In a preferred embodiment the antibody can bind to the extracellular portion of the 8105 protein, e.g., it can bind to a whole cell which expresses the 8105 protein. In another embodiment, the antibody binds an intracellular portion of the 8105 protein. In preferred embodiments antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g., tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g., membrane fractions.

[2923] The anti-8105 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 8105 protein.

[2924] In a preferred embodiment the antibody has effector function and/or can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement.

[2925] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[2926] In a preferred embodiment, an anti-8105 antibody alters (e.g., increases or decreases) the transport of sugar molecules, e.g., glucose, accross cellular membranes, e.g., the plasma membrane. For example, the antibody can bind at or in proximity to the active site, e.g., to an epitope that includes a residue located from about 42 to 69, 122 to 127, 175 to 187, 280 to 289, 343 to 432, or 489 to 495 of SEQ ID NO: 44.

[2927] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[2928] An anti-8105 antibody (e.g., monoclonal antibody) can be used to isolate 8105 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-8105 antibody can be used to detect 8105 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-8105 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[2929] The invention also includes a nucleic acid which encodes an anti-8105 antibody, e.g., an anti-8105 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[2930] The invention also includes cell lines, e.g., hybridomas, which make an anti-8105 antibody, e.g., an antibody described herein, and method of using said cells to make a 8105 antibody.

[2931] 8105 Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[2932] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[2933] A vector can include a 8105 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 8105 proteins, mutant forms of 8105 proteins, fusion proteins, and the like).

[2934] The recombinant expression vectors of the invention can be designed for expression of 8105 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[2935] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[2936] Purified fusion proteins can be used in 8105 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 8105 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[2937] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[2938] The 8105 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[2939] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[2940] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

[2941] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[2942] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[2943] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 8105 nucleic acid molecule within a recombinant expression vector or a 8105 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein: Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[2944] A host cell can be any prokaryotic or eukaryotic cell. For example, a 8105 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182)). Other suitable host cells are known to those skilled in the art.

[2945] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[2946] A host cell of the invention can be used to produce (i.e., express) a 8105 protein. Accordingly, the invention further provides methods for producing a 8105 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a 8105 protein has been introduced) in a suitable medium such that a 8105 protein is produced. In another embodiment, the method further includes isolating a 8105 protein from the medium or the host cell.

[2947] In another aspect, the invention features, a cell or purified preparation of cells which include a 8105 transgene, or which otherwise misexpress 8105. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a 8105 transgene, e.g., a heterologous form of a 8105, e.g., a gene derived from humans (in the case of a non-human cell). The 8105 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 8105, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 8105 alleles or for use in drug screening.

[2948] In another aspect, the invention features, a human cell, e.g., a hepatic, muscle, endothelial, or neural stem cell, transformed with nucleic acid which encodes a subject 8105 polypeptide.

[2949] Also provided are cells, preferably human cells, e.g., human hepatic, neural, pancreatic, endothelial, muscle or fibroblast cells, in which an endogenous 8105 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 8105 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 8105 gene. For example, an endogenous 8105 gene which is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[2950] In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 8105 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Production of 8105 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 8105 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

[2951] 8105 Transgenic Animals

[2952] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 8105 protein and for identifying and/or evaluating modulators of 8105 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 8105 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[2953] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 8105 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 8105 transgene in its genome and/or expression of 8105 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 8105 protein can further be bred to other transgenic animals carrying other transgenes.

[2954] 8105 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[2955] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[2956] Uses of 8105

[2957] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[2958] The isolated nucleic acid molecules of the invention can be used, for example, to express a 8105 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a 8105 mRNA (e.g., in a biological sample) or a genetic alteration in a 8105 gene, and to modulate 8105 activity, as described further below. The 8105 proteins can be used to treat disorders characterized by insufficient or excessive production of a 8105 substrate or production of 8105 inhibitors. In addition, the 8105 proteins can be used to screen for naturally occurring 8105 substrates, to screen for drugs or compounds which modulate 8105 activity, as well as to treat disorders characterized by insufficient or excessive production of 8105 protein or production of 8105 protein forms which have decreased, aberrant or unwanted activity compared to 8105 wild type protein (e.g., metabolic or sugar transport-related disorders, e.g., obesity, and related disorders such as hormonal disorders, hypertension, hyperphagia, and cardiovascular disorders). Moreover, the anti-8105 antibodies of the invention can be used to detect and isolate 8105 proteins, regulate the bioavailability of 8105 proteins, and modulate 8105 activity.

[2959] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 8105 polypeptide is provided. The method includes: contacting the compound with the subject 8105 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 8105 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 8105 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 8105 polypeptide. Screening methods are discussed in more detail below.

[2960] 8105 Screening Assays

[2961] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 8105 proteins, have a stimulatory or inhibitory effect on, for example, 8105 expression or 8105 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 8105 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 8105 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[2962] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 8105 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 8105 protein or polypeptide or a biologically active portion thereof.

[2963] In one embodiment, an activity of a 8105 protein can be assayed by transforming a cell with an expression plasmid containing a 8105 nucleic acid molecule, expressing the 8105 protein, and adding radiolabeled sugars, e.g., radiolabeled glucose, to the cell culture medium. Determination of the activity of the 8105 protein can be performed by measuring the uptake of the radiolabeled sugar molecules form the cell culture medium using standard techniques known in the art.

[2964] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

[2965] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[2966] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[2967] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 8105 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 8105 activity is determined. Determining the ability of the test compound to modulate 8105 activity can be accomplished by monitoring, for example, the transport of sugar molecules, e.g., glucose molecules, across the plasma membrane. The cell, for example, can be of mammalian origin, e.g., human.

[2968] The ability of the test compound to modulate 8105 binding to a compound, e.g., a 8105 substrate, or to bind to 8105 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 8105 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 8105 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 8105 binding to a 8105 substrate in a complex. For example, compounds (e.g., 8105 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[2969] The ability of a compound (e.g., a 8105 substrate) to interact with 8105 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 8105 without the labeling of either the compound or the 8105. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 8105.

[2970] In yet another embodiment, a cell-free assay is provided in which a 8105 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 8105 protein or biologically active portion thereof is, evaluated. Preferred biologically active portions of the 8105 proteins to be used in assays of the present invention include fragments which participate in interactions with non-8105 molecules, e.g., fragments with high surface probability scores.

[2971] Soluble and/or membrane-bound forms of isolated proteins (e.g., 8105 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[2972] Cell-free assays involve preparing a reaction mixture of the target gene protein, test compound, and an 8105 binding partner, e.g., a substrate, e.g., a sugar molecule, e.g., glucose, under conditions and for a time sufficient to allow the three components to interact and bind, thus forming a complex that can be removed and/or detected. For example, in the absence of the test compound, the 8105 binding partner, e.g., substrate, might stably bind to the 8105 protein, while such an interaction might be abrogated in the presence of the test compound.

[2973] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[2974] In another embodiment, determining the ability of the 8105 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[2975] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[2976] It may be desirable to immobilize either 8105, an anti-8105 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 8105 protein, or interaction of a 8105 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/8105 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 8105 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 8105 binding or activity determined using standard techniques.

[2977] Other techniques for immobilizing either a 8105 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 8105 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[2978] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[2979] In one embodiment, this assay is performed utilizing antibodies reactive with 8105 protein or target molecules but which do not interfere with binding of the 8105 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 8105 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 8105 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 8105 protein or target molecule.

[2980] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[2981] In a preferred embodiment, the assay includes contacting the 8105 protein or biologically active portion thereof with a known compound which binds 8105 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 8105 protein, wherein determining the ability of the test compound to interact with a 8105 protein includes determining the ability of the test compound to preferentially bind to 8105 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[2982] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 8105 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a 8105 protein through modulation of the activity of a downstream effector of a 8105 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[2983] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[2984] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[2985] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[2986] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[2987] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[2988] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[2989] In yet another aspect, the 8105 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 8105 (“8105-binding proteins” or “8105-bp”) and are involved in 8105 activity. Such 8105-bps can be activators or inhibitors of signals by the 8105 proteins or 8105 targets as, for example, downstream elements of a 8105-mediated signaling pathway.

[2990] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 8105 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 8105 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 8105-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 8105 protein.

[2991] In another embodiment, modulators of 8105 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 8105 mRNA or protein evaluated relative to the level of expression of 8105 mRNA or protein in the absence of the candidate compound. When expression of 8105 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 8105 mRNA or protein expression. Alternatively, when expression of 8105 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 8105 mRNA or protein expression. The level of 8105 mRNA or protein expression can be determined by methods described herein for detecting 8105 mRNA or protein.

[2992] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a 8105 protein can be confirmed in vivo, e.g., in an animal such as an animal model for obesity.

[2993] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 8105 modulating agent, an antisense 8105 nucleic acid molecule, a 8105-specific antibody, or a 8105-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[2994] 8105 Detection Assays

[2995] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 8105 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[2996] 8105 Chromosome Mapping

[2997] The 8105 nucleotide sequences or portions thereof can be used to map the location of the 8105 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 8105 sequences with genes associated with disease.

[2998] Briefly, 8105 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 8105 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 8105 sequences will yield an amplified fragment.

[2999] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[3000] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 8105 to a chromosomal location.

[3001] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[3002] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[3003] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[3004] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 8105 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[3005] 8105 Tissue Typing

[3006] 8105 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[3007] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 8105 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[3008] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 43 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 45 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[3009] If a panel of reagents from 8105 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[3010] Use of Partial 8105 Sequences in Forensic Biology

[3011] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[3012] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 43 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 43 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[3013] The 8105 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 8105 probes can be used to identify tissue by species and/or by organ type.

[3014] In a similar fashion, these reagents, e.g., 8105 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[3015] Predictive Medicine of 8105

[3016] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[3017] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 8105.

[3018] Such disorders include, e.g., a disorder associated with the misexpression of 8105 gene; a disorder involving the regulation of metabolism, e.g., a disorder associated with obesity, or a related disorders.

[3019] The method includes one or more of the following:

[3020] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 8105 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[3021] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 8105 gene;

[3022] detecting, in a tissue of the subject, the misexpression of the 8105 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[3023] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 8105 polypeptide.

[3024] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 8105 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[3025] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 43, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 8105 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[3026] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 8105 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 8105.

[3027] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[3028] In preferred embodiments the method includes determining the structure of a 8105 gene, an abnormal structure being indicative of risk for the disorder.

[3029] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 8105 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[3030] Diagnostic and Prognostic Assays of 8105

[3031] Diagnostic and prognostic assays of the invention include method for assessing the expression level of 8105 molecules and for identifying variations and mutations in the sequence of 8105 molecules.

[3032] Expression Monitoring and Profiling:

[3033] The presence, level, or absence of 8105 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 8105 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 8105 protein such that the presence of 8105 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 8105 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 8105 genes; measuring the amount of protein encoded by the 8105 genes; or measuring the activity of the protein encoded by the 8105 genes.

[3034] The level of mRNA corresponding to the 8105 gene in a cell can be determined both by in situ and by in vitro formats.

[3035] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 8105 nucleic acid, such as the nucleic acid of SEQ ID NO: 43, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 8105 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[3036] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 8105 genes.

[3037] The level of mRNA in a sample that is encoded by one of 8105 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[3038] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 8105 gene being analyzed.

[3039] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 8105 mRNA, or genomic DNA, and comparing the presence of 8105 mRNA or genomic DNA in the control sample with the presence of 8105 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 8105 transcript levels.

[3040] A variety of methods can be used to determine the level of protein encoded by 8105. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[3041] The detection methods can be used to detect 8105 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 8105 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 8105 protein include introducing into a subject a labeled anti-8105 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-8105 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[3042] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 8105 protein, and comparing the presence of 8105 protein in the control sample with the presence of 8105 protein in the test sample.

[3043] The invention also includes kits for detecting the presence of 8105 in a biological sample. For example, the kit can include a compound or agent capable of detecting 8105 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 8105 protein or nucleic acid.

[3044] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[3045] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[3046] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 8105 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as obesity and/or related disorders such as diabetes, hormonal disorders, hypertension, hyperphagia, and cardiovascular disorders.

[3047] In one embodiment, a disease or disorder associated with aberrant or unwanted 8105 expression or activity is identified. A test sample is obtained from a subject and 8105 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 8105 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 8105 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[3048] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 8105 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a metabolic or sugar transport-related disorders, e.g., obesity and/or related disorders such as diabetes, hormonal disorders, hypertension, hyperphagia, and cardiovascular disorders.

[3049] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 8105 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 8105 (e.g., other genes associated with a 8105-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[3050] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 8105 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose a metabolic or sugar transport-related disorder, e.g., obesity and/or a related disorder such as diabetes, a hormonal disorder, hypertension, hyperphagia, or a cardiovascular disorder in a subject wherein an increase in 8105 expression is an indication that the subject has or is disposed to having a metabolic or sugar transport-related disorder. The method can be used to monitor a treatment for a metabolic or sugar transport-related disorder, e.g., obesity and/or a related disorder such as diabetes, a hormonal disorder, hypertension, hyperphagia, or a cardiovascular disorder in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[3051] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 8105 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[3052] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 8105 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[3053] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[3054] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 8105 expression.

[3055] 8105 Arrays and Uses Thereof

[3056] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a 8105 molecule (e.g., a 8105 nucleic acid or a 8105 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm2, and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[3057] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a 8105 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 8105. Each address of the subset can include a capture probe that hybridizes to a different region of a 8105 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for a 8105 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 8105 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 8105 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[3058] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[3059] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a 8105 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 8105 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-8105 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[3060] In another aspect, the invention features a method of analyzing the expression of 8105. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 8105-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[3061] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 8105. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 8105. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[3062] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 8105 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[3063] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[3064] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 8105-associated disease or disorder; and processes, such as a cellular transformation associated with a 8105-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 8105-associated disease or disorder.

[3065] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 8105) that could serve as a molecular target for diagnosis or therapeutic intervention.

[3066] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon a 8105 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1 999). Anal. Biochem. 270, 103-111 ; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80, 85, 90, 95 or 99% identical to a 8105 polypeptide or fragment thereof. For example, multiple variants of a 8105 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[3067] The polypeptide array can be used to detect a 8105 binding compound, e.g., an antibody in a sample from a subject with specificity for a 8105 polypeptide or the presence of a 8105-binding protein or ligand.

[3068] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 8105 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[3069] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 8105 or from a cell or subject in which a 8105 mediated response has been elicited, e.g., by contact of the cell with 8105 nucleic acid or protein, or administration to the cell or subject 8105 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 8105 (or does not express as highly as in the case of the 8105 positive plurality of capture probes) or from a cell or subject which in which a 8105 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 8105 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[3070] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 8105 or from a cell or subject in which a 8105-mediated response has been elicited, e.g., by contact of the cell with 8105 nucleic acid or protein, or administration to the cell or subject 8105 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 8105 (or does not express as highly as in the case of the 8105 positive plurality of capture probes) or from a cell or subject which in which a 8105 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[3071] In another aspect, the invention features a method of analyzing 8105, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 8105 nucleic acid or amino acid sequence; comparing the 8105 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 8105.

[3072] Detection of 8105 Variations or Mutations

[3073] The methods of the invention can also be used to detect genetic alterations in a 8105 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 8105 protein activity or nucleic acid expression, such as a metabolic or sugar transport-related disorder, e.g., obesity and/or a related disorder such as diabetes, a hormonal disorder, hypertension, hyperphagia, or a cardiovascular disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 8105-protein, or the mis-expression of the 8105 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 8105 gene; 2) an addition of one or more nucleotides to a 8105 gene; 3) a substitution of one or more nucleotides of a 8105 gene, 4) a chromosomal rearrangement of a 8105 gene; 5) an alteration in the level of a messenger RNA transcript of a 8105 gene, 6) aberrant modification of a 8105 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a 8105 gene, 8) a non-wild type level of a 8105-protein, 9) allelic loss of a 8105 gene, and 10) inappropriate post-translational modification of a 8105-protein.

[3074] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 8105-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 8105 gene under conditions such that hybridization and amplification of the 8105-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[3075] In another embodiment, mutations in a 8105 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[3076] In other embodiments, genetic mutations in 8105 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 8105 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 8105 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7:244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations in 8105 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[3077] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 8105 gene and detect mutations by comparing the sequence of the sample 8105 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[3078] Other methods for detecting mutations in the 8105 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[3079] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 8105 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[3080] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 8105 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 8105 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[3081] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[3082] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[3083] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[3084] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 8105 nucleic acid.

[3085] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 43 or the complement of SEQ ID NO: 43. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[3086] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 8105. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[3087] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the Tm of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[3088] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 8105 nucleic acid.

[3089] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 8105 gene.

[3090] Use of 8105 Molecules as Surrogate Markers

[3091] The 8105 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 8105 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 8105 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HW RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35:258-264; and James (1994) AIDS Treatment News Archive 209.

[3092] The 8105 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 8105 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-8105 antibodies may be employed in an immune-based detection system for a 8105 protein marker, or 8105-specific radiolabeled probes may be used to detect a 8105 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90:229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[3093] The 8105 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 8105 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 8105 DNA may correlate 8105 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[3094] Pharmaceutical Compositions of 8105

[3095] The nucleic acid and polypeptides, fragments thereof, as well as anti-8105 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[3096] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[3097] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[3098] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[3099] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[3100] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[3101] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[3102] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[3103] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[3104] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[3105] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[3106] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[3107] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[3108] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[3109] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[3110] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[3111] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactive ions include, but are not limited to iodine, yttrium and praseodymium.

[3112] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[3113] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[3114] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[3115] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[3116] Methods of Treatment for 8105

[3117] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 8105 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[3118] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 8105 molecules of the present invention or 8105 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[3119] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 8105 expression or activity, by administering to the subject a 8105 or an agent which modulates 8105 expression or at least one 8105 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 8105 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 8105 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 8105 aberrance, for example, a 8105, 8105 agonist or 8105 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[3120] It is possible that some 8105 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[3121] The 8105 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of metabolic disorders, hormonal disorders, neurological disorders, pancreatic disorders, liver disorders, kidney disorders, cardiovascular disorders, blood vessel disorders, pain disorders, disorders of bone metabolism, and cellular proliferative and/or differentiative disorders, as discussed above.

[3122] As discussed, successful treatment of 8105 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 8105 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[3123] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[3124] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[3125] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 8105 expression is through the use of aptamer molecules specific for 8105 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1:5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 8105 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[3126] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 8105 disorders. For a description of antibodies, see the Antibody section above.

[3127] In circumstances wherein injection of an animal or a human subject with a 8105 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 8105 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 8105 protein. Vaccines directed to a disease characterized by 8105 expression may also be generated in this fashion.

[3128] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[3129] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 8105 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[3130] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[3131] Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 8105 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 8105 can be readily monitored and used in calculations of IC50.

[3132] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[3133] Another aspect of the invention pertains to methods of modulating 8105 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 8105 or agent that modulates one or more of the activities of 8105 protein activity associated with the cell. An agent that modulates 8105 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a 8105 protein (e.g., a 8105 substrate or receptor), a 8105 antibody, a 8105 agonist or antagonist, a peptidomimetic of a 8105 agonist or antagonist, or other small molecule.

[3134] In one embodiment, the agent stimulates one or 8105 activities. Examples of such stimulatory agents include active 8105 protein and a nucleic acid molecule encoding 8105. In another embodiment, the agent inhibits one or more 8105 activities. Examples of such inhibitory agents include antisense 8105 nucleic acid molecules, anti-8105 antibodies, and 8105 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 8105 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 8105 expression or activity. In another embodiment, the method involves administering a 8105 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 8105 expression or activity.

[3135] Stimulation of 8105 activity is desirable in situations in which 8105 is abnormally downregulated and/or in which increased 8105 activity is likely to have a beneficial effect. For example, stimulation of 8105 activity is desirable in situations in which a 8105 is downregulated and/or in which increased 8105 activity is likely to have a beneficial effect. Likewise, inhibition of 8105 activity is desirable in situations in which 8105 is abnormally upregulated and/or in which decreased 8105 activity is likely to have a beneficial effect.

[3136] 8105 Pharmacogenomics

[3137] The 8105 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 8105 activity (e.g., 8105 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 8105 associated disorders (e.g., metabolic or sugar transport-related disorders, e.g., obesity and/or related disorders such as diabetes, hormonal disorders, hypertension, hyperphagia, and cardiovascular disorders) associated with aberrant or unwanted 8105 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 8105 molecule or 8105 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 8105 molecule or 8105 modulator.

[3138] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[3139] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[3140] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 8105 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[3141] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 8105 molecule or 8105 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[3142] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 8105 molecule or 8105 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[3143] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 8105 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 8105 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[3144] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a 8105 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 8105 gene expression, protein levels, or upregulate 8105 activity, can be monitored in clinical trials of subjects exhibiting decreased 8105 gene expression, protein levels, or downregulated 8105 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 8105 gene expression, protein levels, or downregulate 8105 activity, can be monitored in clinical trials of subjects exhibiting increased 8105 gene expression, protein levels, or upregulated 8105 activity. In such clinical trials, the expression or activity of a 8105 gene, and preferably, other genes that have been implicated in, for example, a 8105-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[3145] 8105 Informatics

[3146] The sequence of a 8105 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 8105. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 8105 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[3147] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[3148] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[3149] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[3150] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[3151] Thus, in one aspect, the invention features a method of analyzing 8105, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 8105 nucleic acid or amino acid sequence; comparing the 8105 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 8105. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[3152] The method can include evaluating the sequence identity between a 8105 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[3153] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[3154] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[3155] Thus, the invention features a method of making a computer readable record of a sequence of a 8105 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[3156] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 8105 sequence, or record, in machine-readable form; comparing a second sequence to the 8105 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 8105 sequence includes a sequence being compared. In a preferred embodiment the 8105 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 8105 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[3157] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 8105-associated disease or disorder or a pre-disposition to a 8105-associated disease or disorder, wherein the method comprises the steps of determining 8105 sequence information associated with the subject and based on the 8105 sequence information, determining whether the subject has a 8105-associated disease or disorder or a pre-disposition to a 8105-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[3158] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 8105-associated disease or disorder or a pre-disposition to a disease associated with a 8105 wherein the method comprises the steps of determining 8105 sequence information associated with the subject, and based on the 8105 sequence information, determining whether the subject has a 8105-associated disease or disorder or a pre-disposition to a 8105-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 8105 sequence of the subject to the 8105 sequences in the database to thereby determine whether the subject as a 8105-associated disease or disorder, or a pre-disposition for such.

[3159] The present invention also provides in a network, a method for determining whether a subject has a 8105 associated disease or disorder or a pre-disposition to a 8105-associated disease or disorder associated with 8105, said method comprising the steps of receiving 8105 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 8105 and/or corresponding to a 8105-associated disease or disorder (e.g., a metabolic or sugar transport-related disorder, e.g., obesity and/or a related disorder such as diabetes, a hormonal disorder, hypertension, hyperphagia, or a cardiovascular disorder), and based on one or more of the phenotypic information, the 8105 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 8105-associated disease or disorder or a pre-disposition to a 8105-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[3160] The present invention also provides a method for determining whether a subject has a 8105-associated disease or disorder or a pre-disposition to a 8105-associated disease or disorder, said method comprising the steps of receiving information related to 8105 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 8105 and/or related to a 8105-associated disease or disorder, and based on one or more of the phenotypic information, the 8105 information, and the acquired information, determining whether the subject has a 8105-associated disease or disorder or a pre-disposition to a 8105-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[3161] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

EXAMPLES

Examples for 52906, 33408, and 12189

Example 1

[3162] Identification and Characterization of Human 52906, 33408, and 12189 cDNAs

[3163] The human 52906 nucleic acid sequence is recited as follows:

3(SEQ ID NO:1)GCGTCCGCAGATTCCAGAGCCTGCCGGCTGGGAAAGATCCGGTCTCGGGGTCGGCTATGATCCCGCAGCGGCCAAGGCAGGGCTCAGGCCCCGGGATTCTCCCCACACGCTGCTGCACTGGCGCAGCCGGTCGCCAAACTTTTTCTCCCCAAAGCCAGTGCCCCCGCAGTTACTTGGCGGGCAGCCGGCAGCCCACTCTCGGCGGGATGATCTGGGAGAAGCGGGCGTGGGACGAGGGGGCTGCTGTTTTGCAGCCCTGCGAGGCGTGCAGTCGGAGAAGTGGTCGGGGTTCCACACCGTCCCTGAGCCTGCCCCCTGGCCAAGGTGGCCCGACGTGCTGCAGTGGCTGGCGCAGGTGATCCGGGCAGCGCGTCCGGCACTAGTCAAGGGGGCAGCGGCACGGGAGGGAGGGGCGCCTTTCTCTTTTCTCCTCCCCCTGCAGCCCAGCTGCACTGCGTGGGGGCTCTCCATCTCCACGCAATCAGCAGGCGGAATCCCTGCCCTGGAGCGCCCTGGCTCTGGACTGCACCCCCCTAGGGTTTGTCCTGCAGATTCTCCTCCCCATCTTTCTCTGCCACACACGCTTCCCTAAGCCGCGCGCGCCGCAAACTCAGTCTCGGTCCCCGCAGGTGATGTCATGCCCATTGTTTTGGTGCGCCCAACCAATCGGACTCGCCGCCTGGATTCTACCGGAGCCGGCATGGGCCCTTCCTCGCACCAGCAGCAGGAGTCCCCGCTCCCGACCATAACGCATTGCGCAGGGTGCACCACCGCTTGGTCTCCCTGCAGCTTTAACAGCCCTGACATGGAAACCCCATTGCAGTTCCAGCGCGGCTTCTTCCCAGAGCAGCCGCCGCCGCCGCCGCGCTCCTCACACCTGCATTGCCAGCAGCAGCAACAGAGCCAGGACAAGCCGTGCCCGCCCTTCGCGCCCCTCCCGCACCCTCACCACCACCCGCACCTCGCGCACCAGCAGCCGGCCAGCGGCGGCAGCAGCCCATGCCTCCGGTGCAACAGCTGCGCCTCCTCCGGTGCCCCGGCGGCGGGGGCGGGAGATAACCTGTCCCTGCTGCTCCGCACCTCCTCGCCCGGCGGCGCCTTCCGGACCCGCACCTCCTCGCCGCTGTCGGGCTCGTCCTGCTGCTGCTGCTGCTGCTCGTCGCGCCGGGGCAGCCAGCTCAATGTGAGCGAGCTGACGCCGTCCAGCCATGCCAGTGCGCTCCGGCAGCAGTACGCGCAGCAGTCCGCGCAGCAGTCGGCGTCCGCCTCCCAGTACCACCAGTGCCACAGCCTGCAGCCCGCCGCCAGCCCCACGGGCAGCCTCGGCAGTCTGGGCTCCGGGCCCCCGCTCTCGCACCACCACCACCACCCGCACCCGGCGCACCACCAGCACCACCAGCCCCAGGCGCGCCGCGAGAGCAACCCCTTCACCGAAATAGCCATGAGCAGCTGCAGGTACAACGGGGGCGTCATGCGGCCGCTCAGCAACTTGAGCGCGTCCCGCCGGAACCTGCACGAGATGGACTCAGAGGCGCAGCCCCTGCAGCCCCCCGCGTCTGTCGGAGGAGGTGGCGGCGCGTCCTCCCCGTCTGCAGCCGCTGCCGCCGCCGCCGCTGTTTCGTCCTCAGCCCCCGAGATCGTGGTGTCTAAGCCCGAGCACAACAACTCCAACAACCTGGCGCTCTATGGAACCGGCGGCGGAGGCAGCACTGGAGGAGGCGGCGGCGGTGGCGGGAGCGGGCACGGCAGCAGCAGTGGCACCAAGTCCAGCAAAAAGAAAAACCAGAACATCGGCTACAAGCTGGGCCACCGGCGCGCCCTGTTCGAAAAGCGCAAGCGGCTCAGCGACTACGCGCTCATCTTCGGCATGTTCGGCATCGTGGTCATGGTCATCGAGACCGAGCTGTCGTGGGGCGCCTACGACAAGGCGTCGCTGTATTCCTTAGCTCTGAAATGCCTTATCAGTCTCTCCACGATCATCCTGCTCGGTCTGATCATCGTGTACCACGCCAGGGAAATACAGTTGTTCATGGTGGACAATGGAGCAGATGACTGGAGAATAGCCATGACTTATGAGCGTATTTTCTTCATCTGCTTGGAAATACTGGTGTGTGCTATTCATCCCATACCTGGGAATTATACATTCACATGGACGGCCCGGCTTGCCTTCTCCTATGCCCCATCCACAACCACCGCTGATGTGGATATTATTTTATCTATACCAATGTTCTTAAGACTCTATCTGATTGCCAGAGTCATGCTTTTACATAGCAAACTTTTCACTGATACCTCCTCTAGAAGCATTGGAGCACTTAATAAGATAAACTTCAATACACGTTTTGTTATGAAGACTTTAATGACTATATGCCCAGGAACTGTACTCTTGGTTTTTAGTATCTCATTATGGATAATTGCCGCATGGACTGTCCGAGCTTGTGAAAGGTACCATGATCAACAGCTCCATTGGTTATGGTGACATGGTACCTAACACATACTGTGGAAAAGGAGTCTGCTTACTTACTGGAATTATGGGTGCTGGTTGCACAGCCCTGGTGGTAGCTGTAGTGGCAAGGAAGCTAGAACTTACCAAAGCAGAAAAACACGTGCACAATTTCATGATGGATACTCAGCTGACTAAAAGAGTAAAAAATGCAGCTGCCAATGTACTCAGGGAAACATGGCTAATTTACAAAAATACAAAGCTAGTGAAAAAGATAGATCATGCAAAAGTAAGAAAACATCAACGAAAATTCCTGCAAGCTATTCATCAATTAAGAAGTGTAAAAATGGAGCAGAGGAAACTGAATGACCAAGCAAACACTTTGGTGGACTTGGCAAAGACCCAGAACATCATGTATGATATGATTTCTGACTTAAACGAAAGGAGTGAAGACTTCGAGAAGAGGATTGTTACCCTGGAAACAAAACTAGAGACTTTGATTGGTAGCATCCACGCCCTCCCTGGGCTCATAAGCCAGACCATCAGGCAGCAGCAGAGAGATTTCATTGAGGCTCAGATGGAGAGCTACGACAAGCACGTCACTTACAATGCTGAGCGGTCCCGGTCCTCGTCCAGGAGGCGGCGGTCCTCTTCCACAGCACCACCAACTTCATCAGAGAGTAGCTAGAAGAGAATAAGTTAACCACAAAATAAGACTTTTTGCCATCATATGGTCAATATTTTAGCTTTTATTGTAAAGCCCCTATGGTTCTAATCAGCGTTATCCGGGTTCTGATGTCAGAATCCTGGGAACCTGAACACTAAGTTTTAGGCCAAAATGAGTGAAAACTCTTTTTTTTTCTTTCAGATGCACAGGGAATGCACCTATTATTGCTATATAGATTGTTCCTCCTGTAATTTCACTAACTTTTTATTCATGCACTTCAAACAAACTTTACTACTACATTATATGATATATAATAAAAAAAGTTAATTTCTGCAAAAAAAAAAAAAAAAAAAAAAACGGACGGG.

[3164] The human 52906 sequence (FIG. 1; SEQ ID NO: 1) is approximately 3525 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and, a termination codon (TAG), which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 2544 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 1; SEQ ID NO: 3). The coding sequence encodes a 847 amino acid protein (SEQ ID NO: 2), which is recited as follows:

4(SEQ ID NO:2)MPIVLVRPTNRTRRLDSTGAGMGPSSHQQQESPLPTITHCAGCTTAWSPCSFNSPDMETPLQFQRGFFPEQPPPPPRSSHLHCQQQQQSQDKPCPPFAPLPHPHHHPHLAHQQPASGGSSPCLRCNSCASSGAPAAGAGDNLSLLLRTSSPGGAFRTRTSSPLSGSSCCCCCSSRRGSQLNVSELTPSSHASALRQQYAQQSAQQSASASQYHQCHSLQPAASPTGSLGSLGSGPPLSHHHHHPHPAHHQHHQPQARRESNPFTEIAMSSCRYNGGVMRPLSNLSASRRNLHEMDSEAQPLQPPASVGGGGGASSPSAAAAAAAAVSSSAPEIVVSKPEHNNSNNLALYGTGGGGSTGGGGGGGGSGHGSSSGTKSSKKKNQNIGYKLGHRRALFEKRKRLSDYALIFGMFGIVVMVIETELSWGAYDKASLYSLALKCLISLSTIILLGLIIVYHAREIQLFMVDNGADDWRIAMTYERIFFICLEILVCAIHPIPGNYTFTWTARLAFSYAPSTTTADVDIILSIPMFLRLYLIARVMLLHSKLFTDTSSRSIGALNKINFNTRFVMKTLMTICPGTVLLVFSISLWIIAAWTVRACERYHDQQDVTSNFLGAMWLISITFLSIGYGDMVPNTYCGKGVCLLTGIMGAGCTALVVAVVARKLELTKAEKHVHNFMMDTQLTKRVKNAAANVLRETWLIYKNTKLVKKIDHAKVRKHQRKFLQAIHQLRSVKMEQRKLNDQANTLVDLAKTQNIMYDMISDLNERSEDFEKRIVTLETKLETLIGSIHALPGLISQTIRQQQRDFIEAQMESYDKHVTYNAERSRSSSRRRRSSSTAPPTSSESS.

[3165] The human 33408 nucleic acid sequence is recited as follows:

5(SEQ ID NO:4)GACCCACGCGTCCGCTCCCCCGTGTGCGGCACCGCCACAGTCTGGGCAGCGGCGGCCGGGGGAGCGCTACTACCATGAACTGCCTGGTCCTCCTCCCCAGAGCTGCTCATCCGGGTCGGGCTGGAGACACAGTCAGGGGACCCCGTCGCCGCCGCCGCGCCCCCTCTTCTTTCGGCTCAATCTTCTCTTCCACCTTTTCCTCCTCTTCCTCCACCTTCTTTGCCTGCATCCCCCCCTCCCCCGCCGCGGATCCTGGCCGCTGCTCTCCAGACCCAGGATGCCGGGGGGCAAGAGAGGGCTGGTGGCACCGCAGAACACATTTTTGGAGAACATCGTCAGGCGCTCCAGTGAATCAAGTTTCTTACTGGGAAATGCCCAGATTGTGGATTGGCCTGTAGTTTATAGTAATGACGGTTTTTGTAAACTCTCTGGATATCATCGAGCTGACGTCATGCAGAAAAGCAGCACTTGCAGTTTTATGTATGGGGAATTGACTGACAAGAAGACCATTGAGAAAGTCAGGCAAACTTTTGACAACTACGAATCAAACTGCTTTGAAGTTCTTCTGTACAAGAAAAACAGAACCCCTGTTTGGTTTTATATGCAAATTGCACCAATAAGAAATGAACAACGGGCTTTGACAAATAGCCGAAGTGTTTTGCAGCAGCTCACGCCAATGAATAAAACAGAGGTGGTCCATAAACATTCAAGACTAGCTGAAGTTCTTCAGCTGGGATCAGATATCCTTCCTCAGTATAAACAAGAAGCGCCAAAGACGCCACCACACATTATTTTACATTATTGTGCTTTTAAAACTACTTGGGATTGGGTGATTTTAATTCTTACCTTCTACACCGCCATTATGGTTCCTTATAATGTTTCCTTCAAAACAAAGCAGAACAACATAGCCTGGCTGGTACTGGATAGTGTGGTGGACGTTATTTTTCTGGTTGACATCGTTTTAAATTTTCACACGACTTTCGTGGGGCCCGGTGGAGAGCGACTGGGCCGTGTGGCTAGGAAACTGGACCATTACCTAGAATATGGAGCAGCAGTCCTCGTGCTCCTGGTGTGTGTGTTTGGACTGGTGGCCCACTGGCTGGCCTGCATATGGTATAGCATCGGAGACTACGAGGTCATTGATGAAGTCACTAACACCATCCAAATAGACAGTTGGCTCTACCAGCTGGCTTTGAGCATTGGGACTCCATATCGCTACAATACCAGTGCTGGGATATGGGAAGGAGGACCCAGCAAGGATTCATTGTACGTGTCCTCTCTCTACTTTACCATGACAAGCCTTACAACCATAGGATTTGGAAACATAGCTCCTACCACAGATGTGGAGAAGATGTTTTCGGTGGCTATGATGATGGTTGGCTCTCTTCTTTATGCAACTATTTTTGGAAATGTTACAACAATTTTCCAGCAAATGTATGCCAACACCAACCGATACCATGAGATGCTGAATAATGTACGGGACTTCCTAAAACTCTATCAGGTCCCAAAAGGCCTTAGTGAGCGAGTCATGGATTATATTGTCTCAACATGGTCCATGTCAAAAGGCATTGATACAGAAAAGGTCCTCTCCATCTGTCCCAAGGACATGAGAGCTGATATCTGTGTTCATCTAAACCGGAAGGTTTTTAATGAACATCCTGCTTTTCGATTGGCCAGCGATGGGTGTCTGCGCGCCTTGGCGGTAGAGTTCCAAACCATTCACTGTGCTCCCGGGGACCTCATTTACCATGCTGGAGAAAGTGTGGATGCCCTCTGCTTTGTGGTGTCAGGATCCTTGGAAGTCATCCAGGATGATGAGGTGGTGGCTATTTTAGGGAAGGGTGATGTATTTGGAGACATCTTCTGGAAGGAAACCACCCTTGCCCATGCATGTGCGAACGTCCGGGCACTGACGTACTGTGACCTACACATCATCAAGCGGGAAGCCTTGCTCAAAGTCCTGGACTTTTATACAGCTTTTGCAAACTCCTTCTCAAGGAATCTCACTCTTACTTGCAATCTGAGGAAACGGATCATCTTTCGTAAGATCAGTGATGTGAAGAAAGAGGAGGAGGAGCGCCTCCGGCAGAAGAATGAGGTGACCCTCAGCATTCCCGTGGACCACCCAGTCAGAAAGCTCTTCCAGAAGTTCAAGCAGCAGAAGGAGCTGCGGAATCAGGGCTCAACACAGGGTGACCCTGAGAGGAACCAACTCCAGGTAGAGAGCCGCTCCTTACAGAATGGAACCTCCATCACCGGAACCAGCGTGGTGACTGTGTCACAGATTACTCCCATTCAGACGTCTCTGGCCTATGTGAAAACCAGTGAATCCCTTAAGCAGAACAACCGTGATGCCATGGAACTCAAGCCCAACGGCGGTGCTGACCAAAAATGTCTCAAAGTCAACAGCCCAATAAGAATGAAGAATGGAAATGGAAAAGGGTGGCTGCGACTCAAGAATAATATGGGAGCCCATGAGGAGAAAAAGGAAGACTGGAATAATGTCACTAAAGCTGAGTCAATGGGGCTATTGTCTGAGGACCCCAAGAGCAGTGATTCAGAGAACAGTGTGACCAAAAACCCACTAAGGAAAACAGATTCTTGTGACAGTGGAATTACAAAAAGTGACCTTCGTTTGGATAAGGCTGGGGAGGCCCGAAGTCCGCTAGAGCACAGTCCCATCCAGGCTGATGCCAAGCACCCCTTTTATCCCATCCCCGAGCAGGCCTTACAGACCACACTGCAGGAAGTCAAACACGAACTCAAAGAGGACATCCAGCTGCTCAGCTGCAGAATGACTGCCCTAGAAAAGCAGGTGGCAGAAATTTTAAAAATACTGTCGGAAAAAAGCGTACCCCAGGCCTCATCTCCCAAATCCCAAATGCCACTCCAAGTACCCCCCCAGATACCATGTCAGGATATTTTTAGTGTCTCAAGGCCTGAATCACCTGAATCTGACAAAGATGAAATCCACTTTTAATATATATACATATATATTTGTTAATATATTAAAACAGTATATACATATGTGTGTATATACAGTATATACATATATATATTTTCACTTGCTTTCAAGATGATGACCACACATGGATTTTGATATGTAAATATTGCATGTCCAGCTGGATTCTGGCCTGCCAAAGAAGATGATGATTAAAAACATAGATATTGCTTGTATATTATGCAGTTGACTGCATGCACACTTTACATTTATTTATAATCTCTATTCTATAATAAAAGAGTATGATTTTTGTTAAAAAAAAAAAAAAAAAAAAAATTCCTCGCCGGA

[3166] The human 33408 sequence (FIG. 3; SEQ ID NO: 4) is approximately 3553 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA), which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 2967 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 4; SEQ ID NO: 6). The coding sequence encodes a 988 amino acid protein (SEQ ID NO: 5), which is recited as follows:

6(SEQ ID NO:5)MPGGKRGLVAPQNTFLENIVRRSSESSFLLGNAQIVDWPVVYSNDGFCKLSGYHRADVMQKSSTCSFMYGELTDKKTIEKVRQTFDNYESNCFEVLLYKKNRTPVWFYMQIAPIRNEHEKVVLFLCTFKDITLFKQPIEDDSTKGWTKFARLTRALTNSRSVLQQLTPMNKTEVVHKHSRLAEVLQLGSDILPQYKQEAPKTPPHIILHYCAFKTTWDWVILILTFYTAIMVPYNVSFKTKQNNIAWLVLDSVVDVIFLVDIVLNFHTTFVGPGGEVISDPKLIRMNYLKTWFVIDLLSCLPYDIINAFENVDEGISSLFSSLKVVRLLRLGRVARKLDHYLEYGAAVLVLLVCVFGLVAHWLACIWYSIGDYEVIDEVTNTIQIDSWLYQLALSIGTPYRYNTSAGIWEGGPSKDSLYVSSLYFTMTSLTTIGFGNIQPTTDVEKMFSVAMMMVGSLLYATIFGNVTTIFQQMYANTNRYHEMLNNVRDFLKLYQVPKGLSERVMDYIVSTWSMSKGIDTEKVLSICPKDMRADICVHLNRKVFNEHPAFRLASDGCLRALAVEFQTIHCAPGDLIYHAGESVDALCFVVSGSLEVIQDDEVVAILGKGDVFGDIFWKETTLAHACANVRALTYCDLHIIKREALLKVLDFYTAFANSFSRNLTLTCNLRKRIIFRKISDVKKEEEERLRQKNEVTLSIPVDHPVRKLFQKFKQQKELRNQGSTQGDPERNQLQVESRSLQNGTSITGTSVVTVSQITPIQTSLAYVKTSESLKQNNRDAMELKPNGGADQKCLKVNSPIRMKNGNGKGWLRLKNNMGAHEEKKEDWNNVTKAESMGLLSEDPKSSDSENSVTKNPLRKTDSCDSGITKSDLRLDKAGEARSPLEHSPIQADAKHPFYPIPEQALQTTLQEVKHELKEDIQLLSCRMTALEKQVAEILKILSEKSVPQASSPKSQMPLQVPPQIPCQDIFSVSRPESPESDKDEIHF

[3167] The human 12189 nucleic acid sequence is recited as follows:

7(SEQ ID NO:7)TGCTGCGAGCGGCTGGTGCTCAACGTGGCCGGGCTGCGCTTCGAGACGCGGGCGCGCACGCTGGGCCGCTTCCCGGACACTCTGCTAGGGGACCCAGCGCGCCGCGGCCGCTTCTACGACGACGCGCGCCGCGAGTATTTCTTCGACCGGCACCGGCCCAGCTTCGACGCCGTGCTCTACTACTACCAGTCCGGTGGGCGGCTGCGGCGGCCGGCGCACGTGCCGCTCGACGTCTTCCTGGAAGAGGTGGCCTTCTACGGGCTGGGCGCGGCGGCCCTGGCACGCCTGCGCGAGGACGAGGGCTGCCCGGTGCCGCCCGAGCGCCCCCTGCCCCGCCGCGCCTTCGCCCGCCAGCTGTGCCTGCTTTTCGAGTTTCCCGAGAGCTCTCAGGCCGCGCGCGTGCTCGCCGTAGTCTCCGTGCTGGTCATCCTCGTCTCCATCGTCGTCTTCTGCCTCGAGACGCTGCCTGACTTCCGCGACGACCGCGACGGCACGGGGCTTGCTGCTGCAGCCGCAGCCGGCCCGTTCCCCGCTCCGCTGAATGGCTCCAGCCAAATGCCTGGAAATCCACCCCGCCTGCCCTTCAATGACCCGTTCTTCGTGGTGGAGACGCTGTGTATTTGTTGGTTCTCCTTTGAGCTGCTGGTACGCCTCCTGGTCTGTCCAAGCAAGGCTATCTTCTTCAAGAACGTGATGAACCTCATCGATTTTGTGGCTATCCTTCCCTACTTTGTGGCACTGGGCACCGAGCTGGCCCGGCAGCGAGGGGTGGGCCAGCAGGCCATGTCACTGGCCATCCTGAGAGTCATCCGATTGGTGCGTGTCTTCCGCATCTTCAAGCTGTCCCGGCACTCAAAGGGCCTGCAAATCTTGGGCCAGACGCTTCGGGCCTCCATGCGTGAGCTGGGCCTCCTCATCTTTTTCCTCTTCATCGGTGTGGTCCTCTTTTCCAGCGCCGTCTACTTTGCCGAAGTTGACCGGGTGGACTCCCATTTCACTAGCATCCCTGAGTCCTTCTGGTGGGCGGTAGTCACCATGACTACAGTTGGCTATGGAGACATGGCACCCGTCACTGTGGGTGGCAAGATAGTGGGCTCTCTGTGTGCCATTGCGGGCGTGCTGACTATTTCCCTGCCAGTGCCCGTCATTGTCTCCAATTTCAGCTACTTTTATCACCGGGAGACAGAGGGCGAAGAGGCTGGGATGTTCAGCCATGTGGACATGCAGCCTTGTGGCCCACTGGAGGGCAAGGCCAATGGGGGGCTGGTGGACGGGGAGGTACCTGAGCTACCACCTCCACTCTGGGCACCCCCAGGGAAACACCTGGTCACCGAAGTGTGA

[3168] The human 12189 sequence (FIG. 5; SEQ ID NO: 7) is approximately 1341 nucleotides long. The nucleic acid sequence includes a termination codon (TGA), which is underscored above. The coding sequence encodes a 446 amino acid protein (SEQ ID NO: 8), which is recited as follows:

8(SEQ ID NO:8)CCERLVLNVAGLRFETRARTLGRFPDTLLGDPARRGRFYDDARREYFFDRHRPSFDAVLYYYQSGGRLRRPAHVPLDVFLEEVAFYGLGAAALARLREDEGCPVPPERPLPRRAFARQLCLLFEFPESSQAARVLAVVSVLVILVSIVVFCLETLPDFRDDRDGTGLAAAAAAGPFPAPLNGSSQMPGNPPRLPFNDPFFVVETLCICWFSFELLVRLLVCPSKAIFFKNVMNLIDFVAILPYFVALGTELARQRGVGQQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGQTLRASMRELGLLIFFLFIGVVLFSSAVYFAEVDRVDSHFTSIPESFWWAVVTMTTVGYGDMAPVTVGGKIVGSLCAIAGVLTISLPVPVIVSNFSYFYHRETEGEEAGMFSHVDMQPCGPLEGKANGGLVDGEVPELPPPLWAPPGKHLVTEV.

Example 2

[3169] Tissue Distribution of 52906 and 33408 mRNA by TagMan Analysis

[3170] Endogenous human 52906 and 33408 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[3171] To determine the level of 52906 and 33408 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Tables 3 and 4. 52906 mRNA was detected in brain, prostate tumor, and heart samples (Table 3). 33408 expression was found in brain, heart, skin, and adipose samples (Table 4).

9TABLE 3Expression of 52906 mRNA in Human Tissues and Cell LinesTissueRelative ExpressionArtery/normal0Aorta/diseased0Vein/normal0Coronary smooth muscle cells0Human umbilical vein endothelial cells0Hemangioma0Heart/normal0Heart/congestive heart failure0.1902Kidney0Skeletal muscle0Adipose/normal0Pancreas0Primary osteoblasts0Osteoclasts (differentiated)0Skin/normal0Spinal cord/normal0Brain Cortex/normal1.6367Brain Hypothalamus/normal0Nerve0Dorsal Root Ganglion0Breast/normal0Breast/tumor0Ovary/normal0Ovary/tumor0Prostate/normal0Prostate/tumor1.1613Salivary glands0Colon/normal0Colon/tumor0Lung/normal0Lung/tumor0Lung/chronic obstructive pulmonary disease0Colon/inflammatory bowel disease0Liver/normal0Liver fibrosis0Spleen/normal0Tonsil/normal0Lymph node/normal0Small intestine/normal0Macrophages0Synovium0Bone marrow/mononuclear cells0Activated peripheral blood mononuclear cells0Neutrophils0Megakaryocytes0Erythroid cells0positive control0

[3172]

10

TABLE 4

Expression of 33408 mRNA in Human Tissues and Cell Lines

Tissue
Relative Expression

Prostate
0.00

Osteoclasts
0.00

Liver
0.00

Breast
0.00

Breast
0.00

Skeletal Muscle
2.60

Skeletal Muscle
0.13

Brain
33.03

Colon
0.06

Colon
0.01

Heart
30.71

Heart
0.00

Ovary
0.00

Ovary
0.00

Kidney
0.00

Kidney
0.01

Lung
0.01

Lung
0.00

Vein
0.10

Vein
0.01

Adipose
0.00

Adipose
4.26

Small Intestine
0.00

Thyroid
0.00

Bone Marrow
0.00

Skin
11.72

Testes
0.37

Placenta
0.01

Fetal Liver
0.00

Fetal Liver
0.00

Fetal Heart
0.00

Fetal Heart
0.00

Osteoblasts/undifferentiated
0.00

Osteoblasts/differentiated
0.00

Osteoblasts/primary culture
0.00

Spinal Cord
0.00

Cervix
0.00

Spleen
0.00

Spinal Cord
0.00

Thymus
0.00

Tonsil
0.00

Lymph Node
0.00

Aorta
0.00

Example 3

[3173] Tissue Distribution of 52906, 33408, or 12189 mRNA by Northern Analysis

[3174] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 52906, 33408, or 12189 cDNA (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7) can be used. The DNA is radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing, for example, mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 4

[3175] Recombinant Expression of 52906, 33408, or 12189 in Bacterial Cells

[3176] In this example, 52906, 33408, or 12189 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 52906, 33408, or 12189 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-52906, 33408, or 12189 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 5

[3177] Expression of Recombinant 52906, 33408, or 12189 Protein in COS Cells

[3178] To express the 52906, 33408, or 12189 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 52906, 33408, or 12189 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[3179] To construct the plasmid, the 52906, 33408, or 12189 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 52906, 33408, or 12189 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 52906, 33408, or 12189 coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 52906, 33408, or 12189 gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[3180] COS cells are subsequently transfected with the 52906, 33408, or 12189-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 52906, 33408, or 12189 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[3181] Alternatively, DNA containing the 52906, 33408, or 12189 coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 52906, 33408, or 12189 polypeptide is detected by radiolabelling and immunoprecipitation using a 52906, 33408, or 12189 specific monoclonal antibody.

Examples for 21784

Example 6

[3182] Identification and Characterization of Human 21784 cDNA

[3183] The human 21784 nucleic acid sequence is recited as follows:

11(SEQ ID NO:14)AGGGAGTCGCCCCACGCGTCCGCCCAGCATGGCCGGGCCGGGCTCGCCGCGCCGCGCGTCCCGGGGGGCCTCGGCGCTTCTCGCTGCCGCGCTTCTCTACGCCGCGCTGGGGGACGTGGTGCGCTCGGAGCAGCAGATACCGCTCTCCGTGGTGAAGCTCTGGGCCTCGGCTTTTGGTGGGGAGATAAAATCCATTGCTGCTAAGTACTCCGGTTCCCAGCTTCTGCAAAAGAAATACAAAGAGTATGAGAAAGACGTTGCCATAGAAGAAATTGATGGCCTCCAACTGGTAAAGAAGCTGGCAAAGAACATGGAAGAGATGTTTCACAAGAAGTCTGAGGCCGTCAGGCGTCTGGTGGAGGCTGCAGAAGAAGCACACCTGAAACATGAATTTGATGCAGACTTACAGTATGAATACTTCAATGCTGTGCTGATAAATGAAAGGGACAAAGACGGGAATTTTTTGGAGCTGGGAAAGGAATTCATCTTAGCCCCAAATGACCATTTTAATAATTTGCCTGTGAACATCAGTCTAAGTGACGTCCAAGTACCAACGAACATGTACAACAAAGACCCTGCAATTGTCAATGGGGTTTATTGGTCTGAATCTCTAAACAAAGTTTTTGTAGATAACTTTGACCGTGACCCATCTCTCATATGGCAGTACTTTGGAAGTGCAAAGGGCTTTTTTAGGCAGTATCCGGGGATTAAATGGGAACCAGATGAGAATGGAGTCATTGCCTTCGACTGCAGGAACCGAAAATGGTACATCCAGGCAGCAACTTCTCCGAAAGACGTGGTCATTTTAGTTGACGTCAGTGGCAGCATGAAAGGACTCCGTCTGACTATCGCGAAGCAAACAGTCTCATCCATTTTGGATACACTTGGGGATGATGACTTCTTCAACATAATTGCTTATAATGAGGAGCTTCACTATGTGGAACCTTGCCTGAATGGAACTTTGGTGCAAGCCGACAGGACAAACAAAGAGCACTTCAGGGAGCATCTGGACAAACTTTTCGCCAAAGGAATTGGAATGTTGGATATAGCTCTGAATGAGGCCTTCAACATTCTGAGTGATTTCAACCACACGGGACAAGGAAGTATCTGCAGTCAGGCCATCATGCTCATAACTGATGGGGCGGTGGACACCTATGATACAATCTTTGCAAAATACAATTGGCCAGATCGAAAGGTTCGCATCTTCACATACCTCATTGGACGAGAGGCTGCGTTTGCAGACAATCTAAAGTGGATGGCCTGTGCCAACAAAGGATTTTTTACCCAGATCTCCACCTTGGCTGATGTGCAGGAGAATGTCATGGAATACCTTCACGTGCTTAGCCGGCCCAAAGTCATCGACCAGGAGCATGATGTGGTGTGGACCGAAGCTTACATTGACAGCACTCTCCCTCAGGCACAAAAGCTGACTGATGATCAGGGCCCCGTCCTGATGACCACTGTAGCCATGCCTGTGTTTAGTAAGCAGAACGAAACCAGATCGAAGGGCATTCTTCTGGGAGTGGTTGGCACAGATGTCCCAGTGAAAGAACTTCTGAAGACCATCCCCAAATACAAGTTAGGGATTCACGGTTATGCCTTTGCAATCACAATAATGGATATATCCTGACGCATCCGGAACTCAGGCTGCTGTACGAAGAAGGAAAAAAGCGAAGGAAACCTAACTATAGTAGCGTTGACCTCTCTGAGGTGGAGTGGGAAGACCGAGATGACGTGTTGAGAAATGCTATGGTGAATCGAAAGACGGGGAAGTTTTCCATGGAGGTGAAGAAGACAGTGGACAAAGGGAAACGGGTTTTGGTGATGACAAATGACTACTATTATACAGACATCAAGGGTACTCCTTTCAGTTTAGGTGTGGCGCTTTCCAGAGGTCATGGGAAATATTTCTTCCGAGGGAATGTAACCATCGAAGAAGGCCTGCATGACTTAGAACATCCCGATGTGTCCTTGGCAGATGAATGGTCCTACTGCAACACTGACCTACACCCTGAGCACCGCCATCTGTCTCAGTTAGAAGCGATTAAGCTCTACCTAAAAGGCAAAGAACCTCTGCTCCAGTGTGATAAAGAATTGATCCAAGAAGTCCTTTTTGACGCGGTGGTGAGTGCCCCCATTGAAGCGTATTGGACCAGCCTGGCCCTCAACAAATCTGAAAATTCTGACAAGGGCGTGGAGGTTGCCTTCCTCGGCACTCGCACGGGCCTCTCCAGAATCAACCTGTTTGTCGGGGCTGAGCAGCTCACCAATCAGGACTTCCTGAAAGCTGGCGACAAGGAGAACATTTTTAACGCAGACCATTTCCCTCTCTGGTACCGAAGAGCCGCTGAGCAGATTCCAGGGAGCTTCGTCTACTCGATCCCATTCAGCACTGGACCAGTCAATAAAAGCAATGTGGTGACAGCAAGTACATCCATCCAGCTCCTGGATGAACGGAAATCTCCTGTGGTGGCAGCTGTAGGCATTCAGATGAAACTTGAATTTTTCCAAAGGAAGTTCTGGACTGCCAGCAGACAGTGTGCTTCCCTGGATGGCAAATGCTCCATCAGCTGTGATGATGAGACTGTGAATTGTTACCTCATAGACAATAATGGATTTATTTTGGTGTCTGAAGACTACACACAGACTGGAGACTTTTTTGGTGAGATCGAGGGAGCTGTGATGAACAAATTGCTAACAATGGGCTCCTTTAAAAGAATTACCCTTTATGACTACCAAGCCATGTGTAGAGCCAACAAGGAAAGCAGCGATGGCGCCCATGGCCTCCTGGATCCTTATAATGCCTTCCTCTCTGCAGTAAAATGGATCATGACAGAACTTGTCTTGTTCCTGGTGGAATTTAACCTCTGCAGTTGGTGGCACTCCGATATGACAGCTAAAGCCCAGAAATTGAAACAGACCCTGGAGCCTTGTGATACTGAATATCCAGCATTCGTCTCTGAGCGCACCATCAAGGAGACTACAGGGAATATTGCTTGTGAAGACTGCTCCAAGTCCTTTGTCATCCAGCAAATCCCAAGCAGCAACCTGTTCATGGTGGTGGTGGACAGCAGCTGCCTCTGTGAATCTGTGGCCCCCATCACCATGGCACCCATTGAAATCAGGTATAATGAATCCCTTAAGTGTGAACGTCTAAAGGCCCAGAAGATCAGAAGGCGCCCAGAATCTTGTCATGGCTTCCATCCTGAGGAGAATGCAAGGAGTGTGGGGGTGCGCCGAGTCTCCAAGCCCAGACAGTCCTCCTTCTGCTCCCTCTGCTTTTGATGCTCTTCTCAAGGTGACACTGACTGAGATGTTCTCTTACTGACTGAGATGTTCTCTTGGCATGCTAAATCATGGATAAACTGTGAACCAAAATATGGTGCAACATACGAGACATGAATATAGTCCAACCATCAGCATCTCATCATGATTTTAAACTGTGCGTGATATAAACTCTTAAAGATATGTTGACAAAAAGTTATCTATCATCTTTTTACTTTGCCAGTCATGCAAATGTGAGTTTGCCACATGATAATCACCCTTCATCAGAAATGGGACCGCAAGTGGTAGGCAGTGTCCCTTCTGCTTGAAACCTATTGAAACCAATTTAAAACTGTGTACTTTTTAAATAAAGTATATTAAAATCATAAAAAAAAAAAAAAAAARRAWWAAAAAAAAAAGGAAA.

[3184] The human 21784 sequence (SEQ ID NO: 14) is approximately 3690 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 3276 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 14; SEQ ID NO: 16). The coding sequence encodes a 1091 amino acid protein (SEQ ID NO: 15), which is recited as follows:

12(SEQ ID NO:15)MAGPGSPRRASRGASALLAAALLYAALGDVVRSEQQIPLSVVKLWASAFGGEIKSIAAKYSGSQLLQKKYKEYEKDVAIEEIDGLQLVKKLAKNMEEMFHKKSEAVRRLVEAAEEAHLKHEFDADLQYEYFNAVLINERDKDGNFLELGKEFILAPNDHFNNLPVNISLSDVQVPTNMYNKDPAIVNGVYWSESLNKVFVDNFDRDPSLIWQYFGSAKGFFRQYPGIKWEPDENGVIAFDCRNRKWYIQAATSPKDVVILVDVSGSMKGLRLTIAKQTVSSILDTLGDDDFFNIIAYNEELHYVEPCLNGTLVQADRTNKEHFREHLDKLFAKGIGMLDIALNEAFNILSDFNHTGQGSICSQAIMLITDGAVDTYDTIFAKYNWPDRKVRIFTYLIGREAAFADNLKWMACANKGFFTQISTLADVQENVMEYLHVLSRPKVIDQEHDVVWTEAYIDSTLPQAQKLTDDQGPVLMTTVAMPVFSKQNETRSKGILLGVVGTDVPVKELLKTIPKYKLGIHGYAFAITNNGYILTHPELRLLYEEGKKRRKPNYSSVDLSEVEWEDRDDVLRNAMVNRKTGKFSMEVKKTVDKGKRVLVMTNDYYYTDIKGTPFSLGVALSRGHGKYFFRGNVTIEEGLHDLEHPDVSLADEWSYCNTDLHPEHRHLSQLEAIKLYLKGKEPLLQCDKELIQEVLFDAVVSAPIEAYWTSLALNKSENSDKGVEVAFLGTRTGLSRINLFVGAEQLTNQDFLKAGDKENIFNADHFPLWYRRAAEQIPGSFVYSIPFSTGPVNKSNVVTASTSIQLLDERKSPVVAAVGIQMKLEFFQRKFWTASRQCASLDGKCSISCDDETVNCYLIDNNGFILVSEDYTQTGDFFGEIEGAVMNKLLTMGSFKRITLYDYQAMCRANKESSDGAHGLLDPYNAFLSAVKWIMTELVLFLVEFNLCSWWHSDMTAKAQKLKQTLEPCDTEYPAFVSERTIKETTGNIACEDCSKSFVIQQIPSSNLFMVVVDSSCLCESVAPITMAPIEIRYNEXLKCERLKAQKIRRRPESCHGFHPEENARECGGAPSLQAQTVLLLLPLLLMLFSR.

Example 7

[3185] Tissue Distribution of 21784 mRNA by TaqMan Analysis

[3186] Endogenous human 21784 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[3187] To determine the level of 21784 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in the following tables.

[3188] Table 5 below depicts the expression of 21784 mRNA in a panel of normal and tumor human tissues. Elevated expression of 21784 was found in the following tissues: heart, kidney, skeletal muscle, dorsal root ganglion, ovary, nerve, and spinal cord. Expression of 21784 was highest in the normal heart, heart CHF, kidney, skeletal muscle, and dorsal root ganglion, brain cortex, and brain hypothalmus.

13TABLE 5TissueExpressionArtery normal8.7288Aorta diseased0.6556Vein normal1.6769Coronary SMC0HUVEC0Hemangioma0Heart normal25.6482Heart CHF36.3979Kidney26.5527Skeletal Muscle47.5306Adipose normal0.1942Pancreas0primary osteoblasts0Osteoclasts (diff)0Skin normal1.7542Spinal cord normal4.0161Brain Cortex normal390.9348Nerve5.8799DRG (Dorsal Root Ganglion)68.8691Breast normal0.2302Breast tumor0.4178Ovary normal3.582Ovary Tumor7.7049Prostate Normal1.73Prostate Tumor0.796Salivary glands0.1969Colon normal0.3289Colon Tumor0.5038Lung normal0.4325Lung tumor1.0539Lung COPD0.4917Liver normal0Liver fibrosis0Spleen normal0.3739Tonsil normal0.6647Lymph node normal0.4163Small intestine normal1.1102Macrophages0Synovium0.0433BM-MNC0Activated PBMC0Neutrophils0Megakaryocytes0Erythroid0Brain Hypothalamus normal87.1715Colon IBD0.0708positive control94026.7925

Example 8

[3189] Tissue Distribution of 21784 mRNA by Northern Analysis

[3190] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 21784 cDNA (SEQ ID NO: 14) can be used. The DNA was radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 9

[3191] Recombinant Expression of 21784 in Bacterial Cells

[3192] In this example, 21784 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 21784 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-21784 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 10

[3193] Expression of Recombinant 21784 Protein in COS Cells

[3194] To express the 21784 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 21784 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[3195] To construct the plasmid, the 21784 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 21784 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 21784 coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 21784-gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[3196] COS cells are subsequently transfected with the 21784-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 21784 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[3197] Alternatively, DNA containing the 21784 coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 21784 polypeptide is detected by radiolabelling and immunoprecipitation using a 21784 specific monoclonal antibody.

Examples for 56201

Example 11

[3198] Identification and Characterization of Human 56201 cDNA

[3199] The human 56201 nucleic acid sequence is recited as follows:

14(SEQ ID NO:20)GGAAAATCCCTAAGCAGAGATTTTCTGTTGGATGCTAAAAGCAAGGAATAAAAGTTGAAAATTTGGAAAATGTCTCAACACCGTCACCAGCGCCACTCGAGAGTCATTTCTAGTTCACCAGTTGACACTACATCGGTGGGATTTTGCCCAACATTCAAGAAATTTAAGAGGAACGATGATGAATGTCGGGCATTTGTGAAGAGAGTCATAATGAGCCGTTTCTTTAAGATAATTATGATTAGCACTGTCACATCGAATGCGTTTTTTATGGCCTTGTGGACCAGTTATGACATAAGGTACCGCTTGTTCAGACTTCTTGAGTTCTCGGAGATCTTCTTTGTGTCCATCTGCACATCTGAGTTGTCCATGAAGGTCTATGTGGACCCCATCAACTACTGGAAGAACGGCTACAACCTGCTGGATGTGATCATTATCATCGTTATGTTTTTACCCTATGCCCTCCGCCAGCTCATGGGCAAACAGTTCACTTACCTGTATATCGCTGATGGCATGCAGTCCCTGCGCATCCTCAAGCTTATCGGCTATAGCCAGGGCATCCGGACGCTGATCACCGCCGTGGGGCAGACAGTCTACACCGTGGCCTCTGTGCTCCTCCTGCTCTTCCTCCTCATGTACATCTTCGCTATCTTGGGCTTCTGCCTGTTTGGATCTCCAGACAATGGTGACCATGATAACTGGGGGAACCTGGCTGCAGCTTTTTTCACCCTCTTCAGCTTGGTGCTTTGAGCCGGGCATTCACCATCATCTTCATCTTGCTCGCCTCTTTCATCTTCCTCAACATGTTCGTGGGTGTGATGATCATGCACACAGAGGACTCCATCAGAAAGTTTGAGCGAGAGCTGATGTTGGAGCAGCAGGAGATGCTCATGGGAGAGAAGCAGGTGATTCTGCAGCGGCAGCAGGAGGAGATCAGCAGGCTGATGCACATACAGAAAAATGCTGACTGCACAAGTTTCAGTGAGCTGGTGGAGAACTTTAAGAAGACCTTGAGCCACACTGACCCAATGGTCTTGGATGATTTTGGCACTAGCTTACCCTTCATCGATATCTACTTTTCCACTCTGGACTACCAGGACACAACTGTCCACAAGCTTCAAGAGCTGTACTATGAGATCGTGCATGTGCTGAGCCTAATGCTGGAAGACTTGCCCCAGGAGAAGCCCCAGTCCTTGGAAAAGGTGGATGAGAAGTAGTGGGCATGGGGCACCCATGTGCCGAGAGCCTTGCAGACCATGACAGGTCCCTATTAAACACAGGCTTTCTGAAAAAAAAAAAAAAAAAA.

[3200] The human 56201 sequence (FIG. 10; SEQ ID NO: 20), which is approximately 1356 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAG) which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 1197 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 20; SEQ ID NO: 22). The coding sequence encodes a 398 amino acid protein (SEQ ID NO: 21), which is recited as follows:

15(SEQ ID NO:21)MSQHRHQRHSRVISSSPVDTTSVGFCPTFKKFKRNDDECRAFVKRVIMSRFFKIIMISTVTSNAFFMALWTSYDIRYRLFRLLEFSEIFFVSICTSELSMKVYVDPINYWKNGYNLLDVIIIIVMFLPYALRQLMGKQFTYLYIADGMQSLRILKLIGYSQGIRTLITAVGQTVYTVASVLLLLFLLMYIFAILGFCLFGSPDNGDHDNWGNLAAAFFTLFSLATVDGWTDLQKQLDNREFALSRAFTIIFILLASFIFLNMFVGVMIMHTEDSIRKFERELMLEQQEMLMGEKQVILQRQQEEISRLMHIQKNADCTSFSELVENFKKTLSHTDPMVLDDFGTSLPFIDIYFSTLDYQDTTVHKLQELYYEIVHVLSLMLEDLPQEKPQSLEKVDEK

Example 12

[3201] Tissue Distribution of 56201 mRNA by TaqMan Analysis

[3202] Endogenous human 56201 gene expression can be determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[3203] To determine the level of 56201 in various human tissues a primer/probe set can be designed. Total RNA can be prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA can be prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA is used per TaqMan reaction. Tissues tested can include human tissues, e.g., neural, muscular, bone, lymph nodes and blood tissues, as well as cell lines derived from such tissues.

Example 13

[3204] Tissue Distribution of 56201 mRNA by Northern Analysis

[3205] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 56201 cDNA (SEQ ID NO: 20) can be used. The DNA was radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 14

[3206] Recombinant Expression of 56201 in Bacterial Cells

[3207] In this example, 56201 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 56201 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-56201 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 15

[3208] Expression of Recombinant 56201 Protein in COS Cells

[3209] To express the 56201 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell 23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 56201 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[3210] To construct the plasmid, the 56201 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 56201 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 56201 coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 56201_gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[3211] COS cells are subsequently transfected with the 56201-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 56201 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[3212] Alternatively, DNA containing the 56201 coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 56201 polypeptide is detected by radiolabelling and immunoprecipitation using a 56201 specific monoclonal antibody.

Examples for 32620

Example 16

[3213] Identification and Characterization of Human 32620 cDNA

[3214] The human 32620 nucleic acid sequence is recited as follows:

16(SEQ ID NO:26)CCACGCGTCCGCCCACGCGTCCGCCCACGCGTCCGCTTGGCTGCAAAGAGAGAGGATCCCGGGTATCTCCCTCCTTACAACCACCGCCACCTCCTAGTGCCTTAGAAGCCACTGACAGCCCCCAGGGCAGGTGAGCCCTGCATCTGGAATAAGGATCCAGAGGTCTCGTTCAGGACCATGGAGAGCGGCACCAGCAGCCCTCAGCCTCCACAGTTAGATCCCCTGGATGCGTTTCCCCAGAAGGGCTTGGAGCCTGGGGACATCGCGGTGCTAGTTCTGTACTTCCTCTTTGTCCTGGCTGTTGGACTATGGTCCACAGTGAAGACCAAAAGAGACACAGTGAAAGGCTACTTCCTGGCTGAAGGGAACATGGTGTGGTGGCCAGTGGGTGCATCCTTGTTTGCCAGCAATGTTGGAAGTGGACATTTCATTGGCCTGGCAGGGTCAGGTGCTGCTACGGGCATTTCTGTATCAGCTTATGAACTTAATGGCTTGTTTTCTGTGCTGATGTTGGCCTGGATCTTCCTACCCATCTACATTGCTGGTCAGGTCACCACGATGCCAGAATACCTACGGAAGCGCTTCGGTGGCATCAGAATCCCCATCATCCTGGCTGTACTCTACCTATTTATCTACATCTTCACCAAGATCTCGGTAGACATGTATGCAGGTGCCATCTTCATCCAGCAGTCTTCGCACCTGGATCTGTACCTGGCCATAGTTGGGCTACTGGCCATCACTGCTGTATACACGGTTGCTGGTGGCCTGGCTGCTGTGATCTACACGGATGCCCTGCAGACGCTGATCATGCTTATAGGAGCGCTCACCTTGATGGGCTACAGTTTTGCCGCGGTTGGTGGGATGGAAGGACTGAAGGAGAAGTACTTCTTGGCCCTGGCTGCAACCGGAGTGAGAACAGCAGCTGCGGCTGCCCCGGGAAGATGCCTTCCATATTTTCCGAGATCCGCTGACATCTGATCTCCCGTGGCCGGGGGTCCTATTTGGAATGTCCATCCCATCCCTCTGGTACTGGTGCACGGATCAGGTGATTGTCCAGCGGACTCTGGCTGCCAAGAACCTGTCCCATGCCAAAGGAGGTGCTCTGATGGCTGCATACCTGAAGGTGCTGCCCCTCTTCATAATGGTGTTCCCTGGGATGGTCAGCCGCATCCTCTTCCCAGATCAAGTGGCCTGTGCAGATCCAGAGATCTGCCAGAAGATCTGCAGCAACCCCTCAGGCTGTTCGGACATCGCGTATCCCAAACTCGTGCTGGAACTCCTGCCCACAGGGCTCCGTGGGCTGATGATGGCTGTGATGGTGGCGGCTCTCATGTCCTCCCTCACCTCCATCTTTAACAGTGCCAGCACCATCTTCACCATGGACCTCTGGAATCACCTCCGGCCTCGGGCATCTGAGAAGGAGCTCATGATTGTGGGCAGGGTGTTTGTGCTGCTGCTGGTCCTGGTCTCCATCCTCTGGATCCCTGTGGTCCAGGCCAGCCAGGGCGGCCAGCTCTTCATCTATATCCAGTCCATCAGCTCCTACCTGCAGCCGCCTGTGGCGGTGGTCTTCATCATGGGATGTTTCTGGAAGAGGACCAATGAAAAGGGTGCCTTCTGGGGCCTGATCTCGGGCCTGCTCCTGGGCTTGGTTAGGCTGGTCCTGGACTTTATTTACGTGCAGCCTCGATGCGACCAGCCAGATGAGCGCCCGGTCCTGGTGAAGAGCATTCACTACCTCTACTTCTCCATGATCCTGTCCACGGTCACCCTCATCACTGTCTCCACCGTGAGCTGGTTCACAGAGCCACCCTCCAAGGAGATGGTCAGCCACCTGACCTGGTTTACTCGTCACGACCCCGTGGTCCAGAAGGAACAAGCACCACCAGCAGCTCCCTTGTCTCTTACCCTCTCTCAGAACGGGATGCCAGAGGCCAGCAGCAGCAGCAGCGTCCAGTTCGAGATGGTTCAAGAAAACACGTCTAAAACCCACAGCTGTGACATGACCCCAAAGCAGTCCAAAGTGGTGAAGGCCATCCTGTGGCTCTGTGGAATACAGGAGAAGGGCAAGGAAGAGCTCCCGGCCAGAGCAGAAGCCATCATAGTTTCCCTGGAAGAAAACCCCTTGGTGAAGACCCTCCTGGACGTCAACCTCATTTTCTGCGTGAGCTGCGCCATCTTTATCTGGGGCTATTTTGCTTAGTGTGGGGTGAACCCAGGGGTCCAAACTCTGTTTCTCTTCAGTGCTCCATTTTTTTAATGAAAGAAAAAATAATAAAGCTTTTGTTTACCACAAAAAAAAAAAAAAAAAAAAGGGCGGCCGC

[3215] The human 32620 sequence (FIG. 13; SEQ ID NO: 26), which is approximately 2326 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA) which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 2028 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 26; SEQ ID NO: 28). The coding sequence encodes a 675 amino acid protein (SEQ ID NO: 27), which is recited as follows:

17(SEQ ID NO:27)MESGTSSPQPPQLDPLDAFPQKGLEPGDIAVLVLYFLFVLAVGLWSTVKTKRDTVKGYFLAEGNMVWWPVGASLFASNVGSGHFIGLAGSGAATGISVSAYELNGLFSVLMLAWIFLPIYIAGQVTTMPEYLRKRFGGIRIPIILAVLYLFIYIFTKISVDMYAGAIFIQQSSHLDLYLAIVGLLAITAVYTVAGGLAAVIYTDALQTLIMLIGALTLMGYSFAAVGGMEGLKEKYFLALASNRSENSSCGLPREDAFHIFRDPLTSDLPWPGVLFGMSIPSLWYWCTDQVIVQRTLAAKNLSHAKGGALMAAYLKVLPLFIMVFPGMVSRILFPDQVACADPEICQKICSNPSGCSDIAYPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIFTMDLWNHLRPRASEKELMIVGRVFVLLLVLVSILWIPVVQASQGGQLFIYIQSISSYLQPPVAVVFIMGCFWKRTNEKGAFWGLISGLLLGLVRLVLDFIYVQPRCDQPDERPVLVKSIHYLYFSMILSTVTLITVSTVSWFTEPPSKEMVSHLTWFTRHDPVVQKEQAPPAAPLSLTLSQNGMPEASSSSSVQFEMVQENTSKTHSCDMTPKQSKVVKAILWLCGIQEKGKEELPARAEAIIVSLEENPLVKTLLDVNLIFCVSCAIFIWGYFA.

Example 17

[3216] Tissue Distribution of 32620 mRNA by TagMan Analysis

[3217] Endogenous human 32620 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[3218] To determine the level of 32620 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Table 6.

18TABLE 632620 Expression LevlesTissue TypeExpressionArtery normal0.68Aorta diseased0.00Vein normal0.00Coronary SMC0.00HUVEC1.13Hemangioma0.25Heart normal0.24Heart CHF0.11Kidney4.20Skeletal Muscle0.00Adipose normal0.00Pancreas1.37primary osteoblasts0.00Osteoclasts (diff)0.02Skin normal0.00Spinal cord normal35.65Brain Cortex normal136.31Brain Hypothalamus normal145.59Nerve0.66DRG (Dorsal Root Ganglion)0.00Breast normal0.27Breast tumor0.14Ovary normal0.48Ovary Tumor0.11Prostate Normal0.29Prostate Tumor0.28Salivary glands0.14Colon normal20.55Colon Tumor0.40Lung normal0.14Lung tumor2.24Lung COPD0.22Colon IBD0.07Liver normal0.30Liver fibrosis1.55Spleen normal0.51Tonsil normal0.45Lymph node normal1.08Small intestine normal4.83Macrophages0.01Synovium0.00BM-MNC0.00Activated PBMC0.17Neutrophils0.00Megakaryocytes0.04Erythroid0.45positive control56.13

[3219] 32620 mRNA was highly abundant in normal spinal cord, normal brain cortex, and normal brain hypothalamus (Table 6). 32620 expression was also found in some normal colon samples.

Example 18

[3220] Tissue Distribution of 32620 mRNA by Northern Analysis

[3221] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 32620 cDNA (SEQ ID NO: 26) can be used. The DNA was radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 19

[3222] Recombinant Expression of 32620 in Bacterial Cells

[3223] In this example, 32620 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 32620 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-32620 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 20

[3224] Expression of Recombinant 32620 Protein in COS Cells

[3225] To express the 32620 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 32620 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[3226] To construct the plasmid, the 32620 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 32620 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 32620 coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 32620-gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[3227] COS cells are subsequently transfected with the 32620-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 32620 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[3228] Alternatively, DNA containing the 32620 coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 32620 polypeptide is detected by radiolabelling and immunoprecipitation using a 32620 specific monoclonal antibody.

Examples for 44589

Example 21

[3229] Identification and Characterization of Human 44589 cDNA

[3230] The human 44589 nucleic acid sequence is recited as follows:

19(SEQ ID NO:33)GATGTTTAAAAAGAGGGATCAAGCACAGGCTAAGGAGAGGAAAGAGCAGGCACCCAAACCTCTGCATGGCCCCAATATGCTCCCTGCAGGGTAGTGCCCCCTCTTCTGGCTGCTCAAGGCGAGATCTAAGCTTCTTCTAACTCCTGCTGTCTTTTCATATTCTCTGATTCTGGGAAACGAAGAATTGGCAGGAACTGAAAATGACTAGGAAGAGGACATACTGGGTGCCCAACTCTTCTGGTGGCCTCGTGAATCGTGGCATCGACATAGGCGATGACATGGTTTCAGGACTTATTTATAAAACCTATACTCTCCAAGATGGCCCCTGGAGTCAGCAAGAGAGAAATCCTGAGGCTCCAGGGAGGGCAGCTGTCCCACCGTGGGGGAAGTATGATGCTGCCTTGAGAACCATGATTCCCTTCCGTCCCAAGCCGAGGTTTCCTGCCCCCCAGCCCCTGGACAATGCTGGCCTGTTCTCCTACCTCACCGTGTCATGGCTCACCCCGCTCATGATCCAAAGCTTACGGAGTCGCTTAGATGAGAACACCATCCCTCCACTGTCAGTCCATGATGCCTCAGACAAAAATGTCCAAAGGCTTCACCGCCTTTGGGAAGAAGAAGTCTCAAGGCGAGGGATTGAAAAAGCTTCAGTGCTTCTGGTGATGCTGAGGTTCCAGAGAACAAGGTTGATTTTCGATGCACTTCTGGGCATCTGCTTCTGCATTGCCAGTGTACTCGGGCCAATATTGATTATACCAAAGATCCTGGAATATTCAGAAGAGCAGTTGGGGAATGTTGTCCATGGAGTGGGACTCTGCTTTGCCCTTTTTCTCTCCGAATGTGTGAAGTCTCTGAGTTTCTCCTCCAGTTGGATCATCAACCAACGCACAGCCATCAGGTTCCGAGCAGCTGTTTCCTCCTTTGCCTTTGAGAAGCTCATCCAATTTAAGTCTGTAATACACATCACCTCAGGAGAGGGAGGTGACATCTGTGCCCATCAACTTGCTGTCTTGCAGGCCATCAGCTTCTTCACCGGTGATGTAAACTACCTGTTTGAAGGGTGTGCTATGGACCCCTAGTACTGATCACCTGCGCATCGCTGGTCATCTGCAGCATTTCTTCCTACTTCATTATTGGATACACTGCATTTATTGCCATCTTATGCTATCTCCTGGTTTTCCCACTGGCGGTATTCATGACAAGAATGGCTGTGAAGGCTCAGCATCACACATCTGAGGTCAGCGACCAGCGCATCCGTGTGACCAGTGAAGTTCTCACTTGCATTAAGCTGATTAAAATGTACACATGGGAGAAACCATTTGCAAAAATCATTGAAGGTATGGAAAGTCTGACTTTCTGCTCCAAACCTGGTGATGGCATGGCCTTCAGCATGCTGGCCTCCTTGAATCTCCTTCGGCTGTCAGTGTTCTTTGTGCCTATTGCAGTCAAAGGTCTCACGAATTCCAAGTCTGCAGTGATGAGGTTCAAGAAGTTTTTCCTCCAGGAGAGCCCTGTTTTCTATGTCCAGACATTACAAGACCCCAGCAAAGCTCTGGTCTTTGAGGAGGCCACCTTGTCATGGCAACAGACCTGTCCCGGGATCGTCAATGGGGCACTGGAGCTGGAGAGGAACGGGCATGCTTCTGAGGGGATGACCAGGCCTAGAGATGCCCTCGGGCCAGAGGAAGAAGGGAACAGCCTGGGCCCAGAGTTGCACAAGATCAACCTGGTGGTGTCCAAGGGGATGATGTTAGGGGTCTGCGGCAACACGGGGAGTGGTAAGAGCAGCCTGTTGTCAGCCATCCTGGAGGAGATGCACTTGCTCGAGGGCTCGGTGGGGGTGCAGGGAAGCCTGGCCTATGTCCCCCAGCAGGCCTGGATCGTCAGCGGGAACATCAGGGAGAACATCCTCATGGGAGGCGCATATGACAAGGCCCGATACCTCCAGGTGCTCCACTGCTGCTCCCTGAATCGGGACCTGGAACTTCTGCCCTTTGGAGACATGACAGAGATTGGAGAGCGGGGCCTCAACCTCTCTGGGGGGCAGAAACAGAGGATCAGCCTGGCCCGCGCCGTCTATTCCGACCGTCAGATCTACCTGCTGGACGACCCCCTGTCTGCTGTGGACGCCCACGTGGGGAAGCACATTTTTGAGGAGTGCATTAAGAAGACACTCAGGGGGAAGACGGTCGTCCTGGTGACCCACCAGCTGCAGTACTTAGAATTTTGTGGCCAGATCATTTTGTTGGAAAATGGGAAAATCTGTGAAAATGGAACTCACAGTGAGTTAATGCAGAAAAAGGGGAAATATGCCCAACTTATCCAGAAGATGCACAAGGAAGCCACTTCGGACATGTTGCAGGACACAGCAAAGATAGCAGAGAAGCCAAAGGTAGAAAGTCAGGCTCTGGCCACCTCCCTGGAAGAGTCTCTCAACGGAAATGCTGTGCCGGAGCATCAGCTCACACAGGAGGAGGAGATGGAAGAAGGCTCCTTGAGTTGGAGGGTCTACCACCACTACATCCAGGCAGCTGGAGGTTACATGGTCTCTTGCATAATTTTCTTCTTTGTGGTGCTGATCGTCTTCTTAACGATCTTCAGCTTCTGGTGGCTGAGCTACTGGTTGGAGCAGGGCTCGGGGACCAATAGCAGCCGAGAGAGCAATGGAACCATGGCAGACCTGGGCAACATTGCAGACAATCCTCAACTGTCCTTCTACCAGCTGGTGTACGGGCTCAACGCCCTGCTCCTCATCTGTGTGGGGGTCTGCTCCTCAGGGATTTTCACCAAAGTCACGAGGAAGGCATCCACGGCCCTGCACAACAAGCTCTTCAACAAGGTTTTCCGCTGCCCCATGAGTTTCTTTGACACCATCCCAATAGGCCGGCTTTTGAACTGCTTCGCAGGGGACTTGGAACAGCTGGACCAGCTCTTGCCCATCTTTTCAGAGCAGTTCCTGGTCCTGTCCTTAATGGTGATCGCCGTCCTGTTGATTGTCAGTGTGCTGTCTCCATATATCCTGTTAATGGGAGCCATAATCATGGTTATTTGCTTCATTTATTATATGATGTTCAAGAAGGCCATCGGTGTGTTCAAGAGACTGGAGAACTATAGCCGGTCTCCTTTATTCTCCCACATCCTCAATTCTCTGCAAGGCCTGAGCTCCATCCATGTCTATGGAAAAACTGAAGACTTCATCAGCCAGTTTAAGAGGCTGACTGATGCGCAGAATAACTACCTGCTGTTGTTTCTATCTTCCACACGATGGATGGCATTGAGGCTGGAGATCATGACCAACCTTGTGACCTTGGCTGTTGCCCTGTTCGTGGCTTTTGGCATTTCCTCCACCCCCTACTCCTTTAAAGTCATGGCTGTCAACATCGTGCTGCAGCTGGCGTCCAGCTTCCAGGCCACTGCCCGGATTGGCTTGGAGACAGAGGCACAGTTCACGGCTGTAGAGAGGATACTGCAGTACATGAAGATGTGTGTCTCGGAAGCTCCTTTACACATGGAAGGCACAAGTTGTCCCCAGGGGTGGCCACAGCATGGGGAAATCATATTTCAGGATTATCACATGAAATACAGAGACAACACACCCACCGTGCTTCACGGCATCAACCTGACCATCCGCGGCCACGAAGTGGTGGGCATCGTGGGAAGGACGGGCTCTGGGAAGTCCTCCTTGGGCATGGCTCTCTTCCGCCTGGTGGAGCCCATGGCAGGCCGGATTCTCATTGACGGCGTGGACATTTGCAGCATCGGCCTGGAGGACTTGCGGTCCAAGCTCTCAGTGATCCCTCAAGATCCAGTGCTGCTCTCAGGAACCATCAGATTCAACCTAGATCCCTTTGACCGTCACACTGACCAGCAGATCTGGGATGCCTTGGAGAGGACATTCCTGACCAAGGCCATCTCAAAGTTCCCCAAAAAGCTGCATACAGATGTGGTGGAAAACGGTGGAAACTTCTCTGTGGGGGAGAGGCAGCTGCTCTGCATTGCCAGGGCTGTGCTTCGCAACTCCAAGATCATCCTTATCGATGAAGCCACAGCCTCCATTGACATGGAGACAGACACCCTGATCCAGCGCACAATCCGTGAAGCCTTCCAGGGCTGCACCGTGCTCGTCATTGCCCACCGTGTCACCACTGTGCTGAACTGTGACCACATCCTGGTTATGGGCAATGGGAAGGTGGTAGAATTTGATCGGCCGGAGGTACTGCGGAAGAAGCCTGGGTCATTGTTCGCAGCCCTCATGGCCACAGCCACTTCTTCACTGAGATAAGGAGATGTGGAGACTTCATGGAGGCTGGCAGCTGAGCTCAGAGGTTCACACAGGTGCAGCTTCGAGGCCCACAGTCTGCGACCTTCTTGTTTGGAGATGAGAACTTCTCCTGGAAGCGCTACTTGATGGCTCTCAAGACCTTAGAACCCCAGAACCATCTAAGACATGGGATTCAGTGATCATGTGGTTCTCCTTTTAACTTACATGCTGAATAATTTTATAATAAGGTAAAAGCTTATAGTTTTCTGATCTGTGTTAGAAGTGTTGCAAATGCTGTACTGACTTTGTAAAATATAAAACTAAG

[3231] The human 44589 sequence (SEQ ID NO: 33) is approximately 4638 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA), which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 4083 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 33; SEQ [[ NO: 35). The coding sequence encodes a 1360 amino acid protein (SEQ ID NO: 34), which is recited as follows:

20(SEQ ID NO:34)MTRKRTYWVPNSSGGLVNRGIDIGDDMVSGLIYKTYTLQDGPWSQQERNPEAPGRAAVPPWGKYDAALRTMIPFRPKPRFPAPQPLDNAGLFSYLTVSWLTPLMIQSLRSRLDENTIPPLSVHDASDKNVQRLHRLWEEEVSRRGIEKASVLLVMLRFQRTRLIFDALLGICFCIASVLGPILIIPKILEYSEEQLGNVVHGVGLCFALFLSECVKSLSFSSSWIINQRTAIRFRAAVSSFAFEKLIQFKSVIHITSGEGGDICAHQLAVLQAISFFTGDVNYLFEGVCYGPLVLITCASLVICSISSYFHGYTAFIAILCYLLVFPLAVFMTRMAVKAQHHTSEVSDQRIRVTSEVLTCIKLIKMYTWEKPFAKIIEGMESLTFCSKPGDGMAFSMLASLNLLRLSVFFVPIAVKGLTNSKSAVMRFKKFFLQESPVFYVQTLQDPSKALVFEEATLSWQQTCPGIVNGALELERNGHASEGMTRPRDALGPEEEGNSLGPELHKINLVVSKGMMLGVCGNTGSGKSSLLSAILEEMHLLEGSVGVQGSLAYVPQQAWIVSGNIRENILMGGAYDKARYLQVLHCCSLNRDLELLPFGDMTEIGERGLNLSGGQKQRISLARAVYSDRQIYLLDDPLSAVDAHVGKHIFEECIKKTLRGKTVVLVTHQLQYLEFCGQIILLENGKICENGTHSELMQKKGKYAQLIQKMHKEATSDMLQDTAKIAEKPKVESQALATSLEESLNGNAVPEHQLTQEEEMEEGSLSWRVYHHYIQAAGGYMVSCIIFFFVVLIVFLTIFSFWWLSYWLEQGSGTNSSRESNGTMADLGNIADNPQLSFYQLVYGLNALLLICVGVCSSGIFTKVTRKASTALHNKLFNKVFRCPMSFFDTIPIGRLLNCFAGDLEQLDQLLPIFSEQFLVLSLMVIAVLLIVSVLSPYILLMGAIIMVICFIYYMMFKKAIGVFKRLENYSRSPLFSHILNSLQGLSSIHVYGKTEDFISQFKRLTDAQNNYLLLFLSSTRWMALRLEIMTNLVTLAVALFVAFGISSTPYSFKVMAVNIVLQLASSFQATARIGLETEAQFTAVERILQYMKMCVSEAPLHMEGTSCPQGWPQHGEIIFQDYHMKYRDNTPTVLHGINLTIRGHEVVGIVGRTGSGKSSLGMALFRLVEPMAGRILIDGVDICSIGLEDLRSKLSVIPQDPVLLSGTIRFNLDPFDRHTDQQIWDALERTFLTKAISKFPKKLHTDVVENGGNFSVGERQLLCIARAVLRNSKIILIDEATASIDMETDTLIQRTIREAFQGCTVLVIAHRVTTVLNCDHILVMGNGKVVEFDRPEVLRKKPGSLFAALMATATSSLR.

Example 22

[3232] Tissue Distribution of 44589 mRNA by TagMan Analysis

[3233] Endogenous human 44589 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[3234] To determine the level of 44589 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested included the human tissues and several cell lines shown in Table 7. 44589 mRNA was detected in breast tumor, liver firbrosis, normal liver, and prostate tumor samples.

21TABLE 7Expression of 44589 in Human TissuesTissue TypeRelative ExpressionArtery normal0Aorta diseased0Vein normal0Coronary Smooth Muscle Cells0Human Umbilical Vein Endothelial Cells0Hemangioma0Heart normal0Heart Congestive Heart Failure0Kidney0Skeletal Muscle0Adipose normal0Pancreas0Primary osteoblasts0Osteoclasts (differentiated)0Skin normal0Spinal cord normal0Brain Cortex normal0Brain Hypothalamus normal0Nerve0Dorsal Root Ganglion0Breast normal0Breast tumor29.6669Ovary normal0Ovary Tumor0Prostate Normal0Prostate Tumor1.5919Salivary glands0Colon normal0Colon Tumor0Lung normal0Lung tumor0Lung Chronic Obstructive Pulmonary Disease0Colon Inflammatory Bowel Disease0Liver normal3.0648Liver fibrosis9.5519Spleen normal0Tonsil normal0Lymph node normal0Small intestine normal0Macrophages0Synovium0Bone Marrow Mononuclear Cells0Activated Peripheral Blood Mononuclear Cells0Neutrophils0Megakaryocytes0Erythroid0Positive control98.4135

Example 23

[3235] Tissue Distribution of 44589 mRNA by Northern Analysis

[3236] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 44589 cDNA (SEQ ID NO: 33) can be used. The DNA was radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 24

[3237] Recombinant Expression of 44589 in Bacterial Cells

[3238] In this example, 44589 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 44589 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-44589 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 25

[3239] Expression of Recombinant 44589 Protein in COS Cells

[3240] To express the 44589 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 44589 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[3241] To construct the plasmid, the 44589 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 44589 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 44589 coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 44589_gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[3242] COS cells are subsequently transfected with the 44589-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 44589 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[3243] Alternatively, DNA containing the 44589 coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 44589 polypeptide is detected by radiolabeling and immunoprecipitation using a 44589 specific monoclonal antibody.

Examples for 84226

Example 26

[3244] Identification and Characterization of Human 84226 cDNA

[3245] The human 84226 nucleic acid sequence is recited as follows:

22(SEQ ID NO:39)CCACGCGTCCGCGAGACACGGGAGCGCTTGGCACGCGGAGCCAGAGCCGGAGCTGCAGCCGCAGCGGGAGCCGGGGGAGCTCAGGGGCCGCAGGAGCCGGGCCGGAGTGAGCGCACCTCGCGGGGCCCTCGGGGCAGGTGGGTGAGCGCCACCCGGAGTCCCGCGCGCAACTTTCAGGGCGCACTCGGCGGGGCGGCTGCGCGGCTGCCGGGACTCGGCGCGGGACTGCATGGAGGCCAAGGAGAAGCAGCATCTGGGGCTGGCTGGATTCCTCTGCCCCGACCTGGCCTGGACTTGCAGGCCATTGAGCTGGCTGCCCAGAGCAACCATCACTGCCATGCTCAGAAGGGTCCTGACAGTCACTGTGACCCCAAGAAGGGGAAGGCCCAGCGCCAGCTGTATGTAGCCTCTGCCATCTGCCTGTTGTTCATGATCGGAGAAGTCGTTGGTGGGTACCTGGCACACAGCTTGGCTGTCATGACTGACGCAGCACACCTGCTCACTGACTTTGCCAGCATGCTCATCAGCCTCTTCTCCCTCTGGATGTCCTCCCGGCCAGCCACCAAGACCATGAACTTTGGCTGGCAGAGAGCTGAGATCTTGGGAGCCCTGGTCTCTGTACTGTCCATCTGGGTCGTGACGGGGGTACTGGTGTACCTGGCTGTGGAGCGGCTGATCTCTGGGGACTATGAAATTGACGGGGGGACCATGCTGATCACGTCGGGCTGCGCTGTGGCTGTGAACATCATAATGGGGTTGACCCTTCACCAGTCTGGCCATGGGCACAGCCACGGCACCACCAACCAGCAGGAGGAGAACCCCAGCGTCCGAGCTGCCTTCATCCATGTGATCGGCGACTTTATGCAGAGCATGGGTGTCCTAGTGGCAGCCTATATTTTATACTTCAAGCCAGAATACAAGTATGTAGACCCCATCTGCACCTTCGTCTTCTCCATCCTGGTCCTGGGGACAACCTTGACCATCCTGAGAGATGTGATCCTGGTGTTGATGGAAGGGACCCCCAAGGGCGTTGACTTCACAGCTGTTCGTGATCTGCTGCTGTCGGTGGAGGGGGTAGAAGCCCTGCACAGCCTGCATATCTGGGCACTGACGGTGGCCCAGCCTGTTCTGTCTGTCCACATCGCCATTGCTCAGAATACAGACGCCCAGGCTGTGCTGAAGACAGCCAGCAGCCGCCTCCAAGGGAAGTTCCACTTCCACACCGTGACCATCCAGATCGAGGACTACTCGGAGGACATGAAGGACTGTCAGGCATGCCAGGGCCCCTCAGACTGACTGCTCAGCCAGGCACCAACTGGGGCATGAACAGGACCTGCAGGTGGCTGGACTGAGTGTCCCCCAGGCCCAGCCAGGACTTTGCCTACCCCAGCTGTGTTATAAACCAGGTCCCCCTCCTGACCTCTGCCCCACTCCAGGAATGGAGCTCTTCCCAGCCTCCCATCTGACTACAGCCAGGGTGGGGACTCAGCGGGTATAAAGCTAGTGTGACCCTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATTGCGGCCGCAAGCTTA.

[3246] The human 84226 sequence (FIG. 19; SEQ ID NO: 39), which is approximately 1630 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 1119 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 39; SEQ ID NO: 41). The coding sequence encodes a 372 amino acid protein (SEQ ID NO: 40), which is recited as follows:

23(SEQ ID NO:40)MEAKEKQHLLDTRPAIRSYTGSLWQEGAGWIPLPRPGLDLQAIELAAQSNHHCHAQKGPDSHCDPKKGKAQRQLYVASAICLLFMIGEVVGGYLAHSLAVMTDAAHLLTDFASMLISLFSLWMSSRPATKTMNFGWQRAEILGALVSVLSIWVVTGVLVYLAVERLISGDYEIDGGTMLITSGCAVAVNIIMGLTLHQSGHGHSHGTTNQQEENPSVRAAFIHVIGDFMQSMGVLVAAYILYFKPEYKYVDPICTFVFSILVLGTTLTILRDVILVLMEGTPKGVDFTAVRDLLLSVEGVEALHSLHIWALTVAQPVLSVHIAIAQNTDAQAVLKTASSRLQGKFHFHTVTIQIEDYSEDMKDCQACQGPSD.

Example 27

[3247] Tissue Distribution of 84226 mRNA by TagMan Analysis

[3248] Endogenous human 84226 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[3249] To determine the level of 84226 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Table 8.

24TABLE 8Phase 1.6.1 Expression of 84226.Tissue TypeMeanβ 2 Mean∂∂ CtExpressionArtery normal38.5223.9612.570Aorta diseased39.6722.7714.910Vein normal4020.6617.350Coronary SMC4023.1514.860HUVEC4021.7916.220Hemangioma38.9520.1716.790Heart normal31.8621.058.812.2281Heart CHF28.0520.285.7818.2621Kidney28.5520.75.8617.277Skeletal Muscle34.3824.547.854.3493Adipose normal4022.0515.960Pancreas27.4923.012.49178.6243primary osteoblasts4022.0815.930Osteoclasts (diff)4018.9819.030Skin normal38.9823.1613.820Spinal cord normal4022.1915.820Brain Cortex normal4023.5214.490Brain Hypothalamus normal4023.6714.340Nerve37.323.6911.620DRG (Dorsal Root Ganglion)38.1423.6212.530Breast normal39.3122.2815.040Breast tumor37.7922.0613.730Ovary normal4021.9716.040Ovary Tumor36.221.712.510Prostate Normal38.1521.4814.670Prostate Tumor38.1721.7614.420Salivary glands35.5721.0612.520Colon normal37.619.6415.970Colon Tumor35.222.5710.640Lung normal39.9319.3218.620Lung tumor36.1621.9312.230Lung COPD37.9920.0415.960Colon IBD38.1319.0917.040Liver normal37.8821.3514.540Liver fibrosis39.1223.1313.990Spleen normal4021.0516.960Tonsil normal39.5218.718.830Lymph node normal37.1720.6314.550Small intestine normal34.4321.6910.750.5807Macrophages4018.419.610Synovium4021.116.910BM-MNC4020.0917.920Activated PBMC4019.2618.750Neutrophils4020.417.610Megakaryocytes4020.0617.950Erythroid36.1322.9611.180positive control27.6221.514.1257.7114

Example 28

[3250] Tissue Distribution of 84226 mRNA by Northern Analysis

[3251] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 84226 cDNA (SEQ ID NO: 39) can be used. The DNA was radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 29

[3252] Recombinant Expression of 84226 in Bacterial Cells

[3253] In this example, 84226 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 84226 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-84226 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 30

[3254] Expression of Recombinant 84226 Protein in COS Cells

[3255] To express the 84226 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell I.23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 84226 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[3256] To construct the plasmid, the 84226 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 84226 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 84226 coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 84226_gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[3257] COS cells are subsequently transfected with the 84226-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 84226 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[3258] Alternatively, DNA containing the 84226 coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 84226 polypeptide is detected by radiolabelling and immunoprecipitation using an 84226 specific monoclonal antibody.

Examples for 8105

Example 31

[3259] Identification and Characterization of Human 8105 cDNA

[3260] The human 8105 nucleic acid sequence is recited as follows:

25(SEQ ID NO:43)CGACCACGCGTCCGGCTGGATAAGGCTGCGCCCATGTGAGTGCTGGGCTTGTACGTGCATTTTTGCCTGAGTGAGCATTAGTGGCAGTGTCCCCAGCCTACCCCTTTCCTGAATCCCAGGCTCATAGCCAACTGCCCACCTATTTCCACGTGGATGCCTGCTGAGCACCTCAAATGTCACACAGCCAAGACAGAACTCTGGATCTCCTTTCCCAGCCACAAGCTGCCCCTCTTCCAGTCTGCCACTCCCCACCTGTCCTGCCTTTGTGTGCCTCTGTGTCTTTGCTGGGTGGCCTGACCTTTGGTTATGAACTGGCAGTCATATCAGGTGCCCTGCTGCCACTGCAGCTTGACTTTGGGCTAAGCTGCTTGGAGCAGGAGTTCCTGGTGGGCAGCCTGCTCCTGGGGGCTCTCCTCGCCTCCCTGGTTGGTGGCTTCCTCATTGACTGCTATGGCAGGAAGCAAGCCATCCTCGGGAGCAACTTGGTGCTGCTGGCAGGCAGCCTGACCCTGGGCCTGGCTGGTTCCCTGGCCTGGCTGGTCCTGGGCCGCGCTGTGGTTGGCTTCGCCATTTCCCTCTCCTCCATGGCTTGCTGTATCTACGTGTCAGAGCTGGTGGGGCCACGGCAGCGGGGAGTGCTGGTGTCCCTCTATGAGGCAGGCATCACCGTGGGCATCCTGCTCTCCTATGCCCTCAACTATGCACTGGCTGGTACCCCCTGGGGATGGAGGCACATGTTCGGCTGGGCCACTGCACCTGCTGTCCTGCAATCCCTCAGCCTCCTCTTCCTCCCTGCTGGTACAGATGAGACTGCAACACACAAGGACCTCATCCCACTCCAGGGAGGTGAGGCCCCCAAGCTGGGCCCGGGGAGGCCACGGTACTCCTTTCTGGACCTCTTCAGGGCACGCGATAACATGCGAGGCCGGACCACAGTGGGCCTGGGGCTGGTGCTCTTCCAGCAACTAACAGGGCAGCCCAACGTGCTGTGCTATGCCTCCACCATCTTCAGCTCCGTTGGTTTCCATGGGGGATCCTCAGCCGTGCTGGCCTCTGTGGGGCTTGGCGCAGTGAAGGTGGCAGCTACCCTGACCGCCATGGGGCTGGTGGACCGTGCAGGCCGCAGGGCTCTGTTGCTAGCTGGCTGTGCCCTCATGGCCCTGTCCGTCAGTGGCATAGGCCTCGTCAGCTTTGCCGTGCCCATGGACTCAGGCCCAAGCTGTCTGGCTGTGCCCAATGCCACCGGGCAGACAGGCCTCCCTGGAGACTCTGGCCTGCTGCAGGACTCCTCTCTACCTCCCATTCCAAGGACCAATGAGGACCAAAGGGAGCCAATCTTGTCCACTGCTAAGAAAACCAAGCCCCATCCCAGATCTGGAGACCCCTCAGCCCCTCCTCGGCTGGCCCTGAGCTCTGCCCTCCCTGGGCCCCCTCTGCCCGCTCGGGGGCATGCACTGCTGCGCTGGACCGCACTGCTGTGCCTGATGGTCTTTGTCAGTGCCTTCTCCTTTGGGTTTGGGCCAGTGACCTGGCTTGTCCTCAGCGAGATCTACCCTGTGGAGATACGAGGAAGAGCCTTCGCCTTCTGCAACAGCTTCAACTGGGCGGCCAACCTCTTCATCAGCCTCTCCTTCCTCGATCTCATTGGCACCATCGGCTTGTCCTGGACCTTCCTGCTCTACGGACTGACCGCTGTCCTCGGCCTGGGCTTCATCTATTTATTTGTTCCTGAAACAAAAGGCCAGTCGTTGGCAGAGATAGACCAGCAGTTCCAGAAGAGACGGTTCACCCTGAGCTTTGGCCACAGGCAGAACTCCACTGGCATCCCGTACAGCCGCATCGAGATCTCTGCGGCCTCCTGAGGAATCCGTCTGCCTGGAAATTCTGGAACTGTGGCTTTGGCAGACCATCTCCAGCATCCTGCTTCCTAGGCCCCAGAGCACAAGTTCCAGCTGGTCTTTTGGGAGTGGCCCCTGCCCCCAAACGTGGTCTGCTTTTGCTGGGGTAAAAAGGATGAAAGTCTGAGAATGCCCAACTCTTCATTTTGAGTCTCAGGCCCTGAAGGTTCCTGAGGATCTAGCTTCATGCCTCAGTTTCCCCATTGACTTGCACATCTCTGCAGTATTTATAAGAAGAATATTCTATGAAGTCTTTGTTGCACCATGGACTTTTCTCAAAGAATCTCAAGGGTACCAATCCTGGCAGGAAGTCTCTCCCGATATCACCCCTAAATCCAAATGAGGATATCATCTTTTCTAATCTCTTTTTTCAACTGGCTGGGACATTTTCGGAAGGGGGAAGTCTCTTTTTTTACTCTTATCATTTTTTTTTTGAGGTGGAGTCTCATTCTGTTGCCCAGGCTGGCCTGATCTTGGCTCACTGCAACCTCCACCTCCTGAGTTCAAGCGATTCTTGTGCCTCAGCCTCCTAAGCAGCTGGGACTACAGGCGCATGCAACCATACCCAGCTAATTTATTTTTAGCAGAGATGGGGTTTCACTGTGTTGGCCAGGCTGGTCGTGAACTCCTGAGCTCAAGTGATCCACCCACCTCAGCCTCCCAGAGTGCTAGGATTACAGGCCTTTTGACTCTTTTATCTGAGTTTTATTGACCCCTCTAATTCTCTTACCCAGAATATTTATCCTTCACCAGCAACTCTGACTCTTTGACGGGAGGCCTCAGTTCTAGTCCTTGGTCTGCTGGTGTCATTGCTGTAGGAATGACCACGGGCCTCAGTTTCCCCATTTGTATAATGGGAAGCCTGTACCAGGTCATTCTTAAGATTTCTCCTGACTCCAGTGAGCTGGAATTCTAAATGCTGGTCTAGGAGCTGTCTCCAGGATGGTGCAGGATGGCTTTGCGGAAAGGAGATGGGTTTGGAGGCCAACAAACCTGCTTGTCAATATTGCCTTTGCCTCTTGGCAGCCCTTGAACTTGAGTAAATAACAACTCCCTGAACCTCAGTTTCCTCATCTGCAGAATGGGGATAATTATGTCCCAGGGGTATATTTAGACCCTGTTTCCTTTCAGGAGGGTCCCCAGCTGGTCCAGGGCCTGGGAAATTTCTACTTATCCTCATTACCCAGGTCCCTCCTTTGGACCCTGTAAAGGGTCAGGGTGAATCAGATGGGGGACTGAGCAAGTAGCTATGACCGCAGATCATGTAAGGAAGGGACTGACAAGAAGCTCCCAGATGCTGGGGAGAATGAAGAGCTAAAATAGATCCTAGGTGCTGGATGCTTTGTCATCCATGCGTGCACATATGGGTGCTGGCAGAGCCCCCAAGGACTCTGGCCTCTCGAGTTCTCCTATCTTCTCCATTCTAGATGCTTCCCTTGTATCCAGTGATGTGCTGGAGCTGGCTTTGCCAAGCTTGTGAGAGCTGGTTGCTACATTTTCAGGATTTTTACAAGTTGGTAAACACAGCCATTATAAAAAATTAAATGATTTAAATTTATAATTAAGTAAATTACATTAAAACAAAAAAATTATACTCAAAATTCATTACTTAATTTTACTACCTGTTACTATTATCTGTGCTTTTGAGGCTATTTCTACATAGTAACTCTTATGGAGACCTAGGGGAGACACCGCGCATCTCTTCCTGATTCCCCACTCAATGACATCATGTTAGTCTTTGGTTGCTTAACTGGCTGTGGGGAGTGTTTTTGTATCACAAAGATTAGAGAGGACTACACATCAGGGCTTGATTTATTGTTTGTTGATTTTCTAGACTTCAGAACATGCTGGATAAAATGTCAGTAATGCAAATTAAACTTTAAAGTATGTCTTGTTTGTAGCCAATACATGGTGTATAGCACCAAAAAATGGAGGGATTATTCTTCCAGTAGTTGAACACTGTCATCCGTTTCAGCTGACAGCTGCTCAAATCATTTAAGAAGGAGTTCTGACATTCATTTTCATTGTTTTACTTTTGTCTTCCTCACTAGTGTAAACAAAAATTTCAACCAGCATTCATGCCGAACCTATACCCATTCTTCAGTGCCTAGCTGTACAGTTATCAGGGATTTTTATTCGTAGTCTAATTTTGTCAAATCATGGCCAAATCGCAGTGATAGTTGACTTTGGATACAAGGTTTGGCAAAAAAAAAAAAAATATTAACAAAATATTCTGTAAGAATCAATTGGCTATATGGAATTTAGGATAAAGAATATTTACAATAAAGAATATTTACAATAAAGAGTTTATTATTATTTGTAAGTTGTGAGCAACAAACATACCCTTTATCTCTGTAAAATTTATACACACAAAAATTAACAAAAGATTCTGTAAGAATTAATTGGCTATATGGAATTTAGGATAGAATATTTACAATAAAGAGTATTTACAATAAAAAAAAAAAAAAAAAGGGCGGCCGCTAGACT.

[3261] The human 8105 sequence (SEQ ID NO: 43, as shown above) is approximately 4385 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 1689 nucleotides (SEQ ID NO: 45). The coding sequence encodes a 562 amino acid protein (SEQ ID NO: 44), which is recited as follows:

26(SEQ ID NO:44)MSHSQDRTLDLLSQPQAAPLPVCHSPPVLPLCASVSLLGGLTFGYELAVISGALLPLQLDFGLSCLEQEFLVGSLLLGALLASLVGGFLIDCYGRKQAILGSNLVLLAGSLTLGLAGSLAWLVLGRAVVGFAISLSSMACCIYVSELVGPRQRGVLVSLYEAGITVGILLSYALNYALAGTPWGWRHMFGWATAPAVLQSLSLLFLPAGTDETATHKDLIPLQGGEAPKLGPGRPRYSFLDLFRARDNMRGRTTVGLGLVLFQQLTGQPNVLCYASTIFSSVGFHGGSSAVLASVGLGAVKVAATLTAMGLVDRAGRRALLLAGCALMALSVSGIGLVSFAVPMDSGPSCLAVPNATGQTGLPGDSGLLQDSSLPPIPRTNEDQREPILSTAKKTKPHPRSGDPSAPPRLALSSALPGPPLPARGHALLRWTALLCLMVFVSAFSFGFGPVTWLVLSEIYPVEIRGRAFAFCNSFNWAANLFISLSFLDLIGTIGLSWTFLLYGLTAVLGLGFIYLFVPETKGQSLAEIDQQFQKRRFTLSFGHRQNSTGIPYSRIEISAAS.

Example 32

[3262] Tissue Distribution of 8105 mRNA by TagMan Analysis

[3263] Endogenous human 8105 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[3264] To determine the level of 8105 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Table 9. Expression was detected in most of the human tissues and cell lines tested.

27TABLE 9RelativeTissue TypeDiagnosisExpressionArteryNormal1.3763AortaDiseased1.1179VeinNormal1.0576SMCCoronary10.8212HUVECCells17.1577HemangiomaTumor1.0216HeartNormal2.8995HeartCHF1.4802KidneyNormal1.4751Skeletal MuscleNormal1.1493LiverNormal2.791Small IntestineNormal0.4021AdiposeNormal1.4957PancreasNormal13.9364OsteoblastsPrimary9.1946BladderNormal0.6763Adrenal GlandNormal0.8862Pituitary GlandNormal1.3294Spinal CordNormal0.9868Brain CortexNormal1.6086Brain HypothalamusNormal1.543NerveNormal1.5646DRG (Dorsal RootNormal1.0649Ganglion)BreastNormal1.7542BreastTumor1.3066OvaryNormal2.6496OvaryTumor0.9049ProstateBPH4.4253ProstateTumor5.6014ColonNormal0.5888ColonTumor0.8924LungNormal0.5727LungTumor0.6881LungCOPD0.7026ColonIBD0.4635SynoviumNormal0.2475TonsilNormal0.2484Lymph NodeNormal0.1127PBLUninfected0PBMCResting0MacrophagesCells0.0044ProgenitorsCells2.2436MegakaryocytesCells1.2797SpleenNormal0.0872NeutrophilsCells0.0142ErythroidCells0.8355PositiveControl1.8542

[3265] The expression of 8105 mRNA in various human tissues and cell lines is shown in Table 9. Highest levels of 8105 expression were detected in the pancreas, endothelial cells (HUVECs), smooth muscle cells, osteoblasts, prostate (both BPH and tumor cells), heart, liver, and ovary. The remaining tissues displayed moderate levels of 8105 expression, with the exception of PBL (uninfected) and PBMC cells (resting) which did not show any expression at all. 8105 mRNA expression was elevated in tumor samples from the lung, colon, and prostate, as compared to the respective normal tissues (benign prostatic hyperplasia (BPH) cells in the case of the prostate), while expression was decreased in ovarian tumors as compared to normal ovarian tissue.

Example 33

[3266] Tissue Distribution of 8105 mRNA by semi-quantitative RT-PCR

[3267] As an alternative to TaqMan ananlysis, the expression of 8105 was analyzed using standard RT-PCR reactions. Briefly, primers were designed for the amplification of a fragment of the 8105 message. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 mg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per PCR reaction. Tissues tested include the human tissues and several cell lines shown in Table 10, as well as in several tissues from both wild-type and obese (ob/ob) mice (Table 11). Expression was detected by agarose gel electrophoresis and ethidium bromide staining. Relative expression levels were determined visually. 8105 expression was observed in most human tissues tested, including the pancreas and hypothalamus. Importantly, 8105 expression was elevated in the brain tissue of wild-type mice as compared to ob/ob mice (Table 11).

28TABLE 10RelativeTissueExpressionHeart+Brain−Placenta+Lung+Liver++Skeletal Muscle−Kidney+/++Pancreas+++Hypothalamus+++

[3268] Table 10 shows the expression of human 8105 mRNA in several human tissues, as determined using semi-quantitative RT-PCR. Highest expression was seen in the hypothalamus, pancreas, and liver, all of which are involved in the regulation of metabolism.

29TABLE 11RelativeTissueMouseExpressionHeartWild-type+Heartob/ob+AdiposeWild-type+Adiposeob/ob+LiverWild-type+Liverob/ob+MuscleWild-type−Muscleob/ob−Brain*Wild-type++Brain*ob/ob++++HypothalamusWild-type++Hypothalamusob/ob++Negative ControlN/A−*Brain tissue samples lacking hypothalamus tissue

[3269] Table 11 shows the expression of mouse 8105 mRNA in tissues from both wild-type and obese (ob/ob) mice, as determined using semi-quantitative RT-PCR. 8105 mRNA expression is absent in muscle tissue (skeletal), low in heart, adipose, and liver tissues, and moderated in brain and hypothalamus tissues. Except for in the brain tissue, which lacks hypothalamus tissue, the level of 8105 mRNA expression is the same in both wild-type and obese mice. In the brain tissue, however, 8105 mRNA expression is much higher in obese mice, as compared to wild-type mice. This indicates that 8105 may be part of the leptin signaling network which is involved in the regulation of metabolism, hunger, and body weight.

Example 34

[3270] Tissue Distribution of 8105 mRNA by Northern Analysis

[3271] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 8105 cDNA (SEQ ID NO: 43) can be used. The DNA was radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 35

[3272] Recombinant Expression of 8105 in Bacterial Cells

[3273] In this example, 8105 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 8105 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-8105 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 36

[3274] Expression of Recombinant 8105 Protein in COS Cells

[3275] To express the 8105 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Cell I23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 8105 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[3276] To construct the plasmid, the 8105 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 8105 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 8105 coding sequence. The PCR amplified fragment and the pcDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 8105_gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[3277] COS cells are subsequently transfected with the 8105-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 8105 polypeptide is detected by radiolabelling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[3278] Alternatively, DNA containing the 8105 coding sequence is cloned directly into the polylinker of the pcDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 8105 polypeptide is detected by radiolabelling and immunoprecipitation using a 8105 specific monoclonal antibody.

[3279] Equivalents

[3280] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Number	Date	Country
PCT/US01/18340	Jun 2001	WO
PCT/US01/18398	Jun 2001	WO
PCT/US01/18247	Jun 2001	WO
PCT/US01/25474	Aug 2001	WO
PCT/US01/26096	Aug 2001	WO
PCT/US02/09728	Mar 2002	WO

Number	Date	Country
60290288	May 2001	US
60279281	Mar 2001	US
60226770	Aug 2000	US
60227068	Aug 2000	US
60209845	Jun 2000	US

	Number	Date	Country
Parent	09875321	Jun 2001	US
Child	10162102	Jun 2002	US
Parent	PCT/US01/18340	Jun 2001	US
Child	10162102	Jun 2002	US

Novel human ion channel and transporter family members

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