Novel human proteins, polynucleotides encoding them and methods of using the same

FIELD OF THE INVENTION

[0002] The present invention relates to nucleic acids encoding proteins that are new members of the following protein families: ubiquitin-conjugating enzyme-like protein, RINGO-like protein, downstream of tyrosine kinase 5 (DOK5)-like protein, sterol regulatory element binding protein cleavage-activating protein (SCAP)-like protein (homo sapiens), mutT-like domain containing protein-like protein, F-box protein-like protein, colonic and hepatic tumor over-expressed protein-like protein, splice variant of dishevelled-3-like protein, and IPAS-like protein.

[0003] Included in the invention are polynucleotides and the polypeptides encoded by such polynucleotides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using the same. Methods of use encompass diagnostic and prognostic assay procedures as well as methods of treating diverse pathological conditions.

BACKGROUND OF THE INVENTION

[0004] The invention generally relates to nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.

SUMMARY OF THE INVENTION

[0005] The present invention is based in part on nucleic acids encoding proteins that are members of the following protein families: ubiquitin-conjugating enzyme-like protein, RINGO-like protein, downstream of tyrosine kinase 5 (DOK5)-like protein, sterol regulatory element binding protein cleavage-activating protein (SCAP)-like protein (homo sapiens), mutT-like domain containing protein-like protein, F-box protein-like protein, colonic and hepatic tumor over-expressed protein-like protein, splice variant of dishevelled-3-like protein, and IPAS-like protein. The novel polynucleotides and polypeptides are referred to herein as NOV1a, NOV2a, NOV3a, NOV4a, NOV4b, NOV5a, NOV6a, NOV7a, NOV8a, and NOV9a. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as “NOVX” nucleic acid or polypeptide sequences.

[0006] In one aspect, the invention provides an isolated NOVX nucleic acid disclosed in SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20. In some embodiments, the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NO: 2n, wherein n is an integer between 1 and 20. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20. Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic acid (e.g., SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20) or a complement of said oligonucleotide.

[0007] The invention also encompasses isolated NOVX polypeptides (SEQ ID NO: 2n, wherein n is an integer between 1 and 20). In certain embodiments, the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.

[0008] The invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.

[0009] In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.

[0010] In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.

[0011] In another aspect, the invention includes a method of detecting the presence of a NOVX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.

[0012] The invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.

[0013] Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.

[0014] In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.

[0015] In another embodiment, the invention involves a method for identifying a potential therapeutic agent for use in treatment of a pathology, wherein the pathology is related to aberrant expression or aberrant physiological interactions of a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, the method including providing a cell expressing the polypeptide of the invention and having a property or function ascribable to the polypeptide; contacting the cell with a composition comprising a candidate substance; and determining whether the substance alters the property or function ascribable to the polypeptide; whereby, if an alteration observed in the presence of the substance is not observed when the cell is contacted with a composition devoid of the substance, the substance is identified as a potential therapeutic agent.

[0016] Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., allergies, Alzheimer's disease, neurodegenerative disorders, autoimmune disorders, inflammatory diseases, stroke, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia, depression, dementia, memory loss, autism, ataxias, diabetes, lung-inflammatory disease, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, glomerulonephritis, systemic lupus erythematosus, psoriasis, asthma, obesity, cancer, squamous cell carcinoma, colon cancer, liver cancer, ovarian cancer, prostate cancer, lung cancer, renal cancer, gastric cancer, breast cancer, melanoma and/or other pathologies and disorders of the like. Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing conditions including, e.g., those associated with homologs of a NOVX sequence, such as those listed in Table A.

[0017] The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or a NOVX-specific antibody, or biologically-active derivatives or fragments thereof.

[0018] For example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof.

[0019] The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said NOVX polypeptide. Binding of the test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.

[0020] Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g. the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.

[0021] In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide, a NOVX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the NOVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the NOVX polypeptide present in a control sample. An alteration in the level of the NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.

[0022] In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g. the diseases and disorders disclosed above and/or other pathologies and disorders of the like.

[0023] In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.

[0024] NOVX nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVX substances for use in therapeutic or diagnostic methods. These NOVX antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. The disclosed NOVX proteins have multiple hydrophilic regions, each of which can be used as an immunogen. These NOVX proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.

[0025] The NOVX nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences, their encoded polypeptides, antibodies, and other related compounds. The sequences are collectively referred to herein as “NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NOVX nucleic acids and their encoded polypeptides.

1TABLE ASequences and Corresponding SEQ ID NumbersSEQ IDSEQ IDNONONOVXInternal(nucleic(aminoAssignmentIdentificationacid)acid)HomologyNOV1aCG160009-0112Ubiquitin-conjugating enzyme-likeproteinNOV2aCG161998-0134RINGO-like proteinNOV3aCG162071-0156Downstream of tyrosine kinase 5(DOK5)-like proteinNOV4aCG162605-0178Sterol regulatory element bindingprotein cleavage-activating protein(SCAP)-like protein (homo sapiens)NOV4bCG162605-02910Sterol regulatory element bindingprotein cleavage-activating protein(SCAP)-like protein (homo sapiens)NOV5aCG163438-011112MutT-like domain containingprotein-like proteinNOV6aCG163701-011314F-box protein-like proteinNOV7aCG164211-011516Colonic and hepatic tumorover-expressed protein-like proteinNOV8aCG164330-011718Splice variant of dishevelled-3-likeproteinNOV9aCG164521-011920IPAS-like protein

[0029] Table A indicates the homology of NOVX polypeptides to known protein families. Thus, the nucleic acids and polypeptides, antibodies and related compounds according to the invention corresponding to a NOVX as identified in column 1 of Table A will be useful in therapeutic and diagnostic applications implicated in, for example, pathologies and disorders associated with the known protein families identified in column 5 of Table A.

[0030] Pathologies, diseases, disorders and condition and the like that are associated with NOVX sequences include, but are not limited to: allergies, Alzheimer's disease, neurodegenerative disorders, autoimmune disorders, inflammatory diseases, stroke, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia, depression, dementia, memory loss, autism, ataxias, diabetes, lung-inflammatory disease, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, glomerulonephritis, systemic lupus erythematosus, psoriasis, asthma, obesity, cancer, squamous cell carcinoma, colon cancer, liver cancer, ovarian cancer, prostate cancer, lung cancer, renal cancer, gastric cancer, breast cancer, and melanoma.

[0031] NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.

[0032] Consistent with other known members of the family of proteins, identified in column 5 of Table A, the NOVX polypeptides of the present invention show homology to, and contain domains that are characteristic of, other members of such protein families. Details of the sequence relatedness and domain analysis for each NOVX are presented in Example A.

[0033] The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit diseases associated with the protein families listed in Table A.

[0034] The NOVX nucleic acids and polypeptides are also useful for detecting specific cell types. Details of the expression analysis for each NOVX are presented in Example C. Accordingly, the NOVX nucleic acids, polypeptides, antibodies and related compounds according to the invention will have diagnostic and therapeutic applications in the detection of a variety of diseases with differential expression in normal vs. diseased tissues, e.g. detection of a variety of cancers.

[0035] Additional utilities for NOVX nucleic acids and polypeptides according to the invention are disclosed herein.

[0036] NOVX Clones

[0037] NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.

[0038] The NOVX genes and their corresponding encoded proteins are useful for preventing, treating or ameliorating medical conditions, e.g., by protein or gene therapy. Pathological conditions can be diagnosed by determining the amount of the new protein in a sample or by determining the presence of mutations in the new genes. Specific uses are described for each of the NOVX genes, based on the tissues in which they are most highly expressed. Uses include developing products for the diagnosis or treatment of a variety of diseases and disorders.

[0039] The NOVX nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) a biological defense weapon.

[0040] In one specific embodiment, the invention includes an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20; (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, wherein any amino acid in the mature form is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) an amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20; (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20 wherein any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; and (e) a fragment of any of (a) through (d).

[0041] In another specific embodiment, the invention includes an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a mature form of the amino acid sequence given SEQ ID NO: 2n, wherein n is an integer between 1 and 20; (b) a variant of a mature form of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20 wherein any amino acid in the mature form of the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence of the mature form are so changed; (c) the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20; (d) a variant of the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, in which any amino acid specified in the chosen sequence is changed to a different amino acid, provided that no more than 15% of the amino acid residues in the sequence are so changed; (e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2n, wherein n is an integer between 1 and 20 or any variant of said polypeptide wherein any amino acid of the chosen sequence is changed to a different amino acid, provided that no more than 10% of the amino acid residues in the sequence are so changed; and (f) the complement of any of said nucleic acid molecules.

[0042] In yet another specific embodiment, the invention includes an isolated nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20; (b) a nucleotide sequence wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed; (c) a nucleic acid fragment of the sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20; and (d) a nucleic acid fragment wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20 is changed from that selected from the group consisting of the chosen sequence to a different nucleotide provided that no more than 15% of the nucleotides are so changed.

[0043] NOVX Nucleic Acids and Polypeptides

[0044] One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.

[0045] A NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a “mature” form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product “mature” form arises, by way of nonlimiting example, as a result of one or more naturally occurring processing steps that may take place within the cell (e.g., host cell) in which the gene product arises. Examples of such processing steps leading to a “mature” form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a “mature” form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.

[0046] The term “probe”, as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), about 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single-stranded or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.

[0047] The term “isolated” nucleic acid molecule, as used herein, is a nucleic acid that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′- and 3′-termini 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 NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the celltissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, or of chemical precursors or other chemicals.

[0048] A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or a complement of this nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.)

[0049] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template with appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0050] As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.

[0051] In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of a NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, thereby forming a stable duplex.

[0052] As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

[0053] A “fragment” provided herein is defined as a sequence of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, and is at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.

[0054] A full-length NOVX clone is identified as containing an ATG translation start codon and an in-frame stop codon. Any disclosed NOVX nucleotide sequence lacking an ATG start codon therefore encodes a truncated C-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 5′ direction of the disclosed sequence. Any disclosed NOVX nucleotide sequence lacking an in-frame stop codon similarly encodes a truncated N-terminal fragment of the respective NOVX polypeptide, and requires that the corresponding full-length cDNA extend in the 3′ direction of the disclosed sequence.

[0055] A “derivative” is a nucleic acid sequence or amino acid sequence formed from the native compounds either directly, by modification or partial substitution. An “analog” is a nucleic acid sequence or amino acid sequence that has a structure similar to, but not identical to, the native compound, e.g. they differs from it in respect to certain components or side chains. Analogs may be synthetic or derived from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. A “homolog” is a nucleic acid sequence or amino acid sequence of a particular gene that is derived from different species.

[0056] Derivatives and analogs may be full length or other than full length. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below.

[0057] A “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences include those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.

[0058] A NOVX polypeptide is encoded by the open reading frame (“ORF”) of a NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG “start” codon and terminates with one of the three “stop” codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bonafide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.

[0059] The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20; or an anti-sense strand nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20; or of a naturally occurring mutant of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20.

[0060] Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe has a detectable label attached, e.g. the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a NOVX protein, such as by measuring a level of a NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.

[0061] “A polypeptide having a biologically-active portion of a NOVX polypeptide” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a “biologically-active portion of NOVX” can be prepared by isolating a portion of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, that encodes a polypeptide having a NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.

[0062] NOVX Nucleic Acid and Polypeptide Variants

[0063] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO: 2n, wherein n is an integer between 1 and 20.

[0064] In addition to the human NOVX nucleotide sequences of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention.

[0065] Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from a human SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

[0066] Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 65% homologous to each other typically remain hybridized to each other.

[0067] Homologs (i.e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.

[0068] As used herein, the phrase “stringent hybridization conditions” refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

[0069] Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al.,,(eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C., followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0070] In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5×Reinhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

[0071] In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g. as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Krieger, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.

[0072] Conservative Mutations

[0073] In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, thereby leading to changes in the amino acid sequences of the encoded NOVX protein, without altering the functional ability of that NOVX protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO: 2n, wherein n is an integer between 1 and 20. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an “essential” amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.

[0074] Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 40% homologous to the amino acid sequences of SEQ ID NO: 2n, wherein n is an integer between 1 and 20. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NO: 2n, wherein n is an integer between 1 and 20; more preferably at least about 70% homologous to SEQ ID NO: 2n, wherein n is an integer between 1 and 20; still more preferably at least about 80% homologous to SEQ ID NO: 2n, wherein n is an integer between 1 and 20; even more preferably at least about 90% homologous to SEQ ID NO: 2n, wherein n is an integer between 1 and 20; and most preferably at least about 95% homologous to SEQ ID NO: 2n, wherein n is an integer between 1 and 20.

[0075] An isolated nucleic acid molecule encoding a NOVX protein homologous to the protein of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

[0076] Mutations can be introduced any one of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. 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 within 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 non-essential amino acid residue in the NOVX protein is 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 NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis of a nucleic acid of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.

[0077] The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved “strong” residues or fully conserved “weak” residues. The “strong” group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the “weak” group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.

[0078] In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form protein:protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and a NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).

[0079] In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).

[0080] Interfering RNA

[0081] In one aspect of the invention, NOVX gene expression can be attenuated by RNA interference. One approach well-known in the art is short interfering RNA (siRNA) mediated gene silencing where expression products of a NOVX gene are targeted by specific double stranded NOVX derived siRNA nucleotide sequences that are complementary to at least a 19-25 nt long segment of the NOVX gene transcript, including the 5′ untranslated (UT) region, the ORF, or the 3′ UT region. See, e.g., PCT applications WO00/44895, WO99/32619, WO01/75164, WO01/92513, WO 01/29058, WO01/89304, WO02/16620, and WO02/29858, each incorporated by reference herein in their entirety. Targeted genes can be a NOVX gene, or an upstream or downstream modulator of the NOVX gene. Nonlimiting examples of upstream or downstream modulators of a NOVX gene include, e.g., a transcription factor that binds the NOVX gene promoter, a kinase or phosphatase that interacts with a NOVX polypeptide, and polypeptides involved in a NOVX regulatory pathway.

[0082] According to the methods of the present invention, NOVX gene expression is silenced using short interfering RNA. A NOVX polynucleotide according to the invention includes a siRNA polynucleotide. Such a NOVX siRNA can be obtained using a NOVX polynucleotide sequence, for example, by processing the NOVX ribopolynucleotide sequence in a cell-free system, such as but not limited to a Drosophila extract, or by transcription of recombinant double stranded NOVX RNA or by chemical synthesis of nucleotide sequences homologous to a NOVX sequence. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999), Genes & Dev. 13: 3191-3197, incorporated herein by reference in its entirety. When synthesized, a typical 0.2 micromolar-scale RNA synthesis provides about 1 milligram of siRNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.

[0083] The most efficient silencing is generally observed with siRNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand, paired in a manner to have a 2-nt 3′ overhang. The sequence of the 2-nt 3′ overhang makes an additional small contribution to the specificity of siRNA target recognition. The contribution to specificity is localized to the unpaired nucleotide adjacent to the first paired bases. In one embodiment, the nucleotides in the 3′ overhang are ribonucleotides. In an alternative embodiment, the nucleotides in the 3′ overhang are deoxyribonucleotides. Using 2′-deoxyribonucleotides in the 3′ overhangs is as efficient as using ribonucleotides, but deoxyribonucleotides are often cheaper to synthesize and are most likely more nuclease resistant.

[0084] A contemplated recombinant expression vector of the invention comprises a NOVX DNA molecule cloned into an expression vector comprising operatively-linked regulatory sequences flanking the NOVX sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands. An RNA molecule that is antisense to NOVX mRNA is transcribed by a first promoter (e.g., a promoter sequence 3′ of the cloned DNA) and an RNA molecule that is the sense strand for the NOVX mRNA is transcribed by a second promoter (e.g., a promoter sequence 5′ of the cloned DNA). The sense and antisense strands may hybridize in vivo to generate siRNA constructs for silencing of the NOVX gene. Alternatively, two constructs can be utilized to create the sense and anti-sense strands of a siRNA construct. Finally, cloned DNA can encode a construct having secondary structure, wherein a single transcript has both the sense and complementary anuisense sequences from the target gene or genes. In an example of this embodiment, a hairpin RNAi product is homologous to all or a portion of the target gene. In another example, a hairpin RNAi product is a siRNA. The regulatory sequences flanking the NOVX sequence may be identical or may be different, such that their expression may be modulated independently, or in a temporal or spatial manner.

[0085] In a specific embodiment, siRNAs are transcribed intracellularly by cloning the NOVX gene templates into a vector containing, e.g., a RNA po1 III transcription unit from the smaller nuclear RNA (snRNA) U6 or the human RNase P RNA H1. One example of a vector system is the GeneSuppressor™ RNA Interference kit (commercially available from Imgenex). The U6 and H1 promoters are members of the type III class of Po1 III promoters. The +1 nucleotide of the U6-like promoters is always guanosine, whereas the +1 for H1 promoters is adenosine. The termination signal for these promoters is defined by five consecutive thymidines. The transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3′ UU overhang in the expressed siRNA, which is similar to the 3′ overhangs of synthetic siRNAs. Any sequence less than 400 nucleotides in length can be transcribed by these promoter, therefore they are ideally suited for the expression of around 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNA stem-loop transcript.

[0086] A siRNA vector appears to have an advantage over synthetic siRNAs where long term knock-down of expression is desired. Cells transfected with a siRNA expression vector would experience steady, long-term mRNA inhibition. In contrast, cells transfected with exogenous synthetic siRNAs typically recover from mRNA suppression within seven days or ten rounds of cell division. The long-term gene silencing ability of siRNA expression vectors may provide for applications in gene therapy.

[0087] In general, siRNAs are chopped from longer dsRNA by an ATP-dependent ribonuclease called DICER. DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases. The siRNAs assemble with cellular proteins into an endonuclease complex. In vitro studies in Drosophila suggest that the siRNAs/protein complex (siRNP) is then transferred to a second enzyme complex, called an RNA-induced silencing complex (RISC), which contains an endoribonuclease that is distinct from DICER. RISC uses the sequence encoded by the antisense siRNA strand to find and destroy mRNAs of complementary sequence. The siRNA thus acts as a guide, restricting the ribonuclease to cleave only mRNAs complementary to one of the two siRNA strands.

[0088] A NOVX mRNA region to be targeted by siRNA is generally selected from a desired NOVX sequence beginning 50 to 100 nt downstream of the start codon. Alternatively, 5′ or 3′ UTRs and regions nearby the start codon can be used but are generally avoided, as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. An initial BLAST homology search for the selected siRNA sequence is done against an available nucleotide sequence library to ensure that only one gene is targeted. Specificity of target recognition by siRNA duplexes indicate that a single point mutation located in the paired region of an siRNA duplex is sufficient to abolish target mRNA degradation. See, Elbashir et al. 2001 EMBO J. 20(23):6877-88. Hence, consideration should be taken to accommodate SNPs, polymorphisms, allelic variants or species-specific variations when targeting a desired gene.

[0089] In one embodiment, a complete NOVX siRNA experiment includes the proper negative control. A negative control siRNA generally has the same nucleotide composition as the NOVX siRNA but lack significant sequence homology to the genome. Typically, one would scramble the nucleotide sequence of the NOVX siRNA and do a homology search to make sure it lacks homology to any other gene.

[0090] Two independent NOVX siRNA duplexes can be used to knock-down a target NOVX gene. This helps to control for specificity of the silencing effect. In addition, expression of two independent genes can be simultaneously knocked down by using equal concentrations of different NOVX siRNA duplexes, e.g., a NOVX siRNA and an siRNA for a regulator of a NOVX gene or polypeptide. Availability of siRNA-associating proteins is believed to be more limiting than target mRNA accessibility.

[0091] A targeted NOVX region is typically a sequence of two adenines (AA) and two thymidines (TT) divided by a spacer region of nineteen (N19) residues (e.g., AA(N19)TT). A desirable spacer region has a G/C-content of approximately 30% to 70%, and more preferably of about 50%. If the sequence AA(N19)TT is not present in the target sequence, an alternative target region would be AA(N21). The sequence of the NOVX sense siRNA corresponds to (N19)TT or N21, respectively. In the latter case, conversion of the 3′ end of the sense siRNA to TT can be performed if such a sequence does not naturally occur in the NOVX polynucleotide. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3′ overhangs. Symmetric 3′ overhangs may help to ensure that the siRNPs are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs. See, e.g., Elbashir, Lendeckel and Tuschl (2001). Genes & Dev. 15: 188-200, incorporated by reference herein in its entirely. The modification of the overhang of the sense sequence of the siRNA duplex is not expected to affect targeted mRNA recognition, as the antisense siRNA strand guides target recognition.

[0092] Alternatively, if the NOVX target mRNA does not contain a suitable AA(N21) sequence, one may search for the sequence NA(N21). Further, the sequence of the sense strand and antisense strand may still be synthesized as 5′ (N19)TT, as it is believed that the sequence of the 3′-most nucleotide of the antisense siRNA does not contribute to specificity. Unlike antisense or ribozyme technology, the secondary structure of the target mRNA does not appear to have a strong effect on silencing. See, Harborth, et al. (2001) J. Cell Science 114: 4557-4565, incorporated by reference in its entirety.

[0093] Transfection of NOVX siRNA duplexes can be achieved using standard nucleic acid transfection methods, for example, OLIGOFECTAMINE Reagent (commercially available from Invitrogen). An assay for NOVX gene silencing is generally performed approximately 2 days after transfection. No NOVX gene silencing has been observed in the absence of transfection reagent, allowing for a comparative analysis of the wild-type and silenced NOVX phenotypes. In a specific embodiment, for one well of a 24-well plate, approximately 0.84 μg of the siRNA duplex is generally sufficient. Cells are typically seeded the previous day, and are transfected at about 50% confluence. The choice of cell culture media and conditions are routine to those of skill in the art, and will vary with the choice of cell type. The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. Low transfection efficiencies are the most frequent cause of unsuccessful NOVX silencing. The efficiency of transfection needs to be carefully examined for each new cell line to be used. Preferred cell are derived from a mammal, more preferably from a rodent such as a rat or mouse, and most preferably from a human. Where used for therapeutic treatment, the cells are preferentially autologous, although non-autologous cell sources are also contemplated as within the scope of the present invention.

[0094] For a control experiment, transfection of 0.84 μg single-stranded sense NOVX siRNA will have no effect on NOVX silencing, and 0.84 μg antisense siRNA has a weak silencing effect when compared to 0.84 μg of duplex siRNAs. Control experiments again allow for a comparative analysis of the wild-type and silenced NOVX phenotypes. To control for transfection efficiency, targeting of common proteins is typically performed, for example targeting of lamin A/C or transfection of a CMV-driven EGFP-expression plasmid (e.g. commercially available from Clontech). In the above example, a determination of the fraction of lamin A/C knockdown in cells is determined the next day by such techniques as immunofluorescence, Western blot, Northern blot or other similar assays for protein expression or gene expression. Lamin A/C monoclonal antibodies may be obtained from Santa Cruz Biotechnology.

[0095] Depending on the abundance and the half life (or turnover) of the targeted NOVX polynucleotide in a cell, a knock-down phenotype may become apparent after 1 to 3 days, or even later. In cases where no NOVX knock-down phenotype is observed, depletion of the NOVX polynucleotide may be observed by immunofluorescence or Western blotting. If the NOVX polynucleotide is still abundant after 3 days, cells need to be split and transferred to a fresh 24-well plate for re-transfection. If no knock-down of the targeted protein is observed, it may be desirable to analyze whether the target mRNA (NOVX or a NOVX upstream or downstream gene) was effectively destroyed by the transfected siRNA duplex. Two days after transfection, total RNA is prepared, reverse transcribed using a target-specific primer, and PCR-amplified with a primer pair covering at least one exon-exon junction in order to control for amplification of pre-mRNAs. RT/PCR of a non-targeted mRNA is also needed as control. Effective depletion of the mRNA yet undetectable reduction of target protein may indicate that a large reservoir of stable NOVX protein may exist in the cell. Multiple transfection in sufficiently long intervals may be necessary until the target protein is finally depleted to a point where a phenotype may become apparent. If multiple transfection steps are required, cells are split 2 to 3 days after transfection. The cells may be transfected immediately after splitting.

[0096] An inventive therapeutic method of the invention contemplates administering a NOVX siRNA construct as therapy to compensate for increased or aberrant NOVX expression or activity. The NOVX ribopolynucleotide is obtained and processed into siRNA fragments, or a NOVX siRNA is synthesized, as described above. The NOVX siRNA is administered to cells or tissues using known nucleic acid transfection techniques, as described above. A NOVX siRNA specific for a NOVX gene will decrease or knockdown NOVX transcription products, which will lead to reduced NOVX polypeptide production, resulting in reduced NOVX polypeptide activity in the cells or tissues.

[0097] The present invention also encompasses a method of treating a disease or condition associated with the presence of a NOVX protein in an individual comprising administering to the individual an RNAi construct that targets the mRNA of the protein (the mRNA that encodes the protein) for degradation. A specific RNAi construct includes a siRNA or a double stranded gene transcript that is processed into siRNAs. Upon treatment, the target protein is not produced or is not produced to the extent it would be in the absence of the treatment.

[0098] Where the NOVX gene function is not correlated with a known phenotype, a control sample of cells or tissues from healthy individuals provides a reference standard for determining NOVX expression levels. Expression levels are detected using the assays described, e.g., RT-PCR, Northern blotting, Western blotting, ELISA, and the like. A subject sample of cells or tissues is taken from a mammal, preferably a human subject, suffering from a disease state. The NOVX ribopolynucleotide is used to produce siRNA constructs, that are specific for the NOVX gene product. These cells or tissues are treated by administering NOVX siRNA's to the cells or tissues by methods described for the transfection of nucleic acids into a cell or tissue, and a change in NOVX polypeptide or polynucleotide expression is observed in the subject sample relative to the control sample, using the assays described. This NOVX gene knockdown approach provides a rapid method for determination of a NOVX minus (NOVX) phenotype in the treated subject sample. The NOVXC phenotype observed in the treated subject sample thus serves as a marker for monitoring the course of a disease state during treatment.

[0099] In specific embodiments, a NOVX siRNA is used in therapy. Methods for the generation and use of a NOVX siRNA are known to those skilled in the art. Example techniques are provided below.

[0100] Production of RNAs

[0101] Sense RNA (ssRNA) and antisense RNA (asRNA) of NOVX are produced using known methods such as transcription in RNA expression vectors. In the initial experiments, the sense and antisense RNA are about 500 bases in length each. The produced ssRNA and asRNA (0.5 μM) in 10 mM Tris-HCl (pH 7.5) with 20 mM NaCl were heated to 95° C. for 1 min then cooled and annealed at room temperature for 12 to 16 h. The RNAs are precipitated and resuspended in lysis buffer (below). To monitor annealing, RNAs are electrophoresed in a 2% agarose gel in TBE buffer and stained with ethidium bromide. See, e.g., Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989).

[0102] Lysate Preparation

[0103] Untreated rabbit reticulocyte lysate (Ambion) are assembled according to the manufacturer's directions. dsRNA is incubated in the lysate at 30° C. for 10 min prior to the addition of mRNAs. Then NOVX mRNAs are added and the incubation continued for an additional 60 min. The molar ratio of double stranded RNA and mRNA is about 200:1. The NOVX mRNA is radiolabeled (using known techniques) and its stability is monitored by gel electrophoresis.

[0104] In a parallel experiment made with the same conditions, the double stranded RNA is internally radiolabeled with a 32P-ATP. Reactions are stopped by the addition of 2×proteinase K buffer and deproteinized as described previously (Tuschl et al., Genes Dev., 13:3191-3197 (1999)). Products are analyzed by electrophoresis in 15% or 18% polyacrylamide sequencing gels using appropriate RNA standards. By monitoring the gels for radioactivity, the natural production of 10 to 25 nt RNAs from the double stranded RNA can be determined.

[0105] The band of double stranded RNA, about 21-23 bps, is eluded. The efficacy of these 21-23 mers for suppressing NOVX transcription is assayed in vitro using the same rabbit reticulocyte assay described above using 50 nanomolar of double stranded 21-23 mer for each assay. The sequence of these 21-23 mers is then determined using standard nucleic acid sequencing techniques.

[0106] RNA Preparation

[0107] 21 nt RNAs, based on the sequence determined above, are chemically synthesized using Expedite RNA phosphoramidites and thymidine phosphoramidite (Proligo, Germany). Synthetic oligonucleotides are deprotected and gel-purified (Elbashir, Lendeckel, & Tuschl, Genes & Dev. 15, 188-200 (2001)), followed by Sep-Pak C18 cartridge (Waters, Milford, Mass., USA) purification (Tuschl, et al., Biochemistry, 32:11658-11668 (1993)).

[0108] These RNAs (20 μM) single strands are incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90° C. followed by 1 h at 37° C.

[0109] Cell Culture

[0110] A cell culture known in the art to regularly express NOVX is propagated using standard conditions. 24 hours before transfection, at approx. 80% confluency, the cells are trypsinized and diluted 1:5 with fresh medium without antibiotics (1-3×105 cells/ml) and transferred to 24-well plates (500 ml/well). Transfection is performed using a commercially available lipofection kit and NOVX expression is monitored using standard techniques with positive and negative control. A positive control is cells that naturally express NOVX while a negative control is cells that do not express NOVX. Base-paired 21 and 22 nt siRNAs with overhanging 3′ ends mediate efficient sequence-specific mRNA degradation in lysates and in cell culture. Different concentrations of siRNAs are used. An efficient concentration for suppression in vitro in mammalian culture is between 25 nM to 100 nM final concentration. This indicates that siRNAs are effective at concentrations that are several orders of magnitude below the concentrations applied in conventional antisense or ribozyme gene targeting experiments.

[0111] The above method provides a way both for the deduction of NOVX siRNA sequence and the use of such siRNA for in vitro suppression. In vivo suppression may be performed using the same siRNA using well known in vivo transfection or gene therapy transfection techniques.

[0112] Antisense Nucleic Acids

[0113] Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that 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). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NOVX protein of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, or antisense nucleic acids complementary to a NOVX nucleic acid sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, are additionally provided.

[0114] In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a NOVX protein. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding the NOVX protein. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0115] Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or 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).

[0116] Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 5-methoxyuracil, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 2-thiouracil, 4-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subdloned 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).

[0117] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, 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 that 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 nucleic acid 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.

[0118] 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. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g. Inoue, et al., 1987. FEBS Lett. 215: 327-330.

[0119] Ribozymes and PNA Moieties

[0120] Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.

[0121] In one embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for a NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of a NOVX cDNA disclosed herein (i.e., SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20). 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 NOVX-encoding mRNA. See, e.g., U.S. Pat. No. 4,987,071 to Cech, et al. and U.S. Pat. No. 5,116,742 to Cech, et al. NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

[0122] Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.

[0123] In various embodiments, the NOVX nucleic acids 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 acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleotide bases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomer can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.

[0124] PNAs of NOVX 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, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (See, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).

[0125] In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their 20 stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleotide bases, and orientation (see, Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment. See, e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.

[0126] 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. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988. BioTechniques 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, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.

[0127] NOVX Polypeptides

[0128] A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in any one of SEQ ID NO: 2n, wherein n is an integer between 1 and 20. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in any one of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.

[0129] In general, a NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.

[0130] One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

[0131] An “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language “substantially free of cellular material” includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX 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 NOVX protein preparation.

[0132] The language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.

[0133] Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence of SEQ ID NO: 2n, wherein n is an integer between 1 and 20) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of a NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A biologically-active portion of a NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.

[0134] 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 NOVX protein.

[0135] In an embodiment, the NOVX protein has an amino acid sequence of SEQ ID NO: 2n, wherein n is an integer between 1 and 20. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NO: 2n, wherein n is an integer between 1 and 20, and retains the functional activity of the protein of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, and retains the functional activity of the NOVX proteins of SEQ ID NO: 2n, wherein n is an integer between 1 and 20.

[0136] Determining Homology Between Two or More Sequences

[0137] To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid 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 homologous at that position (i.e., as used herein amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”).

[0138] The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20.

[0139] The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.

[0140] Chimeric and Fusion Proteins

[0141] The invention also provides NOVX chimeric or fusion proteins. As used herein, a NOVX “chimeric protein” or “fusion protein” comprises a NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An “NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a NOVX protein of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, whereas a “non-NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within a NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of a NOVX protein. In one embodiment, a NOVX fusion protein comprises at least one biologically-active portion of a NOVX protein. In another embodiment, a NOVX fusion protein comprises at least two biologically-active portions of a NOVX protein. In yet another embodiment, a NOVX fusion protein comprises at least three biologically-active portions of a NOVX protein. Within the fusion protein, the term “operatively-linked” is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.

[0142] In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.

[0143] In another embodiment, the fusion protein is a NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.

[0144] In yet another embodiment, the fusion protein is a NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a NOVX ligand and a NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of a NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in'screening assays to identify molecules that inhibit the interaction of NOVX with a NOVX ligand.

[0145] A NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.

[0146] NOVX Agonists and Antagonists

[0147] The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein). An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, 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 NOVX proteins.

[0148] Variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res. 11: 477.

[0149] Polypeptide Libraries

[0150] In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of a NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX proteins.

[0151] Various techniques are known in the art 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. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. 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 NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein Engineering 6:327-331.

[0152] Anti-NOVX Antibodies

[0153] Included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab, and F(ab′)2 fragments, and an Fab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

[0154] An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence of SEQ ID NO: 2n, wherein n is an integer between 1 and 20, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

[0155] In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human NOVX protein sequence will indicate which regions of a NOVX polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

[0156] The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. A NOVX polypeptide or a fragment thereof comprises at least one antigenic epitope. An anti-NOVX antibody of the present invention is said to specifically bind to antigen NOVX when the equilibrium binding constant (KD) is ≦1 μM, preferably ≦100 nM, more preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0157] A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

[0158] Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference). Some of these antibodies are discussed below.

[0159] Polyclonal Antibodies

[0160] For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

[0161] The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0162] Monoclonal Antibodies

[0163] The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[0164] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0165] The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

[0166] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0167] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

[0168] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding,1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0169] The monoclonal antibodies secreted by the subelones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0170] The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0171] Humanized Antibodies

[0172] The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Pat. No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0173] Human Antibodies

[0174] Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0175] In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al,(Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).

[0176] Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

[0177] An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

[0178] A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

[0179] In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.

[0180] Fab Fragments and Single Chain Antibodies

[0181] According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.

[0182] Bispecific Antibodies

[0183] Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

[0184] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0185] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0186] According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0187] Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab′)2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0188] Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0189] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

[0190] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0191] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

[0192] Heteroconjugate Antibodies

[0193] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0194] Effector Function Engineering

[0195] It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

[0196] Immunoconjugates

[0197] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0198] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.

[0199] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

[0200] In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.

[0201] Immunoliposomes

[0202] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

[0203] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).

[0204] Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention

[0205] In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

[0206] Antibodies directed against a NOVX protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of a NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to a NOVX protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as “Therapeutics”).

[0207] An antibody specific for a NOVX protein of the invention (e.g., a monoclonal antibody or a polyclonal antibody) can be used to isolate a NOVX polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. An antibody to a NOVX polypeptide can facilitate the purification of a natural NOVX antigen from cells, or of a recombinantly produced NOVX antigen expressed in host cells. Moreover, such an anti-NOVX antibody can be used to detect the antigenic NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic NOVX protein. Antibodies directed against a NOVX protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. 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, 311I, 35S or 3H.

[0208] Antibody Therapeutics

[0209] Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question. In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.

[0210] Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.

[0211] A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.

[0212] Pharmaceutical Compositions of Antibodies

[0213] Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0214] If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0215] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

[0216] The formulations to be used for in vivo administration must be sterile. This is, readily accomplished by filtration through sterile filtration membranes.

[0217] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0218] ELISA Assay

[0219] An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with 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 another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein 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.

[0220] NOVX Recombinant Expression Vectors and Host Cells

[0221] Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0222] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

[0223] The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that 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, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).

[0224] The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0225] Expression of proteins in prokaryotes is most often carried out in Escherichia 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: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, 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 and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0226] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0227] One strategy 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. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 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 (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0228] In another embodiment, the NOVX expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyce cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0229] Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0230] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, et al., 1987. EMBO J. 6: 187-195). 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. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0231] 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). Tissue-specific regulatory elements are known in the art. 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, e.g., 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).

[0232] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters andlor enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., “Antisense RNA as a molecular tool for genetic analysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.

[0233] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also 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.

[0234] A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX 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). Other suitable host cells are known to those skilled in the art.

[0235] Vector DNA can be introduced into prokaryotic or eukaryotic 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. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[0236] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0237] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.

[0238] Transgenic NOVX Animals

[0239] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein 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, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered 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.

[0240] A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences, i.e., any one of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. 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 the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX 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 NOVX protein can further be bred to other transgenic animals carrying other transgenes.

[0241] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of any one of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector).

[0242] Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5′- and 3′-termini by additional nucleic acid of the NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′- and 3′-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al., 1992. Cell 69:915.

[0243] The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to fomi aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

[0244] In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0245] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.

[0246] Pharmaceutical Compositions

[0247] The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as “active compounds”) of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0248] A pharmaceutical composition of the invention 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 (i.e., 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 (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The 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.

[0249] 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 syringeability exists. It must 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 polyethylene 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.

[0250] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a NOVX protein or anti-NOVX antibody) 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 that 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, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0251] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. 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. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] It is especially 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. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0257] 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, e.g., 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 that produce the gene delivery system.

[0258] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0259] Screening and Detection Methods

[0260] The isolated nucleic acid molecules of the invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in a NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.

[0261] The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.

[0262] Screening Assays

[0263] The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g. peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity. The invention also includes compounds identified in the screening assays described herein.

[0264] In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a NOVX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; 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 approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12:145.

[0265] A “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.

[0266] 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. U.S.A. 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.

[0267] 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), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409), 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. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Pat. No. 5,233,409.).

[0268] In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test 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. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX 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 NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.

[0269] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule. As used herein, a “target molecule” is a molecule with which a NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A NOVX target molecule can be a non-NOVX molecule or a NOVX protein or polypeptide of the invention. In one embodiment, a NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.

[0270] Determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.

[0271] In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX 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 NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.

[0272] In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to a NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate a NOVX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.

[0273] In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein 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 NOVX protein, wherein determining the ability of the test compound to interact with a NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of a NOVX target molecule.

[0274] The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-meihylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).

[0275] In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein 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 NOVX protein, or interaction of NOVX 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 that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is 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, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.

[0276] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX 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 NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.

[0277] In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX 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 NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.

[0278] In yet another aspect of the invention, the NOVX 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 WO 94/10300), to identify other proteins that bind to or interact with NOVX (“NOVX-binding proteins” or “NOVX-bp”) and modulate NOVX activity. Such NOVX-binding proteins are also involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX pathway.

[0279] 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 NOVX 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. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a NOVX-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) that 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 that encodes the protein which interacts with NOVX.

[0280] The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.

[0281] Detection Assays

[0282] Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.

[0283] Chromosome Mapping

[0284] Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome. The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

[0285] Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. 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 NOVX sequences will yield an amplified fragment.

[0286] Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

[0287] PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.

[0288] 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. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. 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 (Pergamon Press, New York 1988).

[0289] 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.

[0290] 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, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987. Nature, 325: 783-787.

[0291] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX 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.

[0292] Tissue Typing

[0293] The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA markers for RFLP (“restriction fragment length polymorphisms,” described in U.S. Pat. No. 5,272,057).

[0294] Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5′- and 3′-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

[0295] 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. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).

[0296] 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 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If coding sequences, such as those of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0297] Predictive Medicine

[0298] The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in a NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.

[0299] Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as “pharmacogenomics”). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)

[0300] Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.

[0301] These and other agents are described in further detail in the following sections.

[0302] Diagnostic Assays

[0303] An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NO: 2n-1, wherein n is an integer between 1 and 20, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[0304] An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with 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 another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX 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.

[0305] In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

[0306] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.

[0307] The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with 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 NOVX protein or nucleic acid.

[0308] Prognostic Assays

[0309] The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

[0310] Furthermore, 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 NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).

[0311] The methods of the invention can also be used to detect genetic lesions in a NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from a NOVX gene; (ii) an addition of one or more nucleotides to a NOVX gene; (iii) a substitution of one or more nucleotides of a NOVX gene, (iv) a chromosomal rearrangement of a NOVX gene; (v) an alteration in the level of a messenger RNA transcript of a NOVX gene, (vi) aberrant modification of a NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a NOVX gene, (viii) a non-wild-type level of a NOVX protein, (ix) allelic loss of a NOVX gene, and (x) inappropriate post-translational modification of a NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in a NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

[0312] In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a NOVX gene under conditions such that hybridization and amplification of the NOVX 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.

[0313] Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0314] In an alternative embodiment, mutations in a NOVX gene from a sample cell can be identified by 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 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, e.g., U.S. Pat. No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0315] In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, 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 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.

[0316] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995. Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al., 1996. Adv. Chromatography 36: 127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).

[0317] Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.

[0318] 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 NOVX 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. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on a NOVX sequence, e.g., a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Pat. No. 5,459,039.

[0319] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX 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 one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al., 1991. Trends Genet. 7: 5.

[0320] 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). See, e.g., 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. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

[0321] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324: 163; Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[0322] 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; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3′-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., 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. See, e.g., 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. See, e.g., 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′-terminus 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.

[0323] 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 NOVX gene.

[0324] Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.

[0325] Pharmacogenomics

[0326] Agents, or modulators that have a stimulatory or inhibitory effect on NOVX activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

[0327] In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual 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, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.

[0328] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharnacol. Physiol., 23: 983-985; Linder, 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 defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0329] As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome pregnancy zone protein precursor enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-forned metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

[0330] Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. 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 NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[0331] Monitoring of Effects During Clinical Trials

[0332] Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a “read out” or markers of the immune responsiveness of a particular cell.

[0333] By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.

[0334] In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a NOVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.

[0335] Methods of Treatment

[0336] The 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 NOVX expression or activity. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

[0337] These methods of treatment will be discussed more fully, below.

[0338] Diseases and Disorders

[0339] Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to “knockout” endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.

[0340] Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.

[0341] Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).

[0342] Prophylactic Methods

[0343] In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by aberrant NOVX 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 NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, a NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are fuirther discussed in the following subsections.

[0344] Therapeutic Methods

[0345] Another aspect of the invention pertains to methods of modulating NOVX expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NOVX protein, a peptide, a NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. 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 invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NOVX 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) NOVX expression or activity. In another embodiment, the method involves administering a NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.

[0346] Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).

[0347] Determination of the Biological Effect of the Therapeutic

[0348] In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.

[0349] In various specific embodiments, in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.

[0350] Prophylactic and Therapeutic Uses of the Compositions of the Invention

[0351] The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders. The disorders include but are not limited to, e.g., those diseases, disorders and conditions listed above, and more particularly include those diseases, disorders, or conditions associated with homologs of a NOVX protein, such as those summarized in Table A.

[0352] As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from diseases, disorders, conditions and the like, including but not limited to those listed herein.

[0353] Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.

[0354] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example A: Polynucleotide and Polypeptide Sequences, and Homology Data

Example 1

[0355] The NOV1 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 1A.

2TABLE 1ANOV1 Sequence AnalysisSEQ ID NO: 12093 bpNOV1a,GGTGGCGGCGGCGCCATCTTGGCGAAGGGGGGATCAGGAAGTGCGGACCGCGGCGGCGGCGGCGCG160009-01DNA SequenceGCGGCGGCGGCGGCGGCGGAGCCCGGAGCGCAGGCCGGAGGCTCCCGGCCCGCCGGCCCCGGAGCGGAGCGGAGCGGAGGATGCAGCAGCCGCAGCCGCAGGGGCAGCAGCAGCCGGGGCCGGGGCAGCAGCTGGGGGGCCAGGGGGCGGCGCCGGGGGCCAAGGGCGGCCCAGGGGGGGGCCCGGGGCCGGGGCCCTGCCTGAGGCGAGAGCTGAAGCTGCTCGAGTCCATCTTCCACCGCGGCCACGAGCGCTTCCGCATTGCCAGCGCCTGCCTGGACGAGCTGAGCTGCGAGTTCCTGCTGGCTGGGGCCGGAGGGGCCGGGGCGGGGGCCGCGCCCGGACCGCATCTCCCCCCACGGGGGTCGGTGCCTGGGGATCCTGTCCGCATCCACTGCAACATCACGGAGTCATACCCTGCTGTGCCCCCCATCTGGTCGGTGGAGTCTGATGACCCTAACTTGGCTGCTGTCTTGGAGAGGCTGGTGGACATAAAGAAAGGGAATACTCTGCTATTGCAGCATCTGAAGAGGATCATCTCCGACCTGTGTAAACTCTATAACCTCCCTCAGCATCCAGATGTGGAGATGCTGGATCAACCCTTGCCAGCAGAGCAGTGCACACAGGAAGACGTGTCTTCAGAAGATGAAGATGAGGAGATGCCTGAGGACACAGAAGACTTAGATCACTATGAAATGAAAGAGGAAGAGCCAGCTGAGGGCAAGAAATCTGAAGATGATGGCATTGGAAAAGAAAACTTGGCCATCCTAGAGAAAATTAAAAAGAACCAGAGGCAAGATTACTTAAATGGTGCAGTGTCTGGCTCGGTGCAGGCCACTGACCGGCTGATGAAGGAGCTCAGGGATATATACCGATCACAGAGTTTCAAAGGCGGAAACTATGCAGTCGAACTCGTGAATGACAGTCTGTATGATTGGAATGTCAAACTCCTCAAAGTTGACCAGGACAGCGCTTTGCACAACGATCTCCAGATCCTCAAAGAGAAAGAAGGAGCCGACTTCATTCTACTTAACTTTTCCTTTAAAGATAACTTTCCCTTTGACCCACCATTTGTCAGGGTTGTGTCTCCAGTCCTCTCTGGAGGGTATGTTCTGGGCGGAGGGGCCATCTGCATGGAACTTCTCACCAAACAGGGCTGGAGCAGTGCCTACTCCATAGAGTCAGTGATCATGCAGATCAGTGCCACACTGGTGAAGGGGAAAGCACGAGTGCAGTTTGGAGCCAACAAATCTCAATACAGTCTGACAAGAGCACAGCAGTCCTACAAGTCCTTGGTGCAGATCCACGAAAAAAACGGCTGGTACACACCCCCAAAAGAAGACGGCTAACCCTGGAGTATCACCCTTCCTCCCTCCCCAGGCACCACTGGACCAATTACCTTTGAATGCTGTATTTGGATCTCACGCTGCCTCTGTGGTTCCCTCCCTCATTTTTCCTGGACGTGATAGCTCTGCCTATTGCAGGACAATGATGGCTATTCTAAACGCTAAGGAAAAAAAACAAACACAGAACTGTTTCAAGTACTCAAGACTGACTTACAGACCAACCAACCACCTTGCTGGAACCCTTGCTAGCAGGCATTCTTATAAAAGAAACTTTCGAGCCTCCTTATATTGCTGGAAACTCAGCTGTGCTCCAGACTAGAGCCTCCTTACCTATGCTATGGATTTTTAATTTATTTTCTCTTATTTCATGTACACTGCTTTTTTTGGTTACAGTGTATGATGGATGTGTATGAAAAAAATGTATCTTTGGGAAAACAATTACAGTTTGTTAATTTGGAAAAAAAAAAAATGACTCATCTTTATTTTTATTCGCACATCCCACCCCATCCCCTGGAACTACTTGGGGAGGTGGGGAGGGGTACGAGTCCACTGTTGGCTTGGTAGGAGATGAGAGGCCGCATTTGCTGCTTAAGCAAGGGGAACTGGGGCTGAGCACACACCGGGGTGCCCCCGGATTTTTCCTCAAGGGCTCTGGAGCAACGGCACCGGCCCCTTGGGATGCAORF Start: ATG at 145ORF Stop: TAA at 1411SEQ ID NO: 2422 aaMW at 46197.6kDNOV1a,MQQPQPQGQQQPGPGQQLGGQGAAPGAKGGPGGGPGPGPCLRRELKLLESIFHRGHERFRIASACG160009-01Protein SequenceCLDELSCEFLLAGAGGAGAGAAPGPHLPPRGSVPGDPVRIHCNITESYPAVPPIWSVESDDPNLAAVLERLVDIKKGNTLLLQHLKRIISDLCKLYNLPQHPDVEMLDQPLPAEQCTQEDVSSEDEDEEMPEDTEDLDHYEMKEEEPAEGKKSEDDGIGKENLAILEKIKKNQRQDYLNGAVSGSVQATDRLMKELRDIYRSQSFKGGNYAVELVNDSLYDWNVKLLKVDQDSALHNDLQILKEKEGADFILLNFSFKDNFPFDPPFVRVVSPVLSGGYVLGGGAICMELLTKQGWSSAYSIESVIMQISATLVKGKARVQFGANKSQYSLTRAQQSYKSLVQIHEKNGWYTPPKEDG

[0356] Further analysis of the NOV1a protein yielded the following properties shown in Table 1B.

3TABLE 1BProtein Sequence Properties NOV1aSignalP analysis:No Known Signal Sequence PredictedPSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 0; pos.chg 0; neg.chg 0H-region: length 27; peak value 2.46PSG score: −1.94GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −7.55possible cleavage site: between 29 and 30>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS(s) for the threshold 0.5: 0number of TMS(s) . . . fixedPERIPHERAL Likelihood = 1.43 (at 334)ALOM score: 1.43 (number of TMSs: 0)MITDISC: discrimination of mitochondrial targeting seqR content: 2Hyd Moment (75): 3.58Hyd Moment (95): 4.63G content: 14D/E content: 1S/T content: 0Score: −10.36Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 52LRR|ELNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: nonebipartite: nonecontent of basic residues: 10.2%NLS Score: −0.47KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals:KKXX-like motif in the C-terminus: PKEDSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: nuclearReliability: 55.5COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 69.6%: nuclear 13.0%: cytoplasmic 8.7%: mitochondrial 8.7%: peroxisomal >> prediction for CG160009-01 is nuc (k = 23)

[0357] A search of the NOV1a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 1C.

4TABLE 1CGENESEQ Results for NOV1aNOV1aIdentities/Residues/Similarities forGENESEQProtein/Organism/Length [Patent #,Matchthe MatchedExpectIdentifierDate]ResiduesRegionValueAAE24493Human RATL1d6 (regulated in 1 . . . 422421/422 (99%)0.0activated T-lymphocyte 1d6) protein - 1 . . . 422421/422 (99%)Homo sapiens, 422 aa.[WO200236741-A2, 10-MAY-2002]AAG73684Human colon cancer antigen protein 59 . . . 422358/364 (98%)0.0SEQ ID NO: 4448 - Homo sapiens, 374 11 . . . 374358/364 (98%)aa. [WO200122920-A2, 05-APR-2001]AAY48312Human prostate cancer-associated132 . . . 422291/291 (100%)e−168protein 9 - Homo sapiens, 291 aa. 1 . . . 291291/291 (100%)[DE19811194-A1, 16-SEP-1999]AAE24494Human RATL1d6 transmembrane 89 . . . 333245/245 (100%)e−143domain - Homo sapiens, 245 aa. 1 . . . 245245/245 (100%)[WO200236741-A2, 10-MAY-2002]AAB41773Human ORFX ORF1537 polypeptide 39 . . . 297246/261 (94%)e−139sequence SEQ ID NO: 3074 - Homo 2 . . . 262250/261 (95%)sapiens, 263 aa. [WO200058473-A2,05-OCT-2000]

[0358] In a BLAST search of public sequence databases, the NOV1a protein was found to have homology to the proteins shown in the BLASTP data in Table 1D.

5TABLE 1DPublic BLASTP Results for NOV1aNOV1aIdentities/ProteinResidues/Similarities forAccessionMatchthe MatchedExpectNumberProtein/Organism/LengthResiduesPortionValueQ8K2Z8Similar to NICE-5 protein - Mus 41 . . . 422285/385 (74%)e−159musculus (Mouse), 378 aa. 6 . . . 378318/385 (82%)Q8WVN8Similar to NICE-5 protein (EC 6.3.2.19) 41 . . . 422286/386 (74%)e−157(Ubiquitin-conjugating enzyme E2) 6 . . . 375319/386 (82%)(Ubiquitin-protein ligase) (Ubiquitincarrier protein) - Homo sapiens (Human),375 aa.CAD34876Sequence 184 from Patent WO0222660 - 41 . . . 422261/385 (67%)e−140Homo sapiens (Human), 340 aa. 6 . . . 340291/385 (74%)Q9BVX5Hypothetical ubiquitin-protein ligase (EC193 . . . 422230/230 (100%)e−1306.3.2.19) (Ubiquitin-conjugating enzyme 1 . . . 230230/230 (100%)E2) (Ubiquitin carrier protein) - Homosapiens (Human), 230 aa (fragment).Q96MV4Hypothetical protein FLJ31826 (EC206 . . . 422217/217 (100%)e−1226.3.2.19) (Ubiquitin-conjugating enzyme 1 . . . 217217/217 (100%)E2) (Ubiquitin-protein ligase) (Ubiquitincarrier protein) - Homo sapiens (Human),217 aa.

[0359] PFam analysis predicts that the NOV1a protein contains the domains shown in the Table 1E.

6TABLE 1EDomain Analysis of NOV1aIdentities/NOV1a MatchSimilaritiesExpectPfam DomainRegionfor the Matched RegionValueUQ_con248 . . . 410 40/184 (22%)0.0067101/184 (55%)

Example 2

[0360] The NOV2 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 2A.

7TABLE 2ANOV2 Sequence AnalysisSEQ ID NO: 31013 bpNOV2a,CCTAGGGTCGCCCTGTCCCATCTCCATCTCCTATGAGATGAGTGACTCCCAAGACGCCACCACTCG161998-01DNA SequenceTCCCCTGTAGTTACCACCCAGGTAGAGCTGGGGGGCTGCAGCCGGCAAGGTGGGGGCAATGGGTTCCTCCGTTTTCGCCAGCACCAGGAGGTCCAGGCCTTCCTCAGCCTTCTGGAGGACAGTTTTGTCCAGGAATTCCTCTCCAAAGACCCCTGCTTCCAGATTTCAGATAAGTATCTCCTGGCCATGGTGCTGGTCTACTTCCAGCGCGCCCACCTGAAGCTCAGCGAGTATACCCACAGCAGCCTGTTCTTGGCCCTGTACCTTGCAAACGACATGGAGGAGGACCTGGAGGGCCCCAAATGTGAGATTTTTCCATGGGCCCTGGGAAAAGATTGGTGTTTACGAGTGGGGAAATTCCTGCACCAGAGGGATAAGCTTTGGGCACGGATGGGTTTCCGGGCTGTTGTGAGCCGCCAGTGCTGTGAGGAGGTGATGGCAAAGGAGCCATTCCACTGGGCTTGGACTCGGGACCGGCGCCCCCACCATGGTGGGGTTCAGAGGGTCTGTCCACAGGTCCCTGTTCGCCTTCCCCGGGGCCCTGGCCTCTCGCCGCCCCACTGTTCCCCCTGTGGTTTGCCCCAGCACTGCAGCAGCCACTTGCTTAAGCCTGTGTCATCCAAGTGCCCTTCTCTGACCTCTGAATGTCATCGCCCTCCCTCCCAAAATTATCTCTCAAGGGTCAAAAACGCCTGGGGTGGGGACTTTCTCATCGTCTTGCCTCCCCAGATGCAACTGGAACCAGGCACCTACTCCCTCCGCATCTTCCCAAAGCCTCCGGCACGCCCTGGGCACTGAAGCTCTGCAGGGTGAAGAACAGCACGCCCGCTGGCCCACTGACTGACACACCATTGGCTGTGCTCCCACCCCTGCCCCTTCCTCCCAGCCTGCAGCTTGTGCCCACTCTGGTGCCAGTCCCCTCTAATAAATTCATGTCCACAGGACACCAORF Start: ATG at 38ORF Stop: TGA at 860SEQ ID NO: 4274 aaMW at 31051.4 kDNOV2a,MSDSQDATTSPVVTTQVELGGCSRQGGGNGFLRFRQHQEVQAFLSLLEDSFVQEFLSKDPCFQICG161998-01Protein SequenceSDKYLLAMVLVYFQRAHLKLSEYTHSSLFLALYLANDMEEDLEGPKCEIFPWALGKDWCLRVGKFLHQRDKLWARMGFRAVVSRQCCEEVMAKEPFHWAWTRDRRPHHGGVQRVCPQVPVRLPRGPGLSPPHCSPCGLPQHCSSHLLKPVSSKCPSLTSECHRPPSQNYLSRVKNAWGGDFLIVLPPQMQLEPGTYSLRIFPKPPARPGH

[0361] Further analysis of the NOV2a protein yielded the following properties shown in Table 2B.

8TABLE 2BProtein Sequence Properties NOV2aSignalP analysis:No Known Signal Sequence PredictedPSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 6; pos.chg 0; neg.chg 2H-region: length 11; peak value 0.00PSG score: −4.40GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −7.70possible cleavage site: between 52 and 53>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS(s) for the threshold 0.5: 0number of TMS(s)..fixedPERIPHERAL Likelihood = 2.70 (at 61)ALOM score: 2.70 (number of TMSs: 0)MITDISC: discrimination of mitochondrial targeting seqR content: 0Hyd Moment(75): 1.71Hyd Moment(95): 7.84G content: 0D/E content: 2S/T content: 2Score: −6.85Gavel: prediction of cleavage sites for mitochondrial preseq cleavage site motifnot foundNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: nonebipartite: nonecontent of basic residues: 11.3%NLS Score: −0.47KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals: noneSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: nuclearReliability: 55.5COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 56.5%: nuclear 7.4%: cytoplasmic 13.0%: mitochondrial 13.0%: peroxisomal >> prediction for CG161998-01 is nuc (k = 23)

[0362] A search of the NOV2a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 2C.

9TABLE 2CGENESEQ Results for NOV2aNOV2aIdentities/Residues/Similarities forGENESEQProtein/Organism/Length [Patent #,Matchthe MatchedExpectIdentifierDate]ResiduesRegionValueAAB07753An oocyte maturation and proliferation 36 . . . 268116/235 (49%)1e−55protein Is27 - Xenopus sp, 298 aa. 60 . . . 282140/235 (59%)[EP1026244-A1, 09-AUG-2000]AAB07752An oocyte maturation and proliferation 36 . . . 268114/236 (48%)4e−52protein Is26 - Xenopus sp, 300 aa. 60 . . . 284140/236 (59%)[EP1026244-A1, 09-AUG-2000]ABG21390Novel human diagnostic protein #21381 - 41 . . . 120 44/80 (55%)2e−15Homo sapiens, 367 aa.197 . . . 276 53/80 (66%)[WO200175067-A2, 11-OCT-2001]ABG16875Novel human diagnostic protein #16866 - 41 . . . 120 44/80 (55%)2e−15Homo sapiens, 434 aa.264 . . . 343 53/80 (66%)[WO200175067-A2, 11-OCT-2001]ABG14384Novel human diagnostic protein #14375 - 41 . . . 120 44/80 (55%)2e−15Homo sapiens, 434 aa.264 . . . 343 53/80 (66%)[WO200175067-A2, 11-OCT-2001]

[0363] In a BLAST search of public sequence databases, the NOV2a protein was found to have homology to the proteins shown in the BLASTP data in Table 2D.

10TABLE 2DPublic BLASTP Results for NOV2aNOV2aProteinResidues/Identities/AccessionMatchSimilarities for theExpectNumberProtein/Organism/LengthResiduesMatched PortionValueQ9PWR2P33 ringo - Xenopus laevis 36 . . . 268116/235 (49%)3e−55(African clawed frog), 298 aa. 60 . . . 282140/235 (59%)Q9YGL1Speedy protein - Xenopus laevis 36 . . . 268115/235 (48%)1e−54(African clawed frog), 298 aa. 60 . . . 282140/235 (58%)Q9PU13P33 ringo - Xenopus laevis 36 . . . 268113/235 (48%)9e−52(African clawed frog), 299 aa. 60 . . . 283139/235 (59%)Q9W6Z0P33 ringo - Xenopus laevis 36 . . . 268114/236 (48%)1e−51(African clawed frog), 300 aa. 60 . . . 284140/236 (59%)Q8R496GS4 - Rattus norvegicus (Rat), 312 36 . . . 201 89/166 (53%)3e−46aa. 66 . . . 231111/166 (66%)

[0364] PFam analysis predicts that the NOV2a protein contains the domains shown in the Table 2E.

11TABLE 2EDomain Analysis of NOV2aNOV2aIdentities/SimilaritiesExpectPfam DomainMatch Regionfor the Matched RegionValue

[0365] The NOV3 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 3A.

12TABLE 3ANOV3 Sequence AnalysisSEQ ID NO: 51750 bpNOV3a,GAGCGGGTTCCCCGTCCGCGGCGGCCCTGCAGGCGACCCCGCGTCCCCACCGGCGGGAGCTCG162071-01DNA SequenceCGGGGAAGAGCGGGCGGCGGCGCTGCTGCTGGCGGCGGCCGGCTGGATGCGAGACCCGCGCAGACCCGGCGGCGGACGGCGGCTCTCGACTCCGGAGAGCGGATCGCGGGGCGCAGGAGCCCGATCGCGCTGGCCATGGCCTCCAACTTTAACGACATAGTCAAGCAGGGCTACGTGAAAATCCGCAGCAGGAAGCTTGGGATTTTCAGACGATGCTGGTTGGTTTTCAAGAAGGCTTCTAGCAAAGGACCCAGAAGGTTAGAAAAATTTCCAGATGAAAAGGCAGCTTATTTCAGAAACTTTCATAAGGTAACTGAACTGCACAATATCAAAAATATAACCAGACTGCCCCGAGAGACAAAGAAGCATGCGGTGGCAATCATCTTTCACGATGAAACATCGAAGACATTTGCCTGTGAGTCAGAGCTGGAGGCCGAGGAGTGGTGCAAGCACCTCTGCATGGAGTGTCTGGGGACCAGGCTCAATGACATCAGCCTTGGGGAGCCCGACCTTCTGGCCGCAGGAGTGCAGCGGGAACAGAATGAGAGATTCAACGTGTATCTTATGCCTACGCCAAACCTGGATATTTATGGTGAATGCACAATGCAGATCACTCATGAAAATATCTATCTCTGGGATATCCACAATGCCAAGGTCAAACTGGTGATGTGGCCTCTCAGCTCACTGAGGAGATACGGTCGGGACTCAACGTGGTTCACGTTTGAGTCAGGAAGAATGTGTGACACAGGAGAAGGACTATTCACTTTTCAAACAAGGGAAGGAGAAATGATCTATCAGAAGGTTCATTCTGCGACACTGGCCATAGCTGAGCAACATGAAAGATTAATGCTAGAAATGGAACAGAAGGCCCGGCTTCAGACAAGCTTGACTGAACCAATGACATTATCCAAATCAATATCTCTTCCTCGCAGCGCGTACTGGCATCACATCACTCGTCAGAACAGCGTTGGTGAAATCTACAGTTTGCAAGGTCATGGGTTTGGTTCGTCAAAGATGTCTCGTGCACAGACATTTCCCAGCTACGCCCCAGAACAGAGTGAAGAGGCCCAGCAGCCGTTGTCGCGGTCCAGCAGCTATGGATTCAGCTACAGCTCCAGCCTCATCCAATGACACACAGAGAGCCGCTGTTGACTAGAGAGACAGTCTGTCCTGGACCCGTCTGTGGGGTCATTACCCTCTGCCTCCTGGAAGACCAATTGCAGTACAAATTGAAATGGGTCGGCTCTAGCCCCAGTCCCATTGGAAGGTTAAAAACTGGAGTTCTGCTTCCTTGATTCAGTGGCTAAGTCCTTTATTTATTGAATTTCTTTGGGGGAATCTATGTTTTACATAAATAGAATCCAAATCGTATGAACAGTTATTTAAATAAACAAAATTCTTTGGGTCTGACAGTAACAGAAACCTCAGCACTGGGAAAAGCTGCCCACACTGGGGTATGCCTGGGTGATGGGCACCTCTCACTGACCTCACAGCGCAGTCCGCCCATCTGAGCACAGGGTCTGTCCTTAGACATAATGGTGACCCTCCACAAGCTCTGTGGCTTTTAAAGTTCTGACAGGGATAAATACAGTAAGCTCTGCAAATGACATCGTAGCTGCATATAAATAAACCCAGTGACAATTAAAGGAACATGAAATTCAGAACACACTATTAAGCAGGGCCCTGTAGORF Start: ATG at 197ORF Stop: TGA at 1190SEQ ID NO: 6331 aaMW at 38317.2 kDNOV3a,MASNFNDIVKQGYVKIRSRKLGIFRRCWLVFKKASSKGPRRLEKFPDEKAAYFRNFHKVTECG162071-01 ProteinSequenceLHNIKNITRLPRETKKHAVAIIFHDETSKTFACESELEAEEWCKHLCMECLGTRLNDISLGEPDLLAAGVQREQNERFNVYLMPTPNLDIYGECTMQITHENIYLWDIHNAKVKLVMWPLSSLRRYGRDSTWFTFESGRMCDTGEGLFTFQTREGEMIYQKVHSATLAIAEQHERLMLEMEQKARLQTSLTEPMTLSKSISLPRSAYWHHITRQNSVGEIYSLQGHGFGSSKMSRAQTFPSYAPEQSEEAQQPLSRSSSYGFSYSSSLIQ

[0366] Further analysis of the NOV3a protein yielded the following properties shown in Table 3B.

13TABLE 3BProtein Sequence Properties NOV3aSignalP analysis:No Known Signal Sequence PredictedPSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 10; pos.chg 1; neg.chg 1H-region: length 4; peak value −9.72PSG score: −14.12GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −10.63possible cleavage site: between 36 and 37>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS(s) for the threshold 0.5: 0number of TMS(s) . . . fixedPERIPHERAL Likelihood = 10.45 (at 78)ALOM score: 10.45 (number of TMSs: 0)MITDISC: discrimination of mitochondrial targeting seqR content: 6Hyd Moment(75): 4.01Hyd Moment(95): 9.12G content: 3D/E content: 2S/T content: 4Score: −1.73Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 51RRL|EKNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: nonebipartite: RRCWLVFKKASSKGPRR at 25content of basic residues: 13.0%NLS Score: 0.02KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals: noneSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: nuclearReliability: 76.7COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 56.5%: mitochondrial 39.1%: nuclear 4.3%: peroxisomal >> prediction for CG162071-01 is mit (k = 23)

[0367] A search of the NOV3a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 3C.

14TABLE 3CGENESEQ Results for NOV3aNOV3aIdentities/Residues/Similarities forGENESEQProtein/Organism/Length [Patent #,Matchthe MatchedExpectIdentifierDate]ResiduesRegionValueAAM41593Human polypeptide SEQ ID NO 6524 - 1 . . . 287286/287 (99%)e−170Homo sapiens, 365 aa. 29 . . . 315287/287 (99%)[WO200153312-A1, 26-JUL-2001]AAM41592Human polypeptide SEQ ID NO 6523 - 1 . . . 287286/287 (99%)e−170Homo sapiens, 365 aa. 29 . . . 315287/287 (99%)[WO200153312-A1, 26-JUL-2001]AAM39806Human polypeptide SEQ ID NO 2951 - 1 . . . 287286/287 (99%)e−170Homo sapiens, 337 aa. 1 . . . 287287/287 (99%)[WO200153312-A1, 26-JUL-2001]AAM39807Human polypeptide SEQ ID NO 2952 - 1 . . . 248246/248 (99%)e−146Homo sapiens, 252 aa. 1 . . . 248248/248 (99%)[WO200153312-A1, 26-JUL-2001]AAM38742Human polypeptide SEQ ID NO 1887 - 1 . . . 306216/307 (70%)e−129Homo sapiens, 306 aa. 1 . . . 305262/307 (84%)[WO200153312-A1, 26-JUL-2001]

[0368] In a BLAST search of public sequence databases, the NOV3a protein was found to have homology to the proteins shown in the BLASTP data in Table 3D.

15TABLE 3DPublic BLASTP Results for NOV3aNOV3aProteinResidues/Identities/AccessionMatchSimilarities for theExpectNumberProtein/Organism/LengthResiduesMatched PortionValueQ96HI2Hypothetical protein - Homo 75 . . . 331257/257 (100%)e−150sapiens (Human), 257 aa 1 . . . 257257/257 (100%)(fragment).Q8TEW7Downstream of tyrosine kinase 5 - 1 . . . 306216/307 (70%)e−128Homo sapiens (Human), 306 aa. 1 . . . 305262/307 (84%)Q8WUZ8Hypothetical protein - Homo109 . . . 331223/223 (100%)e−128sapiens (Human), 223 aa. 1 . . . 223223/223 (100%)Q91ZM9Dok5 - Mus musculus (Mouse), 306 1 . . . 306212/306 (69%)e−128aa. 1 . . . 305256/306 (83%)Q96LU2Hypothetical protein FLJ25066 -109 . . . 331222/223 (99%)e−128Homo sapiens (Human), 223 aa. 1 . . . 223223/223 (99%)

[0369] PFam analysis predicts that the NOV3a protein contains the domains shown in the Table 3E.

16TABLE 3EDomain Analysis of NOV3aIdentities/NOV3aSimilaritiesExpectPfam DomainMatch Regionfor the Matched RegionValuePH 8 . . . 11219/105 (18%)3.6e−0976/105 (72%)IRS130 . . . 23241/110 (37%)2.6e−4391/110 (83%)

Example 4

[0370] The NOV4 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 4A.

17TABLE 4ANOV4 Sequence AnalysisSEQ ID NO: 71224 bpNOV4a,GTACCTGCACATGTTGTTCTTTGTCAGTGCTGTCAAGTGTGTGCCAGGGTGATCCATGGTCACTCG162605-01DNA SequenceTTCCGGGATGGCAGCAAGGTGACTTCGGCTGAGGATGACCCTGACTGAAAGGCTGCGTGAGAAGATATCTCGGGCCTTCTACAACCATGGGCTCCTCTGTGCATCCTATCCCATCCCCATCATCCTCTTCACAGGGTTCTGCATCTTAGCCTGCTGCTACCCACTGCTGAAACTCCCCTTGCCAGGAACAGGACCTGTGGAATTCACCACCCCTGTGAAGGATTACTCGCCCCCACCTGTGGACTCTGACCGCAAACAAGGAGAGCCTACTGAGCAGCCTGAGTGGTATGTGGGTGCCCCGGTGGCTTATGTCCAGCAGATATTTGTGAAGTCCTCAGTGTTTCCCTGGCACAAGAACCTCCTGGCAGTAGATGTATTTCGTTCACCTTTGTCCCGGGCATTCCAACTGGTGGAGGAGATCCGGAACCACGTGCTGAGAGACAGCTCTGGGATCAGGAGCTTGGAGGAGTTGTGTCTGCAAGTGACCGACCTGCTGCAGGGCCACTCAGGGGCCATCACGACCGTGTACATTGACCAGACCATGGTGCTGGCCAGTGGAGGACAAGATGGGGCCATCTGCCTGTGGGATGTACTGACTGGCAGCCGGGTCAGCCATGTGTTTGCTCACCGTGGGGATGTCACCTCCCTTACCTGTACCACCTCCTGTGTCATCAGCAGTGGCCTGGATGACCTCATCAGCATCTGGGACCGCAGCACAGGCATCAAGTTCTACTCCATTCAGCAGGACCTGGGCTGTGGTGCAAGCTTGGGTGTCATCTCAGACAACCTGCTGGTGACTGGCGGCCAGGGCTGTGTCTCCTTTTGGGACCTAAACTACGGGGACCTGTTACAGACAGTCTACCTGGGGAAGAACAGTGAGGCCCAGCCTGCCCGCCAGATCCTGGTGCTGGACAACGCTGCCATTGTCTGCAACTTTGGCAGTGAGCTCAGCCTGGTGTATGTGCCCTCTGTGCTGGAGAAGCTGGACTGAGCGCAGGGCCTCCTTGCCCAGGCAGGAGGCTGGGGTGCTGTGTGGGGGCCAATGCACTGAACCTGGACTTGGGGGAAAGAGCCGAGTATCTTCCAGCCGCTGCCTCCTGACTGTAATAATATTAAACTTTTTTAAAAAACCATATCATCATCTGTCAGGCACTTTGGGAORF Start: ATG at 99ORF Stop: TGA at 1053SEQ ID NO: 8318 aaMW at 34825.5 kDNOV4a,MTLTERLREKISRAFYNHGLLCASYPIPIILFTGFCILACCYPLLKLPLPGTGPVEFTTPVKDYCG162605-01Protein SequenceSPPPVDSDRKQGEPTEQPEWYVGAPVAYVQQIFVKSSVFPWHKNLLAVDVFRSPLSRAFQLVEEIRNHVLRDSSGIRSLEELCLQVTDLLQGHSGAITTVYIDQTMVLASGGQDGAICLWDVLTGSRVSHVFAHRGDVTSLTCTTSCVISSGLDDLISIWDRSTGIKFYSIQQDLGCGASLGVISDNLLVTGGQGCVSFWDLNYGDLLQTVYLGKNSEAQPARQILVLDNAAIVCNFGSELSLVYVPSVLEKLDSEQ ID NO: 92106 bpNOV4b,GTACCTGCACATGTTGTTCTTTGTCAGTGCTGTCAAGTGTGTGCCAGGGTGATCCATGGTCACTCG162605-02DNA SequenceTTCCGGGATGGCAGCAAGGTGACTTCGGCTGAGGATGACCCTGACTGAAAGGCTGCGTGAGAAGATATCTCGGGCCTTCTACAACCATGGGCTCCTCTGTGCATCCTATCCCATCCCCATCATCCTCTTCACAGGGTTCTGCATCTTAGCCTGCTGCTACCCACTGCTGAAACTCCCCTTGCCAGGAACAGGACCTGTGGAATTCACCACCCCTGTGAAGGATTACTCGCCCCCACCTGTGGACTCTGACCGCAAACAAGGAGAGCCTACTGAGCAGCCTGAGTGGTATGTGGGTGCCCCGGTGGCTTATGTCCAGCAGATATTTGTGAAGTCCTCAGTGTTTCCCTGGCACAAGAACCTCCTGGCAGTAGATGTATTTCGTTCACCTTTGTCCCGGGCATTCCAACTGGTGGAGGAGATCCGGAACCACGTGCTGAGAGACAGCTCTGGGATCAGGAGCTTGGAGGAGTTGTGTCTGCAAGTGACCGACCTGCTGCCAGGCCTTAGGAAGCTCAGGAACCTACTCCCTGAGCATGGATGCCTGCTGCTGTCCCCTGGGAACTTCTGGCAGAATGACTGGGAACGCTTCCATGCTGATCCTGACATCATTGGGACCATCCACCAGCACGAGCCTAAAACCCTGCAGACTTCAGCCACACTCAAAGACTTGTTATTTGGTGTTCCTGGGAAGTACAGCGGGGTGAGCCTCTACACCAGGAAGAGGATGGTCTCCTACACCATCACCCTGGTCTTCCAGCACTACCATGCCAAGTTCCTGGGCAGCCTGCGTGCCCGCCTGATGCTTCTGCACCCCAGCCCCAACTGCAGCCTTCGGGCGGAGAGCCTGGTCCACGTGCACTTCAAGGAGGAGATTGGTGTCGCTGAGCTCATCCCCCTTGTGACCACCTACATCATCTTGTTTGCCTACATCTACTTCTCCATCTGGAGCTTGGAGCTGCAGGGCAACCTCATCGTGGTGGGGCGGAGCAGCGGCCGGCTGGAGGTGTGGGACGCCATTGAAGGGGTGCTGTGCTGCAGCAGCGAGGAGGTCTCCTCAGGCATTACCGCTCTGGTGTTCTTGGACAAAAGGATTGTGGCTGCACGGCTCAACGGTTCCCTTGATTTCTTCTCCTTGGAGACCCACACTGCCCTCAGCCCCCTGCAGTTTAGAGGGACCCCAGGGCGGGGCAGTTCCCCTGCCTCTCCAGTGTACAGCAGCAGCGACACAGTGGCCTGTCACCTGACCCACACAGTGCCCTGTGCACACCAAAAACCCATCACAGCCCTGAAAGCCGCTGCTGGGCGCTTGGTGACTGGGAGCCAAGACCACACACTGAGAGTGTTCCGTCTGGAGGACTCGTGCTGCCTCTTCACCCTTCAGGGCCACTCAGGGGCCATCACGACCGTGTACATTGACCAGACCATGGTGCTGGCCAGTGGAGGACAAGATGGGGCCATCTGCCTGTGGGATGTACTGACTGGCAGCCGGGTCAGCCATGTGTTTGCTCACCGTGGGGATGTCACCTCCCTTACCTGTACCACCTCCTGTGTCATCAGCAGTGGCCTGGATGACCTCATCAGCATCTGGGACCGCAGCACAGGCATCAAGTTCTACTCCATTCAGCAGGACCTGGGCTGTGGTGCAAGCTTGGGTGTCATCTCAGACAACCTGCTGGTGACTGGCGGCCAGGGCTGTGTCTCCTTTTGGGACCTAAACTACGGGGACCTGTTACAGACAGTCTACCTGGGGAAGAACAGTGAGGCCCAGCCTGCCCGCCAGATCCTGGTGCTGGACAACGCTGCCATTGTCTGCAACTTTGGCAGTGAGCTCAGCCTGGTGTATGTGCCCTCTGTGCTGGAGAAGCTGGACTGAGCGCAGGGCCTCCTTGCCCAGGCAGGAGGCTGGGGTGCTGTGTGGGGGCCAATGCACTGAACCTGGACTTGGGGGAAAGAGCCGAGTATCTTCCAGCCGCTGCCTCCTGACTGTAATAATATTAAACTTTTTTAAAAAACCATATCATCATCTGTCAGGCACTTTGGGAORF Start: ATG at 99ORF Stop: TGA at 1935SEQ ID NO: 10612 aaMW at 67389.7kDNOV4b,MTLTERLREKISRAFYNHGLLCASYPIPIILFTGFCILACCYPLLKLPLPGTGPVEFTTPVKDYCG162605-02Protein SequenceSPPPVDSDRKQGEPTEQPEWYVGAPVAYVQQIFVKSSVFPWHKNLLAVDVFRSPLSRAFQLVEEIRNHVLRDSSGIRSLEELCLQVTDLLPGLRKLRNLLPEHGCLLLSPGNFWQNDWERFHADPDIIGTIHQHEPKTLQTSATLKDLLFGVPGKYSGVSLYTRKRMVSYTITLVFQHYHAKFLGSLRARLMLLHPSPNCSLRAESLVHVHFKEEIGVAELIPLVTTYIILFAYIYFSIWSLELQGNLIVVGRSSGRLEVWDAIEGVLCCSSEEVSSGITALVFLDKRIVAARLNGSLDFFSLETHTALSPLQFRGTPGRGSSPASPVYSSSDTVACHLTHTVPCAHQKPITALKAAAGRLVTGSQDHTLRVFRLEDSCCLFTLQGHSGAITTVYIDQTMVLASGGQDGAICLWDVLTGSRVSHVFAHRGDVTSLTCTTSCVISSGLDDLISIWDRSTGIKFYSIQQDLGCGASLGVISDNLLVTGGQGCVSFWDLNYGDLLQTVYLGKNSEAQPARQILVLDNAAIVCNFGSELSLVYVPSVLEKLD

[0371] Sequence comparison of the above protein sequences yields the following sequence relationships shown in Table 4B.

18TABLE 4BComparison of NOV4a against NOV4b.ProteinNOV4a Residues/Identities/Similarities forSequenceMatch Residuesthe Matched RegionNOV4b122 . . . 318175/197 (88%)422 . . . 612180/197 (90%)

[0372] Further analysis of the NOV4a protein yielded the following properties shown in Table 4C.

19TABLE 4CProtein Sequence Properties NOV4aSignalP analysis:Cleavage site between residues 42 and 43PSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 10; pos.chg 3; neg.chg 2H-region: length 2; peak value −6.17PSG score: −10.57GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −1.38possible cleavage site: between 41 and 42>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS(s) for the threshold 0.5: 1Number of TMS(s) for threshold 0.5: 1INTEGRAL Likelihood = −6.74 Transmembrane 29-45PERIPHERAL Likelihood = 2.33 (at 289)ALOM score: −6.74 (number of TMSs: 1)MTOP: Prediction of membrane topology (Hartmann et al.)Center position for calculation: 36Charge difference: −2.5 C(0.0) − N(2.5)N >= C: N-terminal side will be inside>>> membrane topology: type 2 (cytoplasmic tail 1 to 29)MITDISC: discrimination of mitochondrial targeting seqR content: 2Hyd Moment (75): 3.34Hyd Moment (95): 6.55G content: 0D/E content: 2S/T content: 2Score: −4.95Gavel: prediction of cleavage sites for mitochondrial preseq cleavage site motifnot foundNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: nonebipartite: nonecontent of basic residues: 6.9%NLS Score: −0.47KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals:KKXX-like motif in the C-terminus: LEKLSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: 15,24Dileucine motif in the tail: foundLL at 20checking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: cytoplasmicReliability: 94.1COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 30.4%: cytoplasmic 30.4%: mitochondrial 8.7%: vacuolar 8.7%: Golgi 8.7%: endoplasmic reticulum 4.3%: extracellular, including cell wall 4.3%: vesicles of secretory system 4.3%: nuclear >> prediction for CG162605-01 is cyt (k = 23)

[0373] A search of the NOV4a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 4D.

20TABLE 4DGENESEQ Results for NOV4aNOV4aIdentities/Residues/Similarities forGENESEQProtein/Organism/Length [Patent #,Matchthe MatchedExpectIdentifierDate]ResiduesRegionValueAAB88444Human membrane or secretory protein122 . . . 318175/197 (88%)1e−93clone PSEC0227 - Homo sapiens, 379189 . . . 379180/197 (90%)aa. [EP1067182-A2, 10-JAN-2001]AAB93996Human protein sequence SEQ ID122 . . . 318175/197 (88%)1e−93NO: 14101 - Homo sapiens, 886 aa.696 . . . 886180/197 (90%)[EP1074617-A2, 07-FEB-2001]AAU01592Human secreted protein #1 encoded by122 . . . 318175/197 (88%)1e−93gene #8 - Homo sapiens, 266 aa. 76 . . . 266180/197 (90%)[WO200123547-A1, 05-APR-2001]AAM24010Hamster EST encoded protein SEQ ID122 . . . 318175/197 (88%)1e−93NO: 1535 - Cricetulus griseus, 972 aa.782 . . . 972180/197 (90%)[WO200154477-A2, 02-AUG-2001]AAM23988Hamster EST encoded protein SEQ ID122 . . . 318175/197 (88%)1e−93NO: 1513 - Cricetulus griseus, 972 aa.782 . . . 972180/197 (90%)[WO200154477-A2, 02-AUG-2001]

[0374] In a BLAST search of public sequence databases, the NOV4a protein was found to have homology to the proteins shown in the BLASTP data in Table 4E.

21TABLE 4EPublic BLASTP Results for NOV4aNOV4aIdentities/ProteinResidues/Similarities forAccessionMatchthe MatchedExpectNumberProtein/Organism/LengthResiduesPortionValueBAA12111KIAA0199 protein - Homo sapiens122 . . . 318175/197 (88%)4e−93(Human), 1283 aa (fragment).1093 . . . 1283180/197 (90%)Q8N2E0Hypothetical protein HEMBA1006019 -122 . . . 318175/197 (88%)4e−93Homo sapiens (Human), 379 aa.189 . . . 379180/197 (90%)Q8WUA1Hypothetical protein - Homo sapiens122 . . . 318175/197 (88%)4e−93(Human), 905 aa.715 . . . 905180/197 (90%)Q12770Sterol regulatory element binding protein122 . . . 318175/197 (88%)4e−93cleavage-activating protein (SREBP1089 . . . 1279180/197 (90%)cleavage-activating protein) (SCAP) -Homo sapiens (Human), 1279 aa.P97260Sterol regulatory element binding protein122 . . . 318172/197 (87%)1e−91cleavage-activating protein (SREBP1086 . . . 1276178/197 (90%)cleavage-activating protein) (SCAP) -Cricetulus griseus (Chinese hamster),1276 aa.

[0375] PFam analysis predicts that the NOV4a protein contains the domains shown in the Table 4F.

22TABLE 4FDomain Analysis of NOV4aIdentities/NOV4aSimilaritiesExpectPfam DomainMatch Regionfor the Matched RegionValueWD40150 . . . 18513/37 (35%)0.01527/37 (73%)WD40191 . . . 225 9/37 (24%)0.4125/37 (68%)

Example 5

[0376] The NOV5 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 5A.

23TABLE 5ANOV5 Sequence AnalysisSEQ ID NO: 11947 bpNOV5a,AGCGGGGCTTCGGGACAGCAGAGGAGCAGTGTCCGGCCATGGCCGGAGCCCGCAGGCTGGAGCTCG163438-01DNA SequenceAGGCGAGGCCCTGGCGCTGGGGTCGGGCTGGCGTCATGCGTGCCACGCTCTCCTCTACGCGCCGGACCCTGGGATGCTCTTCGGCCGCATCCCGCTGCGCTACGCCATACTGATGCAGATGCGCTTCGATGGACGCCTGGGCTTCCCCGGCGGATTCGTGGACACGCAGGACAGAAGCCTAGAGGACGGGCTGAACCGCGAGCTGCGCGAGGAGCTGGGCGAAGCGGCTGCCGCTTTCCGCGTGGAGCGCACTGACTACCGCAGCTCCCACGTCGGGTCAGGGCCACGCGTTGTGGCCCACTTCTATGCCAAGCGTCTGACGCTCGAGGAGCTGTTGGCTGTGGAGGCCGGCGCAACACGCGCCAAGGACCACGGGCTGGAGGTGCTGGGCCTGGTGCGAGTGCCCCTGTATACCCTGCGGGATGGTGTAGGAGGCCTGCCTACCTTCCTGGAGAATTCCTTTATTGGCTCTGCGCGGGAGCAGTTACTTGAAGCTCTCCAGGACTTGGGACTGCTGCAGTCTGGCTCTATTTCAGGCCTTAAGATTCCAGCTCATCACTAGAGGCAGCCCTCCATGGACCCATGAAAACTGAGATGAGGACCTTGGTACTAGGGAGGGAGGGAAGGACGTGGGAATGTTTTCTTATTGGATCTGAGAGATGATACATGATACCAGATGAAAAGAAGGAGAAGTGTGTACCATATGTTTTGAGCAGAGGACCCTCCAACTTATGGCATCAGGGGCAAAAAGTCACAGCTTATCCCAGGCACCCTGGCAGGTTCTCAGAGCCTGCCTCCTCCCTGTTTATATGCGTACAGCCTGGTAACCCCCAGGCATGCAAATATACAATCTGTAACAACACAAAAAAAAAAAAAAAAAAAAORF Start: ATG at 39ORF Stop: TAG at 624SEQ ID NO: 12195 aaMW at 21273.1kDNOV5a,MAGARRLELGEALALGSGWRHACHALLYAPDPGMLFGRIPLRYAILMQMRFDGRLGFPGGFVDTCG163438-01Protein SequenceQDRSLEDGLNRELREELGEAAAAFRVERTDYRSSHVGSGPRVVAHFYAKRLTLEELLAVEAGATRAKDHGLEVLGLVRVPLYTLRDGVGGLPTFLENSFIGSAREQLLEALQDLGLLQSGSISGLKIPAHH

[0377] Further analysis of the NOV5a protein yielded the following properties shown in Table 5B.

24TABLE 5BProtein Sequence Properties NOV5aSignalP analysis:No Known Signal Sequence PredictedPSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 11; pos.chg 2; neg.chg 2H-region: length 8; peak value 6.95PSG score: 2.55GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −2.77possible cleavage site: between 25 and 26>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS(s) for the threshold 0.5: 0number of TMS(s) . . . fixedPERIPHERAL Likelihood = 6.47 (at 137)ALOM score: 6.47 (number of TMSs: 0)MTOP: Prediction of membrane topology (Hartmann et al.)Center position for calculation: 6Charge difference: −3.5 C(−0.5) − N(3.0)N >= C: N-terminal side will be insideMITDISC: discrimination of mitochondrial targeting seqR content: 2Hyd Moment (75): 6.43Hyd Moment (95): 7.76G content: 2D/E content: 2S/T content: 0Score: −5.93Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motif at 16RRL|ELNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: nonebipartite: nonecontent of basic residues: 11.3%NLS Score: −0.47KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals:XXRR-like motif in the N-terminus: AGARnoneSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: cytoplasmicReliability: 94.1COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 22.2%: extracellular, including cell wall 22.2%: mitochondrial 22.2%: vacuolar 22.2%: endoplasmic reticulum 11.1%: nuclear >> prediction for CG163438-01 is exc (k = 9)

[0378] A search of the NOV5a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 5C.

25TABLE 5CGENESEQ Results for NOV5aNOV5aIdentities/Residues/Similarities forGENESEQProtein/Organism/Length [Patent #,Matchthe MatchedExpectIdentifierDate]ResiduesRegionValueAAU99581Human syndecan 4 related protein 47 . . . 195149/149 (100%)3e−8016.39 - Homo sapiens, 149 aa. 1 . . . 149149/149 (100%)[CN1341624-A, 27-MAR-2002]AAB18821Amino acid sequence of a chicken 11 . . . 181109/172 (63%)3e−56syndesmos polypeptide - Gallus sp, 320 44 . . . 211132/172 (76%)aa. [WO200054731-A2, 21-SEP-2000]AAU17002Human novel secreted protein, SEQ ID 11 . . . 181106/172 (61%)4e−54243 - Homo sapiens, 218 aa. 23 . . . 190130/172 (74%)[WO200155441-A2, 02-AUG-2001]AAU16929Human novel secreted protein, SEQ ID 11 . . . 181106/172 (61%)4e−54170 - Homo sapiens, 220 aa. 25 . . . 192130/172 (74%)[WO200155441-A2, 02-AUG-2001]ABP41566Human ovarian antigen HE2KN09, 11 . . . 181105/172 (61%)3e−53SEQ ID NO: 2698 - Homo sapiens, 218 23 . . . 190129/172 (74%)aa. [WO200200677-A1, 03-JAN-2002]

[0379] In a BLAST search of public sequence databases, the NOV5a protein was found to have homology to the proteins shown in the BLASTP data in Table 5D.

26TABLE 5DPublic BLASTP Results for NOV5aNOV5aIdentities/ProteinResidues/Similarities forAccessionMatchthe MatchedExpectNumberProtein/Organism/LengthResiduesPortionValueQ96DE0Hypothetical protein - Homo sapiens 2 . . . 195194/194 (100%) e−108(Human), 194 aa (fragment). 1 . . . 194194/194 (100%)Q96N82Hypothetical protein FLJ3126534 . . . 195162/162 (100%)2e−87(Similar to RIKEN cDNA 1 . . . 162162/162 (100%)2310041H06) - Homo sapiens(Human), 162 aa.Q9D7162310041H06Rik protein - Mus 1 . . . 192155/192 (80%)1e−84musculus (Mouse), 212 aa. 1 . . . 192168/192 (86%)Q9D9G61700080E11Rik protein - Mus 1 . . . 190131/191 (68%)1e−67musculus (Mouse), 206 aa. 1 . . . 191151/191 (78%)Q9IAY5Syndesmos - Gallus gallus (Chicken),11 . . . 181109/172 (63%)7e−56320 aa.44 . . . 211132/172 (76%)

[0380] PFam analysis predicts that the NOV5a protein contains the domains shown in the Table 5E.

27TABLE 5EDomain Analysis of NOV5aPfamNOV5aIdentities/SimilaritiesDomainMatch Regionfor the Matched RegionExpect ValueNUDIX19 . . . 16126/153 (17%)0.001892/153 (60%)

Example 6

[0381] The NOV6 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 6A.

28TABLE 6ANOV6 Sequence AnalysisSEQ ID NO: 131561 bpNOV6a,AGCGGGTCTCTATGGCGACTGGACGCGGTCGGATCTTGCAGCAGCACTGGCTCGGCCTCCAGACCG163701-01DNA SequenceGCTGCGCGGGCCCAGCAGGGGCGGTGGCGCGGCCCGGGGGCGCGCCAGGCCTTTGGGTGCAGAAAGGGGCCAGGGGTCAAGCTTTCTGCAGGCTCTGCTGCCCTGAGGTGCCATGCCGGAGGTGGACAGCACTGGGAGAGCTCTTTCTCCTGCTGTTCTGGGTTCCTGGATGGAATGCCTTCAGAAATCTTGCTGAAGATATTTTCCTACTTGGATGCTGTGAGCCTTCTGTGTACTGGATGTGTGAGCAGGCGCTTTTATCATCTAGCCAATGACAGTTTTATTTGGATCGGAATCTACTCAACTGCTTTTTCACCTGCAAGATCAAATTGGAAATTTAATTCAGTAGAGAAGATAGCTATGTCTATGAGCTTTCTGTCAGTTCAGGATAAAGAAGCTGGTTATTGGAAGAAAGAATATATCACAAAACAAATAGCATCTGTAAAAGCCGCACTAGCTGACATTCTCAAACCTGTCAACCCTTACACAGGCCTTCCAGTTAAGACCAAAGAGGCCCTCAGGATATTTGGTTTAGGTTGGGCAATTATACTGAAAGAAAAAGGTGGAAAAGAATATATCATGGAGCATGTTGATCTTTCCATAAATGACACATCAGTTACTGTTATATGGTATGGCAAAAAATGGCCATGCCTAGCATCATTGTCAACCTTAGATTTATGTGGCATGACACCAGTTTTTACCGACTGGTATAAAACTCCCACCAAACATAGACTCCGATGGCATTCTTTAATTGCAAAGTACAATCTGAGTCATTTGACCATATCTACCATGATTGGCTGTGACAGACTCATTCGGATCTTCTGCCTGCACCCTGGCCTCCTGGTGGGAGTGTGGAAGAAGGAGGAAGAACTGGCTTTTGTTATGGCAAATCTTCATTTTCATCACCTTGTGGAGAGGAGCACATTAGGCTCGGCTACTATGCCCTATGAACTGCCTCCACATAGCCCCTTTTTGGATGATAGCCCCGAGTATGGACTGCACGGCTACCAACTCCATGTTGATCTGCACAGCGGTGGGGTTTTCTACCTATGTGGTACATTTCGCAATCTCTTCACCAAGAGAGGAAATATTGAAAATGGACATGTGAAGCTCATTGTTATACATTTAAAAAATAACAGAGAACACCTACCTCTTATTGGAAAAGTTGGCCTCTCGTGGAAAACTGATATTTTTGATGGCTGTATAAAGAGTTGTTCCATGATGGACGTAACTCTTTTGGATGAACATGGGAAACCCTTTTGGTGTTTCAGTTCCCCGGTGTGCCTGAGATCGCCTGCCACACCCTCTGACAGCTCTAGCTTCTTGGGACAGACATACAACGTGGACTACGTTGATGCGGAAGGAAGAGTGCACGTGGAGCTGGTGTGGATCAGAGAGACCGAAGAATACCTTATTGTCAACCTGGTCCTTTATCTTAGTATCGCAAAAATCAACCATTGGTTTGGGACTGAATATTAGCAGTAGGTGGCAAATTATORF Start: ATG at 239ORF Stop: TAG at 1541SEQ ID NO: 14434 aaMW at 49440.8 kDNOV6a,MPSEILLKIFSYLDAVSLLCTGCVSRRFYHLANDSFIWIGIYSTAFSPARSNWKFNSVEKIAMSCG163701-01Protein SequenceMSFLSVQDKEAGYWKKEYITKQIASVKAALADILKPVNPYTGLPVKTKEALRIFGLGWAIILKEKGGKEYIMEHVDLSINDTSVTVIWYGKKWPCLASLSTLDLCGMTPVFTDWYKTPTKHRLRWHSLIAKYNLSHLTISTMIGCDRLIRIFCLHPGLLVGVWKKEEELAFVMANLHFHHLVERSTLGSATMPYELPPHSPFLDDSPEYGLHGYQLHVDLHSGGVFYLCGTFRNLFTKRGNIENGHVKLIVIHLKNNREHLPLIGKVGLSWKTDIFDGCIKSCSMMDVTLLDEHGKPFWCFSSPVCLRSPATPSDSSSFLGQTYNVDYVDAEGRVHVELVWIRETEEYLIVNLVLYLSIAKINHWFGTEY

[0382] Further analysis of the NOV6a protein yielded the following properties shown in Table 6B.

29TABLE 6BProtein Sequence Properties NOV6aSignalP analysis:Cleavage site between residues 26 and 27PSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 8; pos.chg 1; neg.chg 1H-region: length 5; peak value 2.36PSG score: −2.04GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −1.23possible cleavage site: between 25 and 26>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS (s) for the threshold 0.5: 1Number of TMS(s) for threshold 0.5: 0PERIPHERAL Likelihood = 2.07 (at 212)ALOM score: −0.75 (number of TMSs: 0)MITDISC: discrimination of mitochondrial targeting seqR content: 0Hyd Moment (75): 11.42Hyd Moment (95): 9.29G content: 0D/E content: 2S/T content: 2Score: −5.34Gavel: prediction of cleavage sites for mitochondrial preseq cleavagesite motif not foundNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: nonebipartite: nonecontent of basic residues: 10.1%NLS Score: −0.47KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals: noneSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: cytoplasmicReliability: 94.1COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23); 47.8%: mitochondrial 26.1%: cytoplasmic 13.0%: nuclear 4.3%: vacuolar 4.3%: extracellular, including cell wall 4.3%: peroxisomal >> prediction for CG163701-01 is mit (k = 23)

[0383] A search of the NOV6a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 6C.

30TABLE 6CGENESEQ Results for NOV6aNOV6aIdentities/Residues/Similarities forGENESEQProtein/Organism/LengthMatchthe MatchedExpectIdentifier[Patent #, Date]ResiduesRegionValueABB08271Human myb protein 41 - Homo 63 . . . 425358/363 (98%)0.0sapiens, 369 aa. [CN1321686-A, 1 . . . 362359/363 (98%)14-NOV-2001]AAE08040Mouse F-box protein, F15 Rho - Mus 1 . . . 3830/38 (78%)4e−11musculus, 38 aa. [US6232081-B1, 1 . . . 3835/38 (91%)15-MAY-2001]AAY02268A F-box protein sequence - Mus sp, 38 1 . . . 3830/38 (78%)4e−11aa. [WO9918989-A1, 22-APR-1999] 1 . . . 3835/38 (91%)AAB48292Human ZF4 protein - Homo sapiens, 1 . . . 4922/49 (44%)2e−05192 aa. [WO200075184-A1,140 . . . 18832/49 (64%)14-DEC-2000]AAG66507F-box structure protein 65 - 1 . . . 5022/50 (44%)2e−04Unidentified, 592 aa. 82 . . . 13128/50 (56%)[WO200155417-A1, 02-AUG-2001]

[0384] In a BLAST search of public sequence databases, the NOV6a protein was found to have homology to the proteins shown in the BLASTP data in Table 6D.

31TABLE 6DPublic BLASTP Results for NOV6aNOV6aIdentities/ProteinResidues/Similarities forAccessionMatchthe MatchedExpectNumberProtein/Organism/LengthResiduesPortionValueQ8NCQ5Hypothetical protein - Homo sapiens 1 . . . 434432/434 (99%)0.0(Human), 434 aa. 1 . . . 434434/434 (99%)Q9GKV7Hypothetical 49.4 kDa protein - Macaca 1 . . . 434411/434 (94%)0.0fascicularis (Crab eating macaque) 1 . . . 434423/434 (96%)(Cynomolgus monkey), 434 aa.Q9QZN0F-box protein FBX15 - Mus musculus 1 . . . 433253/437 (57%) e−149(Mouse), 517 aa (fragment). 85 . . . 517319/437 (72%)Q9UHI1F-box protein FBX11 - Homo sapiens 1 . . . 4922/49 (44%)5e−05(Human), 192 aa (fragment).140 . . . 18832/49 (64%)Q9UKC2F-box protein Fbx11 - Homo sapiens 1 . . . 5022/50 (44%)0.001(Human), 197 aa (fragment).144 . . . 19328/50 (56%)

[0385] PFam analysis predicts that the NOV6a protein contains the domains shown in the Table 6E.

32TABLE 6EDomain Analysis of NOV6aPfamNOV6aIdentities/SimilaritiesDomainMatch Regionfor the Matched RegionExpect ValueF-box1 . . . 4312/48 (25%)0.1234/48 (71%)

Example 7

[0386] The NOV7 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 7A.

33TABLE 7ANOV7 Sequence AnalysisSEQ ID NO: 156931 bpNOV7a,CTTAGAGCAGGCCGCTTGGTTCTGACCCAGCTGAGGAAATACTCTTAATTCTAAGGAAAACCTGCG164211-01DNA SequenceGAAGCACAATGGGAGATGACAGTGAGTGGTTGAAACTGCCAGTTGATCAGAAATGTGAACACAAGCTGTGGAAAGCAAGGTTAAGTGGGTATGAAGAGGCCCTGAAGATCTTCCAGAAAATAAAGGATGAAAAGAGCCCAGAGTGGTCCAAATTTTTAGGATTGATCAAAAAATTTGTCACTGATTCCAATGCAGTGGTTCAATTGAAAGGATTAGAAGCTGCACTTGTTTATGTTGAAAATGCCCATGTAGCAGGAAAAACCACAGGAGAAGTTGTGTCAGGTGTTGTAAGTAAGGTGTTCAATCAACCTAAAGCTAAAGCCAAGGAGCTGGGCATAGAGATCTGTCTTATGTACATAGAGATTGAGAAAGGAGAGGCTGTTCAAGAAGAGCTCCTGAAAGGCTTGGACAATAAGAATCCCAAGATCATAGTGGCCTGTATAGAGACACTGAGGAAAGCCTTAAGTGAATTTGGTTCCAAAATCATCTTGCTTAAGCCAATTATCAAAGTGTTGCCAAAACTCTTTGAGTCTCGAGAGAAGGCTGTTCGAGATGAAGCCAAACTAATTGCTGTGGAGATTTACAGATGGATTCGGGATGCTCTGAGACCCCCATTACAAAATATAAACTCTGTTCAGTTGAAAGAACTAGAAGAAGAATGGGTCAAACTGCCAACAAGTGCTCCTAGACCTACTCGATTTCTTCGTTCCCAACAAGAACTAGAAGCTAAATTGGAACAACAACAGTCTGCTGGTGGAGATGCTGAAGGAGGTGGTGATGATGGTGATGAGGTGCCACAAATAGATGCTTATGAGCTTTTAGAAGCTGTAGAAATCCTTTCCAAACTTCCCAAAGACTTTTATGACAAAATTGAGGCAAAAAAATGGCAAGAGAGAAAAGAGGCCCTGGAGTCTGTAGAAGTACTAATAAAAAACCCCAAACTGGAAGCTGGCGATTATGCAGATTTAGTAAAAGCATTAAAGAAGGTTGTTGGAAAGGACACCAATGTCATGTTGGTGGCTTTGGCAGCAAAATGTCTTACTGGCCTGGCTGTTGGGCTAAGGAAGAAATTTGGACAATATGCAGGACATGTTGTGCCAACCATCTTGGAGAAATTCAAAGAGAAGAAACCTCAAGTGGTACAAGCCCTGCAGGAGGCAATTGATGCAATCTTCCTTACTACCACACTACAGAACATCAGTGAGGATGTTTTAGCAGTAATGGATAATAAAAATCCAACCATCAAGCAGCAGACATCTCTTTTTATTGCAAGAAGTTTCCGCCACTGCACTGCTTCTACCCTGCCAAAGAGCTTGCTAAAGCCCTTTTGTGCTGCACTACTTAAGCACATCAATGATTCTGCTCCTGAAGTCAGAGATGCCGCATTTGAAGCATTGGGTACTGCTTTGAAGGTGGTTGGCGAGAAAGCAGTAAAACCATTCCTAGCTGATGTGGACAAACTCAAGCTTGATAAGATCAAAGAATGTTCAGAAAAGGTAGAACTGATACATGGTAAGAAAGCTGGACTAGCTGCTGATAAGAAGGAATTCAAACCTCTGCCTGGAAGGACTGCTGCTTCAGGGGCTGCAGGAGATAAGGACACAAAGGACATTTCTGCACCCAAACCAGGACCTCTAAAAAAGGCACCTGCTGCTAAGGCTGGTGGGCCACCAAAAAAGGGGAAACCAGCTGCACCAGGAGGCGCAGGGAATACTGGAACCAAGAACAAGAAAGGACTGGAGACTAAAGAAATAGTGGAGCCTGAGCTCTCGATAGAAGTATGTGAAGAAAAAGCTTCAGCTGTTCTTCCCCCTACCTGTATACAGCTTCTTGACAGCAGTAACTGGAAAGAAAGGCTGGCTTGTATGGAAGAGTTCCAGAAGGCTGTTGAGCTAATGGACCGAACTGAAATGCCATGCCAGGCATTAGTGAGGATGCTAGCCAAGAAACCTGGATGGAAAGAAACTAATTTTCAGGTGATGCAAATGAAGCTTCATATAGTTGCTTTGATTGCCCAGAAGGGAAATTTTTCCAAAACGTCAGCTCAGGTTGTATTAGATGGCCTTGTGGACAAGATTGGAGATGTGAAATGTGGGGACAATGCAAAAGAAGCTATGACAGCAATAGCCGAAGCCTGTATGTTACCATGGACTGCTGAACAGGTTGTGTCAATGGCTTTCTCACAAAAGAATCCCAAAAATCAGTCAGAAACTCTGAATTGGCTATCAAATGCCATAAAAGAATTTGGTTTTTCTGGGTTGAATGTCAAAGCTTTCATTAGCAATGTGAAGACAGCTCTTGCTGCAACAAACCCAGCTGTGAGGACTGCTGCCATAACCCTGCTTGGCGTGATGTATCTGTATGTTGGTCCCTCTTTGCGAATGTTCTTTGAGGATGAGAAGCCTGCCCTCCTATCCCAGATAGATGCAGAATTTGAGAAGATGCAGGGACAAAGTCCACCTGCTCCAACCAGAGGAATTTCCAAGCATAGCACAAGTGGTACAGATGAAGGAGAAGATGGAGATGAACCAGATGACGGGAGCAATGATGTCGTTGATCTTTTGCCGAGGACGGAGATCAGTGATAAAATCACTTCAGAGTTGGTATCTAAGATTGGTGATAAGAATTGGAAGATTAGGAAAGAAGGCCTAGATGAAGTGGCAGGTATTATTAATGACGCAAAATTTATCCAACCGAATATAGGTGAACTTCCAACTGCCTTGAAGGGTCGACTCAATGATTCAAATAAAATCTTGGTACAGCAAACGCTGAATATCCTGCAACAACTGGCAGTAGCCATGGGCCCAAATATTAAGCAACATGTAAAAAATTTAGGCATCCCTATCATCACAGTCCTTGGAGACAGCAAGAACAATGTTCGAGCTGCTGCCCTAGCGACTGTGAATGCTTGGGCAGAACAGACTGGCATGAAGGAATGGCTGGAAGGAGAAGATCTTTCTGAAGAGCTCAAAAAGGAAAATCCTTTCTTGAGGCAAGAGCTTCTGGGCTGGCTGGCTGAGAAACTACCTACTCTTCGTTCCACCCCTACAGACCTTATCCTTTGTGTTCCTCATCTCTACTCCTGCCTAGAAGATCGAAATGGAGATGTGCGAAAGAAGGCCCAAGATGCCTTGCCATTCTTCATGATGCATTTAGGATATGAAAAAATGGCCAAGGCTACTGGGAAACTAAAGCCAACTTCTAAAGATCAGGTATTGGCCATGCTAGAGAAAGCCAAAGTTAACATGCCAGCCAAGCCTGCTCCACCCACTAAAGCAACTTCTAAACCAATGGGAGGGTCCGCTCCAGCCAAATTCCAGCCTGCATCAGCACCTGCTGAAGATTGTATTTCCAGCAGTACAGAACCCAAACCTGATCCAAAAAAGGCCAAAGCTCCAGGATTATCCTCTAAAGCAAAGAGTGCACAAGGGAAGAAGATGCCAAGCAAAACCAGCTTAAAGGAGGATGAAGACAAATCCGGGCCTATTTTTATTGTTGTTCCAAATGGAAAAGAGCAAAGGATGAAAGATGAAAAAGGATTGAAGGTGCTAAAGTGGAATTTTACTACCCCACGGGATGAATACATTGAGCAACTAAAGACTCAAATGTCTAGCTGTGTGGCTAAATGGTTACAAGATGAGATGTTTCACTCAGACTTTCAGCATCATAACAAAGCCCTTGCTGTTATGGTTGATCACTTGGAGAGTGAAAAAGAAGGAGTTATTGGTTGCCTGGATCTTATCTTAAAGTGGCTTACCCTGAGGTTTTTTGACACCAATACAAGCGTCCTGATGAAAGCACTAGAATATTTAAAATTGCTCTTCACCTTGCTAAGTGAAGAAGAATATCATCTTACTGAGAATGAAGCATCTTCCTTCATCCCCTATCTTGTCGTCAAGGTTGGAGAACCAAAGGATGTCATTCGTAAAGATGTTCGTGCCATCCTGAACCGGATGTGCCTTGTCTACCCAGCTAGCAAGATGTTTCCCTTTATCATGGAAGGAACCAAATCCAAAAACTCTAAGCAGAGAGCAGAGTGCCTGGAAGAGCTGGGATGTCTGGTTGAGTCCTATGGCATGAATGTTTGCCAACCAACCCCAGGAAAAGCCTTAAAGGAAATAGCTGTTCACATAGGAGACCGTGACAATGCTGTACGCAATGCTGCACTCAACACCATTGTAACGGTGTACAATGTACATGGGGATCAGGTGTTCAAACTGATTGGAAATCTTTCTGAAAAGGATATGAGCATGCTCGAGGAGAGGATTAAGCGGTCAGCAAAGAGACCCTCTGCTGCACCAATAAAACAGGTGGAAGAGAAACCTCAGCGTGCACAGAACATAAGCTCCAATGCCAACATGTTACGCAAGGGACCAGCTGAGGACATGTCTTCCAAACTCAAAATTATGTATCGCACTTATAGGATCCAAGCCCGAAGCATGAGTGGGCATCCTGAGGCAGCCCAGATGGTCCGCCGAGAATTCCAGCTGGATCTAGATGAGATTGAGAATGACAATGGTACAGTCCGATGTGAAATGCCAGAACTTGTTCAGCACAAACTGGATGACATTTTTGAGCCAGTCCTTATTCCTGAACCCAAGATCCGGGCTGTTTCTCCACACTTCGATGACATGCACAGTAATACAGCATCCACAATCAATTTCATTATCTCCCAAGTAGCCAGTGGTGACATCAACACAAGTATCCAAGCTCTGACACAGATCGATGAGGTCCTGAGACAGGAAGACAAAGCTGAAGCCATGTCCGGCCATATTGATCAGTTTCTGATAGCCACTTTTATGCAGCTAAGACTCATCTACAACACACACATGGCAGATGAGAAATTGGAGAAGGACGAGATCATCAAGTTGTATAGCTGTATCATTGGCAACATGATTTCGCTGTTTCAGATAGAGAGCCTTGCCCGGGAGGCCTCCACTGGAGTACTAAAAGACCTAATGCATGGCCTCATCACCTTAATGCTGGATTCTCGGATTGAAGATCTTGAGGAAGGACAACAGGTCATCCGCTCTGTGAACCTCTTGGTGGTGAAGGTTCTGGAGAAGTCAGACCAGACCAACATCCTGAGTGCCCTACTTGTTTTGCTCCAAGACAGCCTGCTAGCAACAGCCAGTTCTCCCAAATTCTCAGAGCTTGTTATGAAGTGTCTCTGGAGAATGGTTCGACTGTTGCCTGATACCATCAATAGCATTAACCTAGACAGAATTCTTCTGGATATCCACATTTTCATGAAGGTCTTCCCCAAAGAGAAACTGAAGCAATGCAAAAGTGAATTTCCCATAAGGACCCTAAAGACCCTGCTACACACCTTATGCAAATTAAAAGGGCCCAAGATCCTGGACCACCTAACGATGATCGACAACAAAAACGAGTCTGAGCTGGAGGCCCATCTCTGCCGGATGATGAAGCACAGTATGGACCAGACTGGGAGCAAGTCTGATAAGGAAACAGAAAAGGGAGCATCTCGAATAGATGAAAAATCATCAAAGGCCAAAGTGAATGATTTCTTAGCTGAGATTTTTAAGAAGATTGGCTCTAAAGAAAACACTAAAGAGGGACTAGCAGAGTTATATGAATATAAGAAGAAATACTCAGATGCTGACATTGAACCATTTCTGAAAAATTCCTCACAGTTCTTCCAGAGCTATGTCGAAAGAGGCCTTCGGGTGATTGAGATGGAGAGGGAGGGCAAAGGTCGTATTTCCACTTCAACAGGCATCTCCCCTCAGATGGAAGTCACATGTGTGCCCACGCCCACAAGCACAGTGTCCTCCATAGGTAACACAAATGGGGAAGAAGTGGGGCCATCTGTCTACTTGGAAAGGCTAAAGATCCTCCGACAGCGATGTGGTCTGGACAACACAAAGCAAGATGACCGACCTCCTTTGACCTCTTTGCTCTCCAAACCAGCAGTTCCTACTGTCGCCTCTTCCACAGACATGCTCCACAGCAAACTCTCTCAGCTCCGGGAGTCACGGGAGCAGCACCAGCATTCAGACCTGGATTCTAACCAGACTCACTCTTCAGGAACTGTGACCTCCTCCTCCTCCACAGCTAACATAGACGACTTGAAAAAAAGACTGGAGAGAATAAAGAGCAGTCGCAAATGAAGCTGCCCCACTCCCCCGGCACCCTGCAGCTTTAGTTTACTAAACTAGAAGTCCTCATAGTTTAAAATGGCCTCAGCAGGCCTAGTGTATACAAACTGGTTGTATGTATCATGCCGTGGAGCTAGGGGGAGGAGTCATTGTGGCACAAGTATTTGTACATACTCTGCTTCTCTCTGTCAGCGTCCTGCTGCTCTAGAAGACTGTCCGTGGATGAGTTTAGTGTACAGACTTGTAAACAGCTGCCCCCTCTCTGCTCAGTCTAGTTCCCAGATCCTTTTCTTTTCTTTTTAATTGCTCATTTGTAAAATTGTCCTAATCTTTCCTAGCTTTTTAATAGTTAATATTAGAAACTCTTTAATAGTTTTCCTTTCAGTTTGTGAGCTCTTCTCTGTCGCCCTGAAGGGTCACTGTATTCTGTATGAATGCATGGCATGATACAACTAATTTAAGAGTCTTTTATAAATAAAGTTTGCATTAACTATACCTGACTCCCAAGATGCTCCATATCATGGAGTGTTGGGCAGCAATTTTTTAACTTTTGGTACCAGCTGGGTATTCAGCCAGTTAGAGAAAGGTTAATTACTGCTTACAGAGCTTCCCTTTTTCTTGGCTCACAGACTTGGAGCTGTCTTTTATTCCTTTGGAATGGAAGGAACTCTCCTTCCCTGCTCTCCACAAACAGCTCCTAGGGAGCAGAAAAATCTCATCCTTCCTTGTTTTGAGATTTTGGTCATGAORF Start: ATG at 73ORF Stop: TGA at 6193SEQ ID NO: 162040 aaMW at 226618.4 kDNOV7a,MGDDSEWLKLPVDQKCEHKLWKARLSGYEEALKIFQKIKDEKSPEWSKFLGLIKKFVTDSNAVVCG164211-01Protein SequenceQLKGLEAALVYVENAHVAGKTTGEVVSGVVSKVFNQPKAKAKELGIEICLMYIEIEKGEAVQEELLKGLDNKNPKIIVACIETLRKALSEFGSKIILLKPIIKVLPKLFESREKAVRDEAKLIAVEIYRWIRDALRPPLQNINSVQLKELEEEWVKLPTSAPRPTRFLRSQQELEAKLEQQQSAGGDAEGGGDDGDEVPQIDAYELLEAVEILSKLPKDFYDKIEAKKWQERKEALESVEVLIKNPKLEAGDYADLVKALKKVVGKDTNVMLVALAAKCLTGLAVGLRKKFGQYAGHVVPTILEKFKEKKPQVVQALQEAIDAIFLTTTLQNISEDVLAVMDNKNPTIKQQTSLFIARSFRHCTASTLPKSLLKPFCAALLKHINDSAPEVRDAAFEALGTALKVVGEKAVKPFLADVDKLKLDKIKECSEKVELIHGKKAGLAADKKEFKPLPGRTAASGAAGDKDTKDISAPKPGPLKKAPAAKAGGPPKKGKPAAPGGAGNTGTKNKKGLETKEIVEPELSIEVCEEKASAVLPPTCIQLLDSSNWKERLACMEEFQKAVELMDRTEMPCQALVRMLAKKPGWKETNFQVMQMKLHIVALIAQKGNFSKTSAQVVLDGLVDKIGDVKCGDNAKEAMTAIAEACMLPWTAEQVVSMAFSQKNPKNQSETLNWLSNAIKEFGFSGLNVKAFISNVKTALAATNPAVRTAAITLLGVMYLYVGPSLRMFFEDEKPALLSQIDAEFEKMQGQSPPAPTRGISKHSTSGTDEGEDGDEPDDGSNDVVDLLPRTEISDKITSELVSKIGDKNWKIRKEGLDEVAGIINDAKFIQPNIGELPTALKGRLNDSNKILVQQTLNILQQLAVAMGPNIKQHVKNLGIPIITVLGDSKNNVRAAALATVNAWAEQTGMKEWLEGEDLSEELKKENPFLRQELLGWLAEKLPTLRSTPTDLILCVPHLYSCLEDRNGDVRKKAQDALPFFMMHLGYEKMAKATGKLKPTSKDQVLAMLEKAKVNMPAKPAPPTKATSKPMGGSAPAKFQPASAPAEDCISSSTEPKPDPKKAKAPGLSSKAKSAQGKKMPSKTSLKEDEDKSGPIFIVVPNGKEQRMKDEKGLKVLKWNFTTPRDEYIEQLKTQMSSCVAKWLQDEMFHSDFQHHNKALAVMVDHLESEKEGVIGCLDLILKWLTLRFFDTNTSVLMKALEYLKLLFTLLSEEEYHLTENEASSFIPYLVVKVGEPKDVIRKDVRAILNRMCLVYPASKMFPFIMEGTKSKNSKQRAECLEELGCLVESYGMNVCQPTPGKALKEIAVHIGDRDNAVRNAALNTIVTVYNVHGDQVFKLIGNLSEKDMSMLEERIKRSAKRPSAAPIKQVEEKPQRAQNISSNANMLRKGPAEDMSSKLKIMYRTYRIQARSMSGHPEAAQMVRREFQLDLDEIENDNGTVRCEMPELVQHKLDDIFEPVLIPEPKIRAVSPHFDDMHSNTASTINFIISQVASGDINTSIQALTQIDEVLRQEDKAEAMSGHIDQFLIATFMQLRLIYNTHMADEKLEKDEIIKLYSCIIGNMISLFQIESLAREASTGVLKDLMHGLITLMLDSRIEDLEEGQQVIRSVNLLVVKVLEKSDQTNILSALLVLLQDSLLATASSPKFSELVMKCLWRMVRLLPDTINSINLDRILLDIHIFMKVFPKEKLKQCKSEFPIRTLKTLLHTLCKLKGPKILDHLTMIDNKNESELEAHLCRMMKHSMDQTGSKSDKETEKGASRIDEKSSKAKVNDFLAEIFKKIGSKENTKEGLAELYEYKKKYSDADIEPFLKNSSQFFQSYVERGLRVIEMEREGKGRISTSTGISPQMEVTCVPFPTSTVSSIGNTNGEEVGPSVYLERLKILRQRCGLDNTKQDDRPPLTSLLSKPAVPTVASSTDMLHSKLSQLRESREQHQHSDLDSNQTHSSGTVTSSSSTANIDDLKKRLERIKSSRK

[0387] Further analysis of the NOV7a protein yielded the following properties shown in Table 7B.

34TABLE 7BProtein Sequence Properties NOV7aSignalP analysis:No Known Signal Sequence PredictedPSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 9; pos.chg 1; neg.chg 3H-region: length 3; peak value 0.00PSG score: −4.40GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1) : −12.91possible cleavage site: between 60 and 61>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS (s) for the threshold 0.5: 4INTEGRAL Likelihood = −3.77 Transmembrane 334-350INTEGRAL Likelihood = −0.11 Transmembrane 774-790INTEGRAL Likelihood = 0.26 Transmembrane 1238-1254INTEGRAL Likelihood = −2.39 Transmembrane 1692-1708PERIPHERAL Likelihood = 1.96 (at 1259)ALOM score: −3.77 (number of TMSs: 4)MTOP: Prediction of membrane topology (Hartmann et al.)Center position for calculation: 341Charge difference: 3.0 C ( 4.0) − N( 1.0)C > N: C-terminal side will be inside>>> membrane topology: type 3bMITDISC: discrimination of mitochondrial targeting seqR content: 0Hyd Moment (75): 9.60Hyd Moment (95): 7.96G content: 1D/E content: 2S/T content: 0Score: −6.82Gavel: prediction of cleavage sites for mitochondrial preseq cleavagesite motif not foundNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: PPKKGKP (4) at 554pat7: PKKGKPA (5) at 555pat7: PDPKKAK (3) at 1122pat7: PKKAKAP (5) at 1124bipartite: KKAPAAKAGGPPKKGKP at 544content of basic residues: 14.2%NLS Score: 1.48KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals:KKXX-like motif in the C-terminus: KSSRSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: foundKLPK at 279TLPK at 431RNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern : nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs:Leucine zipper pattern (PS00029): *** found ***LAEKLPTLRSTPTDLILCVPHL at 1002LMKALEYLKLLFTLLSEEEYHL at 1261nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: nuclearReliability: 55.5COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 39.1%: endoplasmic reticulum 30.4%: nuclear 13.0%: vacuolar 4.3%: Golgi 4.3%: mitochondrial 4.3%: vesicles of secretory system 4.3%: cytoplasmic >> prediction for CG164211-01 is end (k = 23)

[0388] A search of the NOV7a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 7C.

35TABLE 7CGENESEQ Results for NOV7aNOV7aIdentities/Residues/Similarities forGENESEQProtein/Organism/Length [Patent #,Matchthe MatchedExpectIdentifierDate]ResiduesRegionValueABB71336Drosophila melanogaster polypeptide 1 . . . 1914 796/2013 (39%)0.0SEQ ID NO 40800 - Drosophila 1 . . . 19301163/2013 (57%)melanogaster, 2018 aa.[WO200171042-A2, 27-SEP-2001]AAB63792Human prostate cancer associated1177 . . . 1428 237/252 (94%) e−131antigen protein sequence SEQ ID 1 . . . 251 238/252 (94%)NO: 1154 - Homo sapiens, 261 aa.[WO200073801-A2, 07-DEC-2000]AAB63791Human prostate cancer associated 581 . . . 789 206/209 (98%) e−114antigen protein sequence SEQ ID 1 . . . 209 208/209 (98%)NO: 1153 - Homo sapiens, 217 aa.[WO200073801-A2, 07-DEC-2000]ABP43215Human ovarian antigen HVVDT44,1888 . . . 2040 151/153 (98%)1e−79SEQ ID NO: 4347 - Homo sapiens, 153 1 . . . 153 151/153 (98%)aa. [WO200200677-A1, 03-JAN-2002]AAO05067Human polypeptide SEQ ID NO 18959 - 858 . . . 886 29/29 (100%)4e−08Homo sapiens, 47 aa. 19 . . . 47 29/29 (100%)[WO200164835-A2, 07-SEP-2001]

[0389] In a BLAST search of public sequence databases, the NOV7a protein was found to have homology to the proteins shown in the BLASTP data in Table 7D.

36TABLE 7DPublic BLASTP Results for NOV7aNOV7aProteinResidues/Identities/AccessionMatchSimilarities for theExpectNumberProtein/Organism/LengthResiduesMatched PortionValueQ14008CH-TOG protein (Colonic and hepatic1 . . . 20402030/2040 (99%)0.0tumor over-expressed protein) - Homo1 . . . 20322031/2040 (99%)sapiens (Human), 2032 aa.S68176TOG protein - human, 1972 aa.1 . . . 20401940/2040 (95%)0.01 . . . 19721950/2040 (95%)Q9DDV7Microtubule associated protein1 . . . 20401649/2052 (80%)0.0XMAP215 isoform Z - Xenopus laevis1 . . . 20301831/2052 (88%)(African clawed frog), 2030 aa.Q9DDV8Microtubule associated protein1 . . . 20401649/2088 (78%)0.0XMAP215 isoform M - Xenopus1 . . . 20661834/2088 (86%)laevis (African clawed frog), 2066 aa.Q9PT63Microtubule associated protein 215 kDa1 . . . 20401650/2088 (79%)0.0(XMAP215) - Xenopus laevis1 . . . 20651833/2088 (87%)(African clawed frog), 2065 aa.

[0390] PFam analysis predicts that the NOV7a protein contains the domains shown in the Table 7E.

37TABLE 7EDomain Analysis of NOV7aPfamNOV7a MatchIdentities/Expect ValueDomainRegionSimilaritiesfor the Matched Region

Example 8

[0391] The NOV8 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 8A.

38TABLE 8ANOV8 Sequence AnalysisSEQ ID NO: 172634 bpNOV8a,CGGCCGCCCGAGCAGGCCGCGCGCGGGCCGCCGGGCCCGAGGCCAGAGCCATGGGCGAGACCAACG164330-01DNA SequenceGATCATCTACCACTTGGATGGGCAGGAGACGCCGTACCTTGTGAAGCTGCCCCTGCCCGCCGAGCGCGTCACCTTGGCGGACTTTAAGGGCGTTTTGCAGCGACCCAGCTATAAGTTCTTCTTCAAGTCTATGGACGACGATTTCGGAGTGGTGAAGGAGGAGATCTCGGATGACAATGCCAAGCTACCATGCTTCAATGGCCGGGTGGTGTCCTGGCTGGTGTCAGCTGAGGGCTCACACCCAGACCCAGCCCCCTTCTGTGCTGATAACCCATCGGAGCTGCCACCACCTATGGAGCGCACGGGAGGCATCGGGGACTCCCGACCCCCATCCTTCCACCCTCATGCTGGTGGGGGCAGCCAGGAGAACCTGGACAATGACACAGAGACGGACTCTTTGGTGTCTGCCCAGCGAGGGCGGCCACGCCGGAGGGATGGCCCAGAGCATGCAACCCGGCTAAATGGAACTGCGAAGGGGGAACGGCGGCGAGGACCAGGGGGTTATGATAGCTCATCCACCCTTATGAGCAGTGAGCTGGAGACCACCAGCTTCTTTGACTCAGATGAGGATGACTCCACCAGCAGGTTCAGCAGCTCCACAGAACAGAGCAGTGCCTCACGCCTGATGAGAAGACACAAGCGGCGGCGGCGGAAGCAGAAGGTTTCTCGGATTGAGCGGTCCTCGTCCTTCAGCAGCATCACGGACTCCACCATGTCACTCAACATCATCACGGTCACTCTCAACATGGAAAAATATAACTTCTTGAGCACCATCACCTCCACCAGCTCCTCCATCACCAGTTCCATCCCTGACACAGAGCGCCTAGACGACTTCCACTTGTCCATCCACAGTGACATGGCTGCCATCGTAAAAGCCATGGCCTCCCCTGAATCAGGGTTGGAGGTCCGTGACCGCATGTGGCTCAAGATTACCATCCCTAATGCTTTCATCGGCTCAGATGTGGTGGACTGGCTGTACCACAATGTGGAAGGCTTCACGGACCGGAGGGAGGCCCGCAAGTATGCCAGCAACCTGCTGAAAGCTGGCTTCATCCGCCATACCGTCAACAAGATCACCTTCTCCGAGCAGTGCTACTACATCTTCGGTGACCTCTGCGGCAACATGGCCAACCTGTCTCTCCACGATCACGATGGCTCCAGTGGCGCCTCTGACCAGGACACACTGGCCCCTTTGCCGCACCCGGGGGCCGCCCCTTGGCCCATGGCTTTCCCGTACCAGTACCCGCCACCCCCGCACCCATACAACCCGCACCCGGGCTTCCCGGAGCTGGGCTACAGCTACGGCGGGGGCAGCGCCAGCAGTCAGCACAGCGAAGGCAGTCGGAGCAGTGGCTCCAACCGTAGCGGCAGCGATCGGAGGAAGGAGAAGGACCCGAAGGCCGGGGACTCCAAGTCCGGGGGCAGCGGCAGCGAATCGGACCACACCACACGCAGCAGCCTGCGGGGGCCGCGGGAGCGGGCGCCCAGCGAGCGCTCAGGGCCGGCGGCCAGCGAGCACAGCCACCGCAGCCACCATTCCCTGGCCAGCAGCCTTCGCAGCCACCACACACACCCGAGCTACGGTCCTCCCGGAGTGCCCCCTCTCTACGGCCCCCCCATGCTGATGATGCCCCCGCCGCCCGCGGCCATGGGGCCCCCAGGAGCCCCTCCGGGCCGCGACCTGGCCTCAGTGCCCCCGGAACTGACCGCCAGCAGACAGTCCTTCCGCATGGCCATGGGAAACCCCAGTGAGTTCTTTGTGGATGTGATGTGAGCAGGGCCCCTCCCCCAGCTCCATTCCGCTCCCACCCCAGCCGGCTGCGTTCCTCTCTCCATCCGTCCGTCTTTTTTACTTTGTCTGGTACCTGAAAGGGAAATAAAAGGAACTAAATCCAGGTGCGCTAACTGCTCGCAGGGTGCTGCGAGGGTGGGGTGCACCTACCGATTGGCTCTGCAGCCCCCTAACCTGCCTCTGGCCCCAGTTCGTTTCCTCTGCCCACTAATCCCTGCGCAGGACTTCCCAGGACCCCTTTTGTCTCTGGGACCAGACTTGTTGGTGCTACCCCTTACTCCCCTCTGCAACCCCCATTTTGGGAGTTGACCCCAGCAATGACCTTGGTGGCACGCTCACTCCCTCATTCTCTCGTTTCCCCTTTAGCTCCCTTTCACCATTTATTCAGCTACATCATCCCTCTATTAACCCCACCCCATCAGGCACGTGTGCAAACCTCTTGACTTTACCCCACATTACTGAAACCAAAATATATTTGCTTCATCTGCCCCTACTAACCATCCCCCTGCCTGCTGCCTCAGTCCTGCAACCTAAAGCTGTAGTCGCCTCCAATAGCCATCCATGCCATCCCTGCCTGTGCCTAGATCAGAGGCCCAGAGGGCCCCCTCAGTTGCCTGAGCAGCTGGTGGCTTCCAGGGAGCATCTCTGCTCTACCCCTGCCCCATGCCTGCCCTGCGTGCTGGTTCCTTCAGACCCCTAACCCTACTAACCAGCAGGCTCATCTCACCTCCAGGCCTGAAACATTTCTTTTCTTTCTTTTTTCCTCCCCCAATTTACCORF Start: ATG at 51ORF Stop: TGA at 1836SEQ ID NO: 18595 aaMW at 65063.2 kDNOV8a,MGETKIIYHLDGQETPYLVKLPLPAERVTLADFKGVLQRPSYKFFFKSMDDDFGVVKEEISDDNCG164330-01Protein SequenceAKLPCFNGRVVSWLVSAEGSHPDPAPFCADNPSELPPPMERTGGIGDSRPPSFHPHAGGGSQENLDNDTETDSLVSAQRGRPRRRDGPEHATRLNGTAKGERRRGPGGYDSSSTLMSSELETTSFFDSDEDDSTSRFSSSTEQSSASRLMRRHKRRRRKQKVSRIERSSSFSSITDSTMSLNIITVTLNMEKYNFLSTITSTSSSITSSIPDTERLDDFHLSIHSDMAAIVKAMASPESGLEVRDRMWLKITIPNAFIGSDVVDWLYHNVEGFTDRREARKYASNLLKAGFIRHTVNKITFSEQCYYIFGDLCGNMANLSLHDHDGSSGASDQDTLAPLPHPGAAPWPMAFPYQYPPPPHPYNPHPGFPELGYSYGGGSASSQHSEGSRSSGSNRSGSDRRKEKDPKAGDSKSGGSGSESDHTTRSSLRGPRERAPSERSGPAASEHSHRSHHSLASSLRSHHTHPSYGPPGVPPLYGPPMLMMPPPPAAMGPPGAPPGRDLASVPPELTASRQSFRMAMGNPSEFFVDVM

[0392] Further analysis of the NOV8a protein yielded the following properties shown in Table 8B.

39TABLE 8BProtein Sequence Properties NOV8aSignalP analysis:No Known Signal Sequence PredictedPSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 11; pos.chg 1; neg.chg 2H-region: length 2; peak value 0.00PSG score: −4.40GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −7.84possible cleavage site: between 31 and 32>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS(s) for the threshold 0.5: 0number of TMS(s) .. fixedPERIPHERAL Likelihood = 5.30 (at 311)ALOM score: 5.30 (number of TMSs: 0)MITDISC: discrimination of mitochondrial targeting seqR content: 0Hyd Moment(75): 6.16Hyd Moment (95): 0.51G content: 1D/E content: 2S/T content: 1Score: −7.87Gavel: prediction of cleavage sites for mitochondrial preseq cleavagesite motif not foundNUCDISC: discrimination of nuclear localization signalspat4: RPRR (4) at 145pat4: PRRR (4) at 146pat4: RRHK (3) at 215pat4: RHKR (3) at 216pat4: HKRR (3) at 217pat4: KRRR (5) at 218pat4: RRRR (5) at 219pat4: RRRK (5) at 220pat7: PRRRDGP (5) at 146bipartite: nonecontent of basic residues: 11.1%NLS Score: 1.96KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals: noneSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: nuclearReliability: 94.1COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 95.7%: nuclear 4.3%: cytoskeletal >> prediction for CG164330-01 is nuc (k = 23)

[0393] A search of the NOV8a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 8C.

40TABLE 8CGENESEQ Results for NOV8aNOV8aIdentities/Residues/Similarities forGENESEQProtein/Organism/Length [Patent #,Matchthe MatchedExpectIdentifierDate]ResiduesRegionValueABB57329Mouse ischaemic condition related264 . . . 595218/348 (62%) e−105protein sequence SEQ ID NO: 913 - Mus396 . . . 736252/348 (71%)musculus, 736 aa. [WO200188188-A2,22-NOV-2001]AAY97560Mouse Dishevelled-1 protein sequence -261 . . . 595192/338 (56%)5e−93Mus musculus, 695 aa.384 . . . 695233/338 (68%)[WO200074706-A1, 14-DEC-2000]ABP63528Human dishevelled 1 (DVL1) PDZ 1 . . . 260175/263 (66%)8e−91domain related amino acid sequence #1 - 1 . . . 261212/263 (80%)Homo sapiens, 341 aa.WO200242422-A2, 30-MAY-2002]ABP63529Human dishevelled 1 (DVL1) PDZ 1 . . . 195129/199 (64%)2e−63domain related amino acid sequence #2 - 1 . . . 197152/199 (75%)Homo sapiens, 198 aa.[WO200242422-A2, 30-MAY-2002]ABB77054Human protein sequence encoded by380 . . . 580123/214 (57%)2e−48gene no: 14 from cDNA clone HCOOS80 - 6 . . . 211144/214 (66%)Homo sapiens, 293 aa.[WO200222638-A1, 21-MAR-2002]

[0394] In a BLAST search of public sequence databases, the NOV8a protein was found to have homology to the proteins shown in the BLASTP data in Table 8D.

41TABLE 8DPublic BLASTP Results for NOV8aNOV8aIdentities/ProteinResidues/Similarities forAccessionMatchthe MatchedExpectNumberProtein/Organism/LengthResiduesPortionValueJC5763dishevelled protein 3 - human, 716 aa.260 . . . 595336/336 (100%) 0.0381 . . . 716336/336 (100%)Q8WVA8Hypothetical protein - Homo sapiens260 . . . 595336/336 (100%)0.0(Human), 608 aa (fragment).273 . . . 608336/336 (100%)Q9UG07Hypothetical protein - Homo sapiens260 . . . 595336/336 (100%)0.0(Human), 548 aa.213 . . . 548336/336 (100%)Q92997Segment polarity protein dishevelled260 . . . 595336/336 (100%)0.0homolog DVL-3 (Dishevelled-3) (DSH381 . . . 716336/336 (100%)homolog 3) - Homo sapiens (Human),716 aa.Q8N5E9Dishevelled, dsh homolog 3260 . . . 595335/336 (99%)0.0(Drosophila) - Homo sapiens (Human),381 . . . 716335/336 (99%)716 aa.

[0395] PFam analysis predicts that the NOV8a protein contains the domains shown in the Table 8E.

42TABLE 8EDomain Analysis of NOV8aIdentities/PfamNOV8aSimilaritiesDomainMatch Regionfor the Matched RegionExpect ValueDIX 1 . . . 8261/86 (71%)1.7e−5481/86 (94%)Dishevelled142 . . . 21349/74 (66%)4.5e−3770/74 (95%)DEP301 . . . 37535/89 (39%) 2e−3270/89 (79%)

Example 9

[0396] The NOV9 clone was analyzed, and the nucleotide and encoded polypeptide sequences are shown in Table 9A.

43TABLE 9ANOV9 Sequence AnalysisSEQ ID NO: 191006 bpNOV9a,TCCGGAGCGG CGACTGGCGA GCCATGGCGCTGGGGCTGCAGCGCGCAAGGTCGACCACGGAGCTCG164521-01DNA SequenceGCGCAAGGAAAAGTCCCGGGATGCGGCCCGCAGCCGGCGCAGCCAGGAGACCGAGGTGCTGTACCAGCTGGCTCACACGCTGCCCTTCGCCCGCGGCGTCAGCGCCCACCTGGACAAGGCCTCTATCATGCGCCTCACCATCAGCTACCTGCGCATGCACCGCCTCTGCGCCGCAGGTGGGAGCAGGGGGAGAACCACTGGATGCCTGCTACCTGAAGGCCCTGGAGGGCTTCGTCATGGTGCTCACCGCCGAGGGAGACATGGCTTACCTGTCGGAGAATGTCAGCAAACACCTGGGCCTCAGTCAGTTGGTATCTGTTCCTTTTCTCTGCCTCGTTACCCCACTCCTGGTACCAATTTCTCTCTGGAGCTCATTGGACACAGCATCTTTGATTTCATCCACCCCTGTGACCAAGAGGAGCTTCAGGACGCCCTGACCCCCCAGCCAGCCCTGTCCAGGAGGAAGGTGGAGGCCCCCACGGAGCGGTGCTTCTCCTTGCGCATGAAGAGTACACTCACCAGCCGCGGGCGCACCCTCAACCTCAAGGCGGCCACCTGGAAGGTGCTGAACTGCTCTGGACATATGAGGGCCTACAAGCCACCTGCGCAGACTTCTCCAGCTGGGAGCCCTGACTCAGAGCCCCCGCTGCAGTGCCTGGTGCTCATCTGCGAAGCCATCCCCAGCTTTCTTTCTACAGATGGTGCTACTCTTGGTCTCCCACAGGAAAAGGCCTCCCCCCTTCTTAGCCCCATTTACCCCGTTTGTGGAAGGCACTGCTCGCTCTGTTTTGTCAGAGAGTGGCCTATCCAGATTGGTGCTATGGGGGGGTCTGACCCCTCCCTCCTCCCTCTGGAGGTGATGTGGGCCCTCAATGGAGGGAATTGTGCTGGGCTAGGGAAAGGGGAGGGACTAGACTGGCCACACTGGCTCTGAAACTCACCORF Start: ATG at 24ORF Stop: TGA at 996SEQ ID NO: 20324 aaMW at 35232.1kDNOV9a,MALGLQRARSTTELRKEKSRDAARSRRSQETEVLYQLAHTLPFARGVSAHLDKASIMRLTISYLCG164521-01Protein SequenceRMHRLCAAGGSRGRTTGCLLPEGPGGLRHGAHRRGRHGLPVGECQQTPGPQSVGICSFSLPRYPTPGTNFSLELIGHSIFDFIHPCDQEELQDALTPQPALSRRKVEAPTERCFSLRMKSTLTSRGRTLNLKAATWKVLNCSGHMRAYKPPAQTSPAGSPDSEPPLQCLVLICEAIPSFLSTDGATLGLPQEKASPLLSPIYPVCGRHCSLCFVREWPIQIGAMGGSDPSLLPLEVMWALNGGNCAGLGKGEGLDWPHWL

[0397] Further analysis of the NOV9a protein yielded the following properties shown in Table 9B.

44TABLE 9BProtein Sequence Properties NOV9aSignalP analysis:No Known Signal Sequence PredictedPSORT II analysis:PSG: a new signal peptide prediction methodN-region: length 9; pos.chg 2; neg.chg 0H-region: length 3; peak value −16.59PSG score: −20.99GvH: von Heijne's method for signal seq. recognitionGvH score (threshold: −2.1): −2.21possible cleavage site: between 49 and 50>>> Seems to have no N-terminal signal peptideALOM: Klein et al's method for TM region allocationInit position for calculation: 1Tentative number of TMS(s) for the threshold 0.5: 1Number of TMS(s) for threshold 0.5: 0PERIPHERAL Likelihood = 5.46 (at 262)ALOM score: −0.11 (number of TMSs: 0)MITDISC: discrimination of mitochondrial targeting seqR content: 3Hyd Moment(75): 7.11Hyd Moment (95): 8.81G content: 1D/E content: 2S/T content: 3Score: −3.42Gavel: prediction of cleavage sites for mitochondrial preseq R-2 motifat 19 ARS|TTNUCDISC: discrimination of nuclear localization signalspat4: nonepat7: PALSRRK (3) at 163bipartite: nonecontent of basic residues: 11.4%NLS Score: −0.22KDEL: ER retention motif in the C-terminus: noneER Membrane Retention Signals: noneSKL: peroxisomal targeting signal in the C-terminus: nonePTS2: 2nd peroxisomal targeting signal: noneVAC: possible vacuolar targeting motif: noneRNA-binding motif: noneActinin-type actin-binding motif:type 1: nonetype 2: noneNMYR: N-myristoylation pattern: nonePrenylation motif: nonememYQRL: transport motif from cell surface to Golgi: noneTyrosines in the tail: noneDileucine motif in the tail: nonechecking 63 PROSITE DNA binding motifs: nonechecking 71 PROSITE ribosomal protein motifs: nonechecking 33 PROSITE prokaryotic DNA binding motifs: noneNNCN: Reinhardt's method for Cytoplasmic/NucleardiscriminationPrediction: nuclearReliability: 89COIL: Lupas's algorithm to detect coiled-coil regionstotal: 0 residuesFinal Results (k = 9/23): 69.6%: nuclear 26.1%: mitochondrial 4.3%: cytoplasmic >> prediction for CG164521-01 is nuc (k = 23)

[0398] A search of the NOV9a protein against the GENESEQ database, a proprietary database that contains sequences published in patents and patent publication, yielded several homologous proteins shown in Table 9C.

45TABLE 9CGENESEQ Results for NOV9aNOV9aIdentities/Residues/Similarities forGENESEQProtein/Organism/LengthMatchthe MatchedExpectIdentifier[Patent #, Date]ResiduesRegionValueAAU75902Mouse inhibitory PAS domain (IPAS) 1 . . . 311250/313 (79%) e−140protein - Mus musculus, 307 aa. 1 . . . 307264/313 (83%)[WO200202609-A1, 10-JAN-2002]AAE24222Human HIF-3 protein - Homo sapiens, 6 . . . 241177/242 (73%)1e−87667 aa. [WO200234291-A2, 4 . . . 225183/242 (75%)02-MAY-2002]ABG66737Human novel polypeptide #72 - Homo 10 . . . 241175/238 (73%)7e−87sapiens, 407 aa. [WO200244340-A2, 54 . . . 271181/238 (75%)06-JUN-2002]AAB94934Human protein sequence SEQ ID 57 . . . 241128/191 (67%)5e−60NO: 16415 - Homo sapiens, 648 aa. 1 . . . 171134/191 (70%)[EP1074617-A2, 07-FEB-2001]AAB93710Human protein sequence SEQ ID114 . . . 241108/128 (84%)1e−56NO: 13303 - Homo sapiens, 199 aa. 40 . . . 158111/128 (86%)[EP1074617-A2, 07-FEB-2001]

[0399] In a BLAST search of public sequence databases, the NOV9a protein was found to have homology to the proteins shown in the BLASTP data in Table 9D.

46TABLE 9DPublic BLASTP Results for NOV9aNOV9aIdentities/ProteinResidues/Similarities forAccessionMatchthe MatchedExpectNumberProtein/Organism/LengthResiduesPortionValueQ8VHR1Inhibitory PAS domain protein - Mus 1 . . . 311250/313 (79%) e−140musculus (Mouse), 307 aa. 1 . . . 307264/313 (83%)Q8WXA1Inhibitory PAS domain protein - Homo 1 . . . 241184/247 (74%)2e−91sapiens (Human), 632 aa. 1 . . . 227190/247 (76%)Q9Y2N7Putative homolog of hypoxia inducible 6 . . . 241177/242 (73%)4e−87factor three alpha (Hypoxia- inducible 4 . . . 225183/242 (75%)factor-3 alpha) - Homo sapiens (Human),667 aa.Q9HAI2Hypothetical protein - Homo sapiens 57 . . . 241128/191 (67%)1e−59(Human), 648 aa. 1 . . . 171134/191 (70%)Q9HAM5Hypothetical protein - Homo sapiens114 . . . 241108/128 (84%)4e−56(Human), 199 aa. 40 . . . 158111/128 (86%)

[0400] PFam analysis predicts that the NOV9a protein contains the domains shown in the Table 9E.

47TABLE 9EDomain Analysis of NOV9aPfamNOV9a MatchIdentities/Expect ValueDomainRegionSimilaritiesfor the Matched Region

Example B: Sequencing Methodology and Identification of NOVX Clones

[0401] 1. GeneCalling™ Technology: This is a proprietary method of performing differential gene expression profiling between two or more samples developed at CuraGen and described by Shimkets, et al., “Gene expression analysis by transcript profiling coupled to a gene database query” Nature Biotechnology 17:198-803 (1999). cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then digested with up to as many as 120 pairs of restriction enzymes and pairs of linker-adaptors specific for each pair of restriction enzymes were ligated to the appropriate end. The restriction digestion generates a mixture of unique cDNA gene fragments. Limited PCR amplification is performed with primers homologous to the linker adapter sequence where one primer is biotinylated and the other is fluorescently labeled. The doubly labeled material is isolated and the fluorescently labeled single strand is resolved by capillary gel electrophoresis. A computer algorithm compares the electropherograms from an experimental and control group for each of the restriction digestions. This and additional sequence-derived information is used to predict the identity of each differentially expressed gene fragment using a variety of genetic databases. The identity of the gene fragment is confirmed by additional, gene-specific competitive PCR or by isolation and sequencing of the gene fragment.

[0402] 2. SeqCalling™ Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

[0403] 3. PathCalling™ Technology: The NOVX nucleic acid sequences are derived by laboratory screening of cDNA library by the two-hybrid approach. cDNA fragments covering either the full length of the DNA sequence, or part of the sequence, or both, are sequenced. In silico prediction was based on sequences available in CuraGen Corporation's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.

[0404] The laboratory screening was performed using the methods summarized below:

[0405] cDNA libraries were derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, primary cells or tissue cultured primary cells or cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression, for example, growth factors, chemokines or steroids. The cDNA thus derived was then directionally cloned into the appropriate two-hybrid vector (Ga14-activation domain (Ga14-AD) fusion). Such cDNA libraries as well as commercially available cDNA libraries from Clontech (Palo Alto, Calif.) were then transferred from E.coli into a CuraGen Corporation proprietary yeast strain (disclosed in U.S. Pat. Nos. 6,057,101 and 6,083,693, incorporated herein by reference in their entireties).

[0406] Ga14-binding domain (Ga14-BD) fusions of a CuraGen Corporation proprietary library of human sequences was used to screen multiple Ga14-AD fusion cDNA libraries resulting in the selection of yeast hybrid diploids in each of which the Ga14-AD fusion contains an individual cDNA. Each sample was amplified using the polymerase chain reaction (PCR) using non-specific primers at the cDNA insert boundaries. Such PCR product was sequenced; sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled together, sometimes including public human sequences, using bioinformatic programs to produce a consensus sequence for each assembly. Each assembly is included in CuraGen Corporation's database. Sequences were included as components for assembly when the extent of identity with another component was at least 95% over 50 bp. Each assembly represents a gene or portion thereof and includes information on variants, such as splice forms single nucleotide polymorphisms (SNPs), insertions, deletions and other sequence variations.

[0407] Physical clone: the cDNA fragment derived by the screening procedure, covering the entire open reading frame is, as a recombinant DNA, cloned into pACT2 plasmid (Clontech) used to make the cDNA library. The recombinant plasmid is inserted into the host and selected by the yeast hybrid diploid generated during the screening procedure by the mating of both CuraGen Corporation proprietary yeast strains N106′ and YULH (U.S. Pat. Nos. 6,057,101 and 6,083,693).

[0408] 4. RACE: Techniques based on the polymerase chain reaction such as rapid amplification of cDNA ends (RACE), were used to isolate or complete the predicted sequence of the cDNA of the invention. Usually multiple clones were sequenced from one or more human samples to derive the sequences for fragments. Various human tissue samples from different donors were used for the RACE reaction. The sequences derived from these procedures were included in the SeqCalling Assembly process described in preceding paragraphs.

[0409] 5. Exon Linking: The NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain —amygdala, brain —cerebellum, brain —hippocampus, brain —substantia nigra, brain —thalamus, brain —whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma —Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.

[0410] 6. Physical Clone: Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.

[0411] The PCR product derived by exon linking, covering the entire open reading frame, was cloned into the pCR2.1 vector from Invitrogen to provide clones used for expression and screening purposes.

Example C: Quantitative Expression Analysis of Clones in Various Cells and Tissues

[0412] The quantitative expression of various clones was assessed using microfiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on an Applied Biosystems ABI PRISM® 7700 or an ABI PRISM® 7900 HT Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), AI_comprehensive_panel (containing normal tissue and samples from autoinflammatory diseases), Panel CNSD.01 (containing samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and Alzheimer's diseased brains).

[0413] RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s: 18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.

[0414] First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (Applied Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions.

[0415] In other cases, non-normalized RNA samples were converted to single strand cDNA (sscDNA) using Superscript II (Invitrogen Corporation; Catalog No. 18064-147) and random hexamers according to the manufacturer's instructions. Reactions containing up to 10 μg of total RNA were performed in a volume of 20 μl and incubated for 60 minutes at 42° C. This reaction can be scaled up to 50 μg of total RNA in a final volume of 100 μl. sscDNA samples are then normalized to reference nucleic acids as described previously, using 1×TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions.

[0416] Probes and primers were designed for each assay according to Applied Biosystems Primer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration=250 nM, primer melting temperature (Tm) range=58′-60° C., primer optimal Tm=59° C., maximum primer difference=2° C., probe does not have 5′G, probe Tm must be 10° C. greater than primer Tm, amplicon size 75bp to I00bp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, Tex., USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5′ and 3′ ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200 nM.

[0417] PCR conditions: When working with RNA samples, normalized RNA from each tissue and each cell line was spotted in each well of either a 96 well or a 384-well PCR plate (Applied Biosystems). PCR cocktails included either a single gene specific probe and primers set, or two multiplexed probe and primers sets (a set specific for the target clone and another gene-specific set multiplexed with the target probe). PCR reactions were set up using TaqMan® One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No. 4313803) following manufacturer's instructions. Reverse transcription was performed at 48° C. for 30 minutes followed by amplification/PCR cycles as follows: 95° C. 10 min, then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.

[0418] When working with sscDNA samples, normalized sscDNA was used as described previously for RNA samples. PCR reactions containing one or two sets of probe and primers were set up as described previously, using 1×TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions. PCR amplification was performed as follows: 95° C. 10 min, then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute. Results were analyzed and processed as described previously.

[0419] Panels 1, 1.1, 1.2, and 1.3D

[0420] The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in these panels are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in these panels are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on these panels are comprised of samples derived from all major organ systems from single adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.

[0421] In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used:

[0422] ca.=carcinoma,

[0423] *=established from metastasis,

[0424] met=metastasis,

[0425] s cell var=small cell variant,

[0426] non-s=non-sm=non-small,

[0427] squam=squamous,

[0428] pl. eff=pl effusion=pleural effusion,

[0429] glio=glioma,

[0430] astro=astrocytoma, and

[0431] neuro=neuroblastoma.

[0432] General_screening_panel_v1.4, v1.5, v1.6 and 1.7

[0433] The plates for Panels 1.4, 1.5, 1.6 and 1.7 include 2 control wells (genomic DNA control and chemistry control) and 88 to 94 wells containing cDNA from various samples. The samples in Panels 1.4, 1.5, 1.6 and 1.7 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panels 1.4, 1.5, 1.6 and 1.7 are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panels 1.4, 1.5, 1.6 and 1.7 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose. Abbreviations are as described for Panels 1, 1.1, 1.2, and 1.3D.

[0434] Panels 2D, 2.2, 2.3 and 2.4

[0435] The plates for Panels 2D, 2.2, 2.3 and 2.4 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI) or from Ardais or Clinomics). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have “matched margins” obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted “NAT” in the results below. The tumor tissue and the “matched margins” are evaluated by two independent pathologists (the surgical pathologists and again by a pathologist at NDRI/CHTN/Ardais/Clinomics). Unmatched RNA samples from tissues without malignancy (normal tissues) were also obtained from Ardais or Clinomics. This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue surrounding (i.e. immediately proximal) to the zone of surgery (designated “NAT”, for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, Calif.), Research Genetics, and Invitrogen.

[0436] HASS Panel v 1.0

[0437] The HASS panel v 1.0 plates are comprised of 93 cDNA samples and two controls. Specifically, 81 of these samples are derived from cultured human cancer cell lines that had been subjected to serum starvation, acidosis and anoxia for different time periods as well as controls for these treatments, 3 samples of human primary cells, 9 samples of malignant brain cancer (4 medulloblastomas and 5 glioblastomas) and 2 controls. The human cancer cell lines are obtained from ATCC (American Type Culture Collection) and fall into the following tissue groups: breast cancer, prostate cancer, bladder carcinomas, pancreatic cancers and CNS cancer cell lines. These cancer cells are all cultured under standard recommended conditions. The treatments used (serum starvation, acidosis and anoxia) have been previously published in the scientific literature. The primary human cells were obtained from Clonetics (Walkersville, Md.) and were grown in the media and conditions recommended by Clonetics. The malignant brain cancer samples are obtained as part of a collaboration (Henry Ford Cancer Center) and are evaluated by a pathologist prior to CuraGen receiving the samples. RNA was prepared from these samples using the standard procedures. The genomic and chemistry control wells have been described previously.

[0438] ARDAIS Panel v 1.0

[0439] The plates for ARDAIS panel v 1.0 generally include 2 control wells and 22 test samples composed of RNA isolated from human tissue procured by surgeons working in close cooperation with Ardais Corporation. The tissues are derived from human lung malignancies (lung adenocarcinoma or lung squamous cell carcinoma) and in cases where indicated many malignant samples have “matched margins” obtained from noncancerous lung tissue just adjacent to the tumor. These matched margins are taken from the tissue surrounding (i.e. immediately proximal) to the zone of surgery (designated “NAT”, for normal adjacent tissue) in the results below. The tumor tissue and the “matched margins” are evaluated by independent pathologists (the surgical pathologists and again by a pathologist at Ardais). Unmatched malignant and non-malignant RNA samples from lungs were also obtained from Ardais. Additional information from Ardais provides a gross histopathological assessment of tumor differentiation grade and stage. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical state of the patient.

[0440] Panel 3D, 3.1 and 3.2

[0441] The plates of Panel 3D, 3.1, and 3.2 are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma of the tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D, 3.1, 3.2, 1, 1.1., 1.2, 1.3D, 1.4, 1.5, and 1.6 are of the most common cell lines used in the scientific literature.

[0442] Panels 4D, 4R, and 4.1D

[0443] Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, Calif.) and thymus and kidney (Clontech) was employed. Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc., Hayward, Calif.). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, Pa.).

[0444] Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, Md.) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.

[0445] Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using Ficoll. LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco/Life Technologies, Rockville, Md.), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20 ng/ml PMA and 1-2 μg/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2×106cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5×10−5M) (Gibco), and 10 mM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1-7 days for RNA preparation.

[0446] Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions.

[0447] Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 10 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 μg/ml for 6 and 12-14 hours.

[0448] CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. CD45RO beads were then used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and plated at 106 cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 μg/ml anti-CD28 (Pharmingen) and 3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days of the second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.

[0449] To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 nM Hepes (Gibco). To activate the cells, we used PWM at 5 g/ml or anti-CD40 (Pharmingen) at approximately 10 μg/ml and IL-4 at 5-10 ng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.

[0450] To prepare the primary and secondary Th1/Th2 and Tr1 cells, six-well Falcon plates were coated overnight with 10 μg/ml anti-CD28 (Pharmingen) and 2 μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, German Town, Md.) were cultured at 105-106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL4 (1 μg/ml) were used to direct to Th1, while IL-4 (5 ng/ml) and anti-IFN gamma (1 μg/ml) were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct to Tr1. After 4-5 days, the activated Th1, Th2 and Tr1 lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 M non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated Th1, Th2 and Tr1 lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (1 μg/ml) to prevent apoptosis. After 4-5 days, the Th1, Th2 and Tr1 lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Th1 and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Th1, Th2 and Tr1 after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.

[0451] The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5×10−5 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5×10−5 cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 1 mM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 μg/ml for 6 and 14 hours. Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 1 mM Hepes (Gibco). CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.

[0452] For these cell lines and blood cells, RNA was prepared by lysing approximately 107 cells/ml using Trizol (Gibco BRL). Briefly, {fraction (1/10)} volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at −20° C. overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300 μl of RNAse-free water and 35 μl buffer (Promega) 5 μl DTT, 7 μl RNAsin and 8 μl DNAse were added. The tube was incubated at 37° C. for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with {fraction (1/10)} volume of 3M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at −80° C.

[0453] AI_comprehensive Panel_v1.0

[0454] The plates for Al_comprehensive panel_v1.0 include two control wells and 89 test samples comprised of cDNA isolated from surgical and postmortem human tissues obtained from the Backus Hospital and Clinomics (Frederick, Md.). Total RNA was extracted from tissue samples from the Backus Hospital in the Facility at CuraGen. Total RNA from other tissues was obtained from Clinomics.

[0455] Joint tissues including synovial fluid, synovium, bone and cartilage were obtained from patients undergoing total knee or hip replacement surgery at the Backus Hospital. Tissue samples were immediately snap frozen in liquid nitrogen to ensure that isolated RNA was of optimal quality and not degraded. Additional samples of osteoarthritis and rheumatoid arthritis joint tissues were obtained from Clinomics. Normal control tissues were supplied by Clinomics and were obtained during autopsy of trauma victims.

[0456] Surgical specimens of psoriatic tissues and adjacent matched tissues were provided as total RNA by Clinomics. Two male and two female patients were selected between the ages of 25 and 47. None of the patients were taking prescription drugs at the time samples were isolated.

[0457] Surgical specimens of diseased colon from patients with ulcerative colitis and Crohn's disease and adjacent matched tissues were obtained from Clinomics. Bowel tissue from three female and three male Crohn's patients between the ages of 41-69 were used. Two patients were not on prescription medication while the others were taking dexamethasone, phenobarbital, or tylenol. Ulcerative colitis tissue was from three male and four female patients. Four of the patients were taking lebvid and two were on phenobarbital.

[0458] Total RNA from post mortem lung tissue from trauma victims with no disease or with emphysema, asthma or COPD was purchased from Clinomics. Emphysema patients ranged in age from 40-70 and all were smokers, this age range was chosen to focus on patients with cigarette-linked emphysema and to avoid those patients with alpha-lanti-trypsin deficiencies. Asthma patients ranged in age from 36-75, and excluded smokers to prevent those patients that could also have COPD. COPD patients ranged in age from 35-80 and included both smokers and non-smokers. Most patients were taking corticosteroids, and bronchodilators.

[0459] In the labels employed to identify tissues in the AI_comprehensive panel_v1.0 panel, the following abbreviations are used:

[0460] AI=Autoimmunity

[0461] Syn=Synovial

[0462] Normal=No apparent disease

[0463] Rep22/Rep20=individual patients

[0464] RA=Rheumatoid arthritis

[0465] Backus=From Backus Hospital

[0466] OA=Osteoarthritis

[0467] (SS) (BA) (MF)=Individual patients

[0468] Adj=Adjacent tissue

[0469] Match control=adjacent tissues

[0470] -M=Male

[0471] -F=Female

[0472] COPD=Chronic obstructive pulmonary disease

[0473] AI.05 Chondrosarcoma The AI.05 chondrosarcoma plates are comprised of SW1353 cells that had been subjected to serum starvation and treatment with cytokines that are known to induce MMP (1, 3 and 13) synthesis (e.g. IL1beta). These treatments include: IL-1beta (10 ng/ml), IL-1beta+ TNF-alpha (50 ng/ml), IL-1beta+ Oncostatin (50 ng/ml) and PMA (100 ng/ml). The SW1353 cells were obtained from the ATCC (American Type Culture Collection) and were all cultured under standard recommended conditions. The SW1353 cells were plated at 3×10−5 cells/ml (in DMEM medium-10% FBS) in 6-well plates. The treatment was done in triplicate, for 6 and 18 h. The supernatants were collected for analysis of MMP 1, 3 and 13 production and for RNA extraction. RNA was prepared from these samples using the standard procedures.

[0474] Panels 5D and 5I

[0475] The plates for Panel 5D and 5I include two control wells and a variety of cDNAs isolated from human tissues and cell lines with an emphasis on metabolic diseases. Metabolic tissues were obtained from patients enrolled in the Gestational Diabetes study. Cells were obtained during different stages in the differentiation of adipocytes from human mesenchymal stem cells. Human pancreatic islets were also obtained.

[0476] In the Gestational Diabetes study subjects are young (18-40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective) Caesarean section. After delivery of the infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<1 cc) of the exposed metabolic tissues during the closure of each surgical level. The biopsy material was rinsed in sterile saline, blotted and fast frozen within 5 minutes from the time of removal. The tissue was then flash frozen in liquid nitrogen and stored, individually, in sterile screw-top tubes and kept on dry ice for shipment to or to be picked up by CuraGen. The metabolic tissues of interest include uterine wall (smooth muscle), visceral adipose, skeletal muscle (rectus) and subcutaneous adipose. Patient descriptions are as follows:

[0477] Patient 2: Diabetic Hispanic, overweight, not on insulin

[0478] Patient 7-9: Nondiabetic Caucasian and obese (BMI>30)

[0479] Patient 10: Diabetic Hispanic, overweight, on insulin

[0480] Patient 11: Nondiabetic African American and overweight

[0481] Patient 12: Diabetic Hispanic on insulin

[0482] Adiocyte differentiation was induced in donor progenitor cells obtained from Osirus (a division of Clonetics/BioWhittaker) in triplicate, except for Donor 3U which had only two replicates. Scientists at Clonetics isolated, grew and differentiated human mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol found in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells Science Apr 21999: 143-147. Clonetics provided Trizol lysates or frozen pellets suitable for mRNA isolation and ds cDNA production. A general description of each donor is as follows:

[0483] Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated Adipose

[0484] Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated

[0485] Donor 2 and 3 AD: Adipose, Adipose Differentiated

[0486] Human cell lines were generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: kidney proximal convoluted tubule, uterine smooth muscle cells, small intestine, liver HepG2 cancer cells, heart primary stromal cells, and adrenal cortical adenoma cells. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. All samples were processed at CuraGen to produce single stranded cDNA.

[0487] Panel 5I contains all samples previously described with the addition of pancreatic islets from a 58 year old female patient obtained from the Diabetes Research Institute at the University of Miami School of Medicine. Islet tissue was processed to total RNA at an outside source and delivered to CuraGen for addition to panel 5I.

[0488] In the labels employed to identify tissues in the 5D and 5I panels, the following abbreviations are used:

[0489] GO Adipose=Greater Omentum Adipose

[0490] SK=Skeletal Muscle

[0491] UT=Uterus

[0492] PL=Placenta

[0493] AD=Adipose Differentiated

[0494] AM=Adipose Midway Differentiated

[0495] U=Undifferentiated Stem Cells

[0496] Panel CNSD.01

[0497] The plates for Panel CNSD.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at −80° C. in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.

[0498] Disease diagnoses are taken from patient records. The panel contains two brains from each of the following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy, Depression, and “Normal controls”. Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration of the substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.

[0499] In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:

[0500] PSP=Progressive supranuclear palsy

[0501] Sub Nigra=Substantia nigra

[0502] Glob Palladus=Globus palladus

[0503] Temp Pole=Temporal pole

[0504] Cing Gyr=Cingulate gyrus

[0505] BA 4=Brodman Area 4

[0506] Panel CNS_Neurodegeneration_V1.0

[0507] The plates for Panel CNS_Neurodegeneration_V1.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at −80° C. in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.

[0508] Disease diagnoses are taken from patient records. The panel contains six brains from Alzheimer's disease (AD) patients, and eight brains from “Normal controls” who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0=no evidence of plaques, 3=severe AD senile plaque load). Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Brodman Area 21), parietal cortex (Brodman area 7), and occipital cortex (Brodman area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages of the disease; the occipital cortex is spared in AD and therefore acts as a “control” region within AD patients. Not all brain regions are represented in all cases.

[0509] In the labels employed to identify tissues in the CNS_Neurodegeneration_V1.0 panel, the following abbreviations are used:

[0510] AD=Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy

[0511] Control=Control brains; patient not demented, showing no neuropathology

[0512] Control (Path)=Control brains; patient not demented but showing sever AD-like pathology

[0513] SupTemporal Ctx=Superior Temporal Cortex

[0514] Inf Temporal Ctx=Inferior Temporal Cortex

[0515] A. CG161998-01: Ringo.

[0516] Expression of gene CG 161998-01 was assessed using the primer-probe set Ag5768, described in Table AA. Results of the RTQ-PCR runs are shown in Table AB.

48TABLE AAProbe Name Ag5768StartSEQ IDPrimersLengthPositionNoForward5′-ccaggaattcctctccaaag-3′2019321ProbeTET-5′-cccctgcttccagatttcagataagt-3′-TAMRA2621422Reverse5′-tggaagtagaccagcaccat-3′2025123

[0517]

49

TABLE AB

General screening panel v1.5

Rel.

Rel.

Exp.(%)

Exp.(%)

Ag5768,

Ag5768,

Run

Run

Tissue Name
246263988
Tissue Name
246263988

Adipose
0.0
Renal ca. TK-10
27.0

Melanoma* Hs688(A).T
0.0
Bladder
1.4

Melanoma* Hs688(B).T
0.5
Gastric ca. (liver met.) NCI-N87
0.0

Melanoma* M14
0.0
Gastric ca. KATO III
30.4

Melanoma* LOXIMVI
0.0
Colon ca. SW-948
5.7

Melanoma* SK-MEL-5
0.5
Colon ca. SW480
2.1

Squamous cell carcinoma SCC-4
100.0
Colon ca.* (SW480 met) SW620
38.4

Testis Pool
6.4
Colon ca. HT29
0.0

Prostate ca.* (bone met) PC-3
0.0
Colon ca. HCT-116
47.6

Prostate Pool
0.0
Colon ca. CaCo-2
1.7

Placenta
5.3
Colon cancer tissue
0.4

Uterus Pool
0.0
Colon ca. SW1116
0.0

Ovarian ca. OVCAR-3
0.3
Colon ca. Colo-205
0.7

Ovarian ca. SK-OV-3
0.0
Colon ca. SW-48
1.5

Ovarian ca. OVCAR-4
0.4
Colon Pool
0.0

Ovarian ca. OVCAR-5
2.1
Small Intestine Pool
0.0

Ovarian ca. IGROV-1
9.5
Stomach Pool
0.0

Ovarian ca. OVCAR-8
0.0
Bone Marrow Pool
0.0

Ovary
0.0
Fetal Heart
0.0

Breast ca. MCF-7
0.3
Heart Pool
0.0

Breast ca. MDA-MB-231
0.0
Lymph Node Pool
0.0

Breast ca. BT 549
0.0
Fetal Skeletal Muscle
0.0

Breast ca. T47D
4.4
Skeletal Muscle Pool
0.0

Breast ca. MDA-N
0.0
Spleen Pool
0.0

Breast Pool
0.0
Thymus Pool
0.0

Trachea
0.0
CNS cancer (glio/astro) U87-MG
0.0

Lung
0.0
CNS cancer (glio/astro) U-118-MG
0.0

Fetal Lung
1.1
CNS cancer (neuro;met) SK-N-AS
0.3

Lung ca. NCI-N417
0.0
CNS cancer (astro) SF-539
0.0

Lung ca. LX-1
32.8
CNS cancer (astro) SNB-75
0.0

Lung ca. NCI-H146
0.5
CNS cancer (glio) SNB-19
6.8

Lung ca. SHP-77
0.0
CNS cancer (glio) SF-295
0.0

Lung ca. A549
0.0
Brain (Amygdala) Pool
0.0

Lung ca. NCI-H526
0.0
Brain (cerebellum)
0.4

Lung ca. NCI-H23
0.4
Brain (fetal)
0.0

Lung ca. NCI-H460
0.0
Brain (Hippocampus) Pool
0.0

Lung ca. HOP-62
0.0
Cerebral Cortex Pool
0.0

Lung ca. NCI-H522
0.5
Brain (Substantia nigra) Pool
0.0

Liver
6.9
Brain (Thalamus) Pool
0.3

Fetal Liver
4.6
Brain (whole)
0.4

Liver ca. HepG2
68.3
Spinal Cord Pool
0.0

Kidney Pool
0.0
Adrenal Gland
0.0

Fetal Kidney
0.4
Pituitary gland Pool
0.0

Renal ca. 786-0
0.0
Salivary Gland
0.0

Renal ca. A498
0.0
Thyroid (female)
0.0

Renal ca. ACHN
0.0
Pancreatic ca. CAPAN2
0.0

Renal ca. UO-31
0.0
Pancreas Pool
0.0

[0518] CNS_neurodegeneration_v1.0 Summary: Ag5768 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

[0519] General_screening_panel_v1.5 Summary: Ag5768 Highest expression of this gene is seen in a squamous cell carcinoma cell line (CT=30). Moderate levels of expression are also seen in cell lines derived from colon cancer, liver cancer, renal cancer, lung cancer, and gastric cancer. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of these cancers.

[0520] Panel 4.1D Summary: Ag5768 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

[0521] B. CG162071-01: Downstream of Tyrosine Kinase 5 (DOK5).

[0522] Expression of gene CG162071-01 was assessed using the primer-probe set Ag5822, described in Table BA. Results of the RTQ-PCR runs are shown in Tables BB, BC and BD.

50TABLE BAProbe Name Ag5822StartSEQPrimersLengthPositionID NoForward5′-ccagatgaaaaggcagctta-3′2033224ProbeTET-5′-cagaaactttcataaggtaactgaactgca-3′-TAMRA3035525Reverse5′-cggggcagtctggttatatt-3′2039526

[0523]

51

TABLE BB

CNS neurodegeneration v1.0

Rel.

Rel.

Exp.(%)

Exp.(%)

Ag5822,

Ag5822,

Run

Run

Tissue Name
247108837
Tissue Name
247108837

AD 1 Hippo
17.3
Control (Path) 3 Temporal Ctx
5.2

AD 2 Hippo
44.4
Control (Path) 4 Temporal Ctx
37.1

AD 3 Hippo
18.3
AD 1 Occipital Ctx
8.8

AD 4 Hippo
13.5
AD 2 Occipital Ctx (Missing)
0.0

AD 5 Hippo
56.6
AD 3 Occipital Ctx
3.1

AD 6 Hippo
58.2
AD 4 Occipital Ctx
15.8

Control 2 Hippo
58.6
AD 5 Occipital Ctx
50.0

Control 4 Hippo
9.1
AD 6 Occipital Ctx
9.3

Control (Path) 3 Hippo
6.3
Control 1 Occipital Ctx
1.7

AD 1 Temporal Ctx
11.3
Control 2 Occipital Ctx
39.2

AD 2 Temporal Ctx
38.2
Control 3 Occipital Ctx
15.4

AD 3 Temporal Ctx
5.2
Control 4 Occipital Ctx
3.6

AD 4 Temporal Ctx
23.8
Control (Path) 1 Occipital Ctx
71.7

AD 5 Inf Temporal Ctx
73.7
Control (Path) 2 Occipital Ctx
7.4

AD 5 Sup Temporal Ctx
51.1
Control (Path) 3 Occipital Ctx
1.4

AD 6 Inf Temporal Ctx
26.6
Control (Path) 4 Occipital Ctx
10.8

AD 6 Sup Temporal Ctx
24.0
Control 1 Parietal Ctx
5.4

Control 1 Temporal Ctx
5.8
Control 2 Parietal Ctx
31.2

Control 2 Temporal Ctx
62.9
Control 3 Parietal Ctx
16.4

Control 3 Temporal Ctx
27.5
Control (Path) 1 Parietal Ctx
100.0

Control 3 Temporal Ctx
9.0
Control (Path) 2 Parietal Ctx
19.9

Control (Path) 1 Temporal Ctx
97.9
Control (Path) 3 Parietal Ctx
2.4

Control (Path) 2 Temporal Ctx
40.9
Control (Path) 4 Parietal Ctx
45.4

[0524]

52

TABLE BC

General screening panel v1.5

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5822,

Ag5822,

Run

Run

Tissue Name
246273264
Tissue Name
246273264

Adipose
3.3
Renal ca. TK-10
23.3

Melanoma* Hs688(A).T
25.9
Bladder
2.7

Melanoma* Hs688(B).T
7.4
Gastric ca. (liver met.) NCI-N87
0.0

Melanoma* M14
0.0
Gastric ca. KATO III
0.0

Melanoma* LOXIMVI
0.0
Colon ca. SW-948
0.0

Melanoma* SK-MEL-5
3.1
Colon ca. SW480
0.0

Squamous cell carcinoma SCC-4
0.0
Colon ca.* (SW480 met) SW620
0.0

Testis Pool
1.4
Colon ca. HT29
0.0

Prostate ca.* (bone met) PC-3
0.0
Colon ca. HCT-116
0.0

Prostate Pool
2.3
Colon ca. CaCo-2
0.0

Placenta
4.7
Colon cancer tissue
2.4

Uterus Pool
3.0
Colon ca. SW1116
0.0

Ovarian ca. OVCAR-3
0.1
Colon ca. Colo-205
0.0

Ovarian ca. SK-OV-3
0.1
Colon ca. SW-48
0.0

Ovarian ca. OVCAR-4
0.0
Colon Pool
16.7

Ovarian ca. OVCAR-5
0.0
Small Intestine Pool
4.3

Ovarian ca. IGROV-1
0.5
Stomach Pool
4.8

Ovarian ca. OVCAR-8
3.9
Bone Marrow Pool
3.0

Ovary
12.7
Fetal Heart
8.5

Breast ca. MCF-7
0.3
Heart Pool
5.5

Breast ca. MDA-MB-231
0.0
Lymph Node Pool
11.0

Breast ca. BT 549
46.3
Fetal Skeletal Muscle
2.4

Breast ca. T47D
0.0
Skeletal Muscle Pool
1.4

Breast ca. MDA-N
0.0
Spleen Pool
2.2

Breast Pool
11.9
Thymus Pool
5.2

Trachea
2.4
CNS cancer (glio/astro) U87-MG
3.2

Lung
2.5
CNS cancer (glio/astro) U-118-MG
19.3

Fetal Lung
59.0
CNS cancer (neuro; met) SK-N-AS
43.8

Lung ca. NCI-N417
10.6
CNS cancer (astro) SF-539
7.1

Lung ca. LX-1
0.0
CNS cancer (astro) SNB-75
4.1

Lung ca. NCI-H146
44.8
CNS cancer (glio) SNB-19
0.5

Lung ca. SHP-77
100.0
CNS cancer (glio) SF-295
0.8

Lung ca. A549
0.0
Brain (Amygdala) Pool
46.3

Lung ca. NCI-H526
4.9
Brain (cerebellum)
31.2

Lung ca. NCI-H23
0.2
Brain (fetal)
96.6

Lung ca. NCI-H460
25.2
Brain (Hippocampus) Pool
52.9

Lung ca. HOP-62
5.7
Cerebral Cortex Pool
46.3

Lung ca. NCI-H522
0.1
Brain (Substantia nigra) Pool
40.3

Liver
0.7
Brain (Thalamus) Pool
68.8

Fetal Liver
2.7
Brain (whole)
44.1

Liver ca. HepG2
52.5
Spinal Cord Pool
9.5

Kidney Pool
10.8
Adrenal Gland
1.5

Fetal Kidney
34.9
Pituitary gland Pool
1.6

Renal ca. 786-0
0.0
Salivary Gland
0.8

Renal ca. A498
10.7
Thyroid (female)
2.6

Renal ca. ACHN
0.0
Pancreatic ca. CAPAN2
0.0

Renal ca. UO-31
0.1
Pancreas Pool
14.5

[0525]

53

TABLE BD

Panel 4.1D

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5822,

Ag5822,

Run

Run

Tissue Name
246921320
Tissue Name
246921320

Secondary Th1 act
0.0
HUVEC IL-1beta
0.0

Secondary Th2 act
0.0
HUVEC IFN gamma
0.0

Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN gamma
0.0

Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
0.0

Secondary Th2 rest
0.0
HUVEC IL-11
0.0

Secondary Tr1 rest
0.0
Lung Microvascular EC none
1.8

Primary Th1 act
0.0
Lung Microvascular EC TNFalpha + IL-1beta
0.0

Primary Th2 act
0.0
Microvascular Dermal EC none
0.0

Primary Tr1 act
0.0
Microsvasular Dermal EC TNFalpha + IL-1beta
0.0

Primary Th1 rest
0.0
Bronchial epithelium TNFalpha + IL-1beta
0.0

Primary Th2 rest
0.0
Small airway epithelium none
0.9

Primary Tr1 rest
0.0
Small airway epithelium TNFalpha + IL-1beta
0.0

CD45RA CD4 lymphocyte act
4.6
Coronary artery SMC rest
0.5

CD45RO CD4 lymphocyte act
0.0
Coronary artery SMC TNFalpha + IL-1beta
1.9

CD8 lymphocyte act
0.0
Astrocytes rest
3.9

Secondary CD8 lymphocyte rest
0.0
Astrocytes TNFalpha + IL-1beta
2.7

Secondary CD8 lymphocyte act
0.0
KU-812 (Basophil) rest
0.0

CD4 lymphocyte none
0.0
KU-812 (Basophil) PMA/ionomycin
0.0

2ry Th1/Th2/Tr1_anti-CD95 CH11
0.0
CCD1106 (Keratinocytes) none
0.0

LAK cells rest
0.0
CCD1106 (Keratinocytes) TNFalpha + IL-1beta
0.0

LAK cells IL-2
0.0
Liver cirrhosis
6.5

LAK cells IL-2 + IL-12
0.0
NCI-H292 none
0.0

LAK cells IL-2 + IFN gamma
0.0
NCI-H292 IL-4
0.0

LAK cells IL-2 + IL-18
0.0
NCI-H292 IL-9
0.0

LAK cells PMA/ionomycin
0.0
NCI-H292 IL-13
0.0

NK Cells IL-2 rest
0.0
NCI-H292 IFN gamma
0.0

Two Way MLR 3 day
0.0
HPAEC none
0.0

Two Way MLR 5 day
0.0
HPAEC TNF alpha + IL-1beta
0.0

Two Way MLR 7 day
0.0
Lung fibroblast none
62.4

PBMC rest
0.0
Lung fibroblast TNF alpha + IL-
26.8

1beta

PBMC PWM
0.0
Lung fibroblast IL-4
42.0

PBMC PHA-L
0.0
Lung fibroblast IL-9
54.7

Ramos (B cell) none
0.0
Lung fibroblast IL-13
2.2

Ramos (B cell) ionomycin
0.0
Lung fibroblast IFN gamma
100.0

B lymphocytes PWM
0.9
Dermal fibroblast CCD1070 rest
4.8

B lymphocytes CD40L and IL-4
0.0
Dermal fibroblast CCD1070 TNF
8.5

alpha

EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070 IL-1
17.8

beta

EOL-1 dbcAMP PMA/ionomycin
0.0
Dermal fibroblast IFN gamma
6.9

Dendritic cells none
0.0
Dermal fibroblast IL-4
17.1

Dendritic cells LPS
0.0
Dermal Fibroblasts rest
24.7

Dendritic cells anti-CD40
0.0
Neutrophils TNFa + LPS
0.0

Monocytes rest
0.0
Neutrophils rest
0.0

Monocytes LPS
0.0
Colon
0.9

Macrophages rest
0.0
Lung
3.6

Macrophages LPS
0.0
Thymus
0.0

HUVEC none
0.0
Kidney
69.3

HUVEC starved
0.0

[0526] CNS_neurodegeneration_v1.0 Summary: Ag5822 This panel confirms the expression of this gene at low levels in the brain in an independent group of individuals. This gene appears to be slightly down-regulated in the temporal cortex of Alzheimer's disease patients. Therefore, up-regulation of this gene or its protein product, or treatment with specific agonists for this receptor may be of use in reversing the dementia, memory loss, and neuronal death associated with this disease.

[0527] General_screening_panel_v1.5 Summary: Ag5822 This gene appears to have a brain-preferential distribution, with moderate levels of expression across all regions of the CNS examined. Thus, this gene would be useful for distinguishing brain tissue from non-neural tissue, and may be beneficial as a drug target in neurodegenerative disease.

[0528] Among tissues with metabolic function, this gene is expressed at moderate to low levels in pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal skeletal muscle, heart, and liver. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

[0529] Moderate levels of expression are also seen in brain, liver, lung, renal, breast, and melanoma cancer cell lines. Thus, expression of this gene could be used to differentiate between these cell lines and other cell lines on this panel and as a marker of these cancers.

[0530] Panel 4.1D Summary: Ag5822 Highest expression of this gene is seen in IFN treated lung fibroblasts (CT=31.5). Moderate to low levels of expression are also seen in a cluster of cytokine activated lung fibroblasts samples, as well as in resting and IL-4 treated dermal fibroblasts, and kidney. Thus, expression of this gene could be used to differentiate lung fibroblasts from other samples on this panel and as a marker of these cells. Furthermore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of lung-inflammatory diseases.

[0531] C. CG162605-01 and CG162605-02: SREBP Cleavage-activating Protein.

[0532] Expression of gene CG162605-01 and CG162605-02 was assessed using the primer-probe sets Ag3883, Ag5850, Ag5952 and Ag7342, described in Tables CA, CB, CC and CD. Results of the RTQ-PCR runs are shown in Tables CE, CF, CG and CH. Please note that probe-primer set Ag5952 is specific for CG162605-01 and probe-primer set Ag5850 is specific for CG162605-02.

54TABLE CAProbe Name Ag3883StartSEQ IDPrimersLengthPositionNoForward5′-tggcttatgtccagcagatatt-3′2236727ProbeTET-5′-agtcctcagtgtttccctggcacaag-3′-TAMRA2639428Reverse5′-ggacaaaggtgaacgaaataca-3′2243729

[0533]

55

TABLE CB

Probe Name Ag5850

Start
SEQ ID

Primers

Length
Position
No

Forward
5′-gtgcacttcaaggaggagatt-3′
21
918
30

Probe
TET-5′-ccccttgtgaccacctacatcatcttg-3′-TAMRA
27
957
31

Reverse
5′-caagctccagatggagaagtag-3′
22
995
32

[0534]

56

TABLE CC

Probe Name Ag5952

Primers

Length
Start Position
SEQ ID No

Forward
5′-atcaggagcttggaggagtt-3′
20
516
33

Probe
TET-5′-acctgctgcagggccactca-3′-TAMRA
20
553
34

Reverse
5′-tcaatgtacacggtcgtgat-3′
20
579
35

[0535]

57

TABLE CD

Probe Name Ag7342

SEQ ID

Primers

Length
Start Position
No

Forward
5′-ctgctgcagggccact-3′
16
555
36

Probe
TET-5′-ccatcacgaccgtgtacattgac-3′-TAMRA
23
577
37

Reverse
5′-tggccccatcttgtcct-3′
17
623
38

[0536]

58

TABLE CE

General screening panel v1.4

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag3883,

Ag3883,

Run

Run

Tissue Name
217334263
Tissue Name
217334263

Adipose
2.7
Renal ca. TK-10
31.0

Melanoma* Hs688(A).T
19.3
Bladder
14.4

Melanoma* Hs688(B).T
21.5
Gastric ca. (liver met.) NCI-N87
17.1

Melanoma* M14
24.8
Gastric ca. KATO III
58.6

Melanoma* LOXIMVI
22.5
Colon ca. SW-948
9.9

Melanoma* SK-MEL-5
29.5
Colon ca. SW480
40.1

Squamous cell carcinoma SCC-4
9.8
Colon ca.* (SW480 met) SW620
20.4

Testis Pool
15.4
Colon ca. HT29
12.9

Prostate ca.* (bone met) PC-3
48.6
Colon ca. HCT-116
33.4

Prostate Pool
5.7
Colon ca. CaCo-2
40.1

Placenta
28.9
Colon cancer tissue
16.7

Uterus Pool
2.4
Colon ca. SW1116
7.4

Ovarian ca. OVCAR-3
37.1
Colon ca. Colo-205
8.8

Ovarian ca. SK-OV-3
23.0
Colon ca. SW-48
11.8

Ovarian ca. OVCAR-4
10.5
Colon Pool
18.0

Ovarian ca. OVCAR-5
32.5
Small Intestine Pool
13.2

Ovarian ca. IGROV-1
33.9
Stomach Pool
7.8

Ovarian ca. OVCAR-8
25.5
Bone Marrow Pool
5.0

Ovary
23.8
Fetal Heart
9.9

Breast ca. MCF-7
15.9
Heart Pool
6.1

Breast ca. MDA-MB-231
34.9
Lymph Node Pool
16.8

Breast ca. BT 549
35.6
Fetal Skeletal Muscle
5.7

Breast ca. T47D
64.2
Skeletal Muscle Pool
9.0

Breast ca. MDA-N
10.2
Spleen Pool
17.8

Breast Pool
16.6
Thymus Pool
14.3

Trachea
12.2
CNS cancer (glio/astro) U87-MG
20.9

Lung
2.5
CNS cancer (glio/astro) U-118-MG
52.5

Fetal Lung
23.2
CNS cancer (neuro; met) SK-N-AS
18.7

Lung ca. NCI-N417
9.0
CNS cancer (astro) SF-539
16.6

Lung ca. LX-1
29.1
CNS cancer (astro) SNB-75
58.2

Lung ca. NCI-H146
7.9
CNS cancer (glio) SNB-19
27.7

Lung ca. SHP-77
19.2
CNS cancer (glio) SF-295
65.5

Lung ca. A549
19.9
Brain (Amygdala) Pool
12.6

Lung ca. NCI-H526
13.6
Brain (cerebellum)
62.4

Lung ca. NCI-H23
33.0
Brain (fetal)
40.1

Lung ca. NCI-H460
15.4
Brain (Hippocampus) Pool
11.7

Lung ca. HOP-62
16.8
Cerebral Cortex Pool
15.1

Lung ca. NCI-H522
21.9
Brain (Substantia nigra) Pool
16.3

Liver
12.0
Brain (Thalamus) Pool
18.3

Fetal Liver
34.4
Brain (whole)
31.2

Liver ca. HepG2
33.7
Spinal Cord Pool
11.3

Kidney Pool
22.7
Adrenal Gland
100.0

Fetal Kidney
9.9
Pituitary gland Pool
4.0

Renal ca. 786-0
18.0
Salivary Gland
10.4

Renal ca. A498
4.2
Thyroid (female)
11.8

Renal ca. ACHN
13.6
Pancreatic ca. CAPAN2
15.3

Renal ca. UO-31
25.5
Pancreas Pool
23.7

[0537]

59

TABLE CF

General screening panel v1.5

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5850,

Ag5850,

Run

Run

Tissue Name
246273487
Tissue Name
246273487

Adipose
3.3
Renal ca. TK-10
48.6

Melanoma* Hs688(A).T
40.1
Bladder
11.4

Melanoma* Hs688(B).T
35.1
Gastric ca. (liver met.) NCI-N87
20.4

Melanoma* M14
35.1
Gastric ca. KATO III
47.6

Melanoma* LOXIMVI
36.3
Colon ca. SW-948
20.2

Melanoma* SK-MEL-5
33.2
Colon ca. SW480
47.0

Squamous cell carcinoma SCC-4
14.0
Colon ca.* (SW480 met) SW620
22.8

Testis Pool
15.7
Colon ca. HT29
17.9

Prostate ca.* (bone met) PC-3
22.5
Colon ca. HCT-116
65.5

Prostate Pool
4.2
Colon ca. CaCo-2
54.0

Placenta
23.3
Colon cancer tissue
11.0

Uterus Pool
2.7
Colon ca. SW1116
10.9

Ovarian ca. OVCAR-3
58.6
Colon ca. Colo-205
15.8

Ovarian ca. SK-OV-3
34.9
Colon ca. SW-48
20.6

Ovarian ca. OVCAR-4
6.4
Colon Pool
5.6

Ovarian ca. OVCAR-5
38.7
Small Intestine Pool
9.7

Ovarian ca. IGROV-1
36.6
Stomach Pool
6.1

Ovarian ca. OVCAR-8
25.7
Bone Marrow Pool
0.0

Ovary
16.5
Fetal Heart
11.8

Breast ca. MCF-7
15.3
Heart Pool
12.2

Breast ca. MDA-MB-231
25.0
Lymph Node Pool
17.0

Breast ca. BT 549
27.7
Fetal Skeletal Muscle
8.4

Breast ca. T47D
6.3
Skeletal Muscle Pool
14.4

Breast ca. MDA-N
11.4
Spleen Pool
11.9

Breast Pool
13.5
Thymus Pool
17.7

Trachea
14.6
CNS cancer (glio/astro) U87-MG
17.6

Lung
0.0
CNS cancer (glio/astro) U-118-MG
62.0

Fetal Lung
27.2
CNS cancer (neuro; met) SK-N-AS
33.9

Lung ca. NCI-N417
12.6
CNS cancer (astro) SF-539
13.2

Lung ca. LX-1
26.6
CNS cancer (astro) SNB-75
74.2

Lung ca. NCI-H146
21.8
CNS cancer (glio) SNB-19
46.7

Lung ca. SHP-77
18.8
CNS cancer (glio) SF-295
73.2

Lung ca. A549
27.4
Brain (Amygdala) Pool
11.0

Lung ca. NCI-H526
38.7
Brain (cerebellum)
31.9

Lung ca. NCI-H23
47.3
Brain (fetal)
27.2

Lung ca. NCI-H460
36.9
Brain (Hippocampus) Pool
6.3

Lung ca. HOP-62
23.8
Cerebral Cortex Pool
28.3

Lung ca. NCI-H522
26.1
Brain (Substantia nigra) Pool
23.2

Liver
5.4
Brain (Thalamus) Pool
28.1

Fetal Liver
25.0
Brain (whole)
36.6

Liver ca. HepG2
35.8
Spinal Cord Pool
6.3

Kidney Pool
20.6
Adrenal Gland
100.0

Fetal Kidney
12.1
Pituitary gland Pool
5.4

Renal ca. 786-0
27.7
Salivary Gland
6.5

Renal ca. A498
4.1
Thyroid (female)
8.2

Renal ca. ACHN
17.1
Pancreatic ca. CAPAN2
29.5

Renal ca. UO-31
37.4
Pancreas Pool
21.9

[0538]

60

TABLE CG

Panel 4.1D

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag7342,

Ag7342,

Run

Run

Tissue Name
305065595
Tissue Name
305065595

Secondary Th1 act
58.2
HUVEC IL-1beta
59.9

Secondary Th2 act
75.8
HUVEC IFN gamma
26.6

Secondary Tr1 act
40.9
HUVEC TNF alpha + IFN gamma
10.7

Secondary Th1 rest
8.3
HUVEC TNF alpha + IL4
15.3

Secondary Th2 rest
13.9
HUVEC IL-11
47.3

Secondary Tr1 rest
8.0
Lung Microvascular EC none
45.7

Primary Th1 act
15.4
Lung Microvascular EC TNFalpha + IL-1beta
7.3

Primary Th2 act
70.7
Microvascular Dermal EC none
20.3

Primary Tr1 act
70.7
Microsvasular Dermal EC TNFalpha + IL-1beta
5.9

Primary Th1 rest
6.5
Bronchial epithelium TNFalpha + IL-1beta
19.9

Primary Th2 rest
4.4
Small airway epithelium none
25.5

Primary Tr1 rest
5.1
Small airway epithelium TNFalpha + IL-1beta
56.6

CD45RA CD4 lymphocyte act
41.8
Coronary artery SMC rest
28.1

CD45RO CD4 lymphocyte act
30.8
Coronery artery SMC TNFalpha + IL-1beta
32.1

CD8 lymphocyte act
15.6
Astrocytes rest
16.6

Secondary CD8 lymphocyte rest
7.6
Astrocytes TNFalpha + IL-1beta
14.0

Secondary CD8 lymphocyte act
4.6
KU-812 (Basophil) rest
60.7

CD4 lymphocyte none
5.3
KU-812 (Basophil) PMA/ionomycin
19.9

2ry Th1/Th2/Tr1_anti-CD95
21.2
CCD1106 (Keratinocytes) none
61.1

CH11

LAK cells rest
3.0
CCD1106 (Keratinocytes) TNFalpha + IL-1beta
15.2

LAK cells IL-2
10.3
Liver cirrhosis
6.7

LAK cells IL-2 + IL-12
0.0
NCI-H292 none
54.0

LAK cells IL-2 + IFN gamma
10.2
NCI-H292 IL-4
62.0

LAK cells IL-2 + IL-18
6.3
NCI-H292 IL-9
95.9

LAK cells PMA/ionomycin
17.2
NCI-H292 IL-13
73.7

NK Cells IL-2 rest
54.3
NCI-H292 IFN gamma
36.1

Two Way MLR 3 day
14.7
HPAEC none
20.3

Two Way MLR 5 day
4.5
HPAEC TNF alpha + IL-1beta
34.4

Two Way MLR 7 day
7.9
Lung fibroblast none
38.4

PBMC rest
5.8
Lung fibroblast TNF alpha + IL-1beta
46.0

PBMC PWM
21.9
Lung fibroblast IL-4
19.3

PBMC PHA-L
17.7
Lung fibroblast IL-9
42.3

Ramos (B cell) none
23.3
Lung fibroblast IL-13
46.0

Ramos (B cell) ionomycin
100.0
Lung fibroblast IFN gamma
76.8

B lymphocytes PWM
20.2
Dermal fibroblast CCD1070 rest
60.3

B lymphocytes CD40L and IL-4
77.4
Dermal fibroblast CCD1070 TNF
79.0

alpha

EOL-1 dbcAMP
71.2
Dermal fibroblast CCD1070 IL-1beta
35.8

EOL-1 dbcAMP
9.2
Dermal fibroblast IFN gamma
25.9

PMA/ionomycin

Dendritic cells none
39.5
Dermal fibroblast IL-4
24.3

Dendritic cells LPS
4.1
Dermal Fibroblasts rest
28.9

Dendritic cells anti-CD40
3.5
Neutrophils TNFa + LPS
6.0

Monocytes rest
17.4
Neutrophils rest
73.2

Monocytes LPS
11.9
Colon
7.9

Macrophages rest
16.4
Lung
3.8

Macrophages LPS
5.7
Thymus
4.8

HUVEC none
52.5
Kidney
44.4

HUVEC starved
76.8

[0539]

61

TABLE CF

Panel 5D

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag3883,

Ag3883,

Run

Run

Tissue Name
170221180
Tissue Name
170221180

97457_Patient-02go_adipose
24.8
94709_Donor 2 AM - A_adipose
42.9

97476_Patient-07sk_skeletal
10.8
94710_Donor 2 AM - B_adipose
27.2

muscle

97477_Patient-07ut_uterus
29.9
94711_Donor 2 AM - C_adipose
26.6

97478_Patient-07pl_placenta
34.6
94712_Donor 2 AD - A_adipose
42.0

97481_Patient-08sk_skeletal
12.2
94713_Donor 2 AD - B_adipose
58.6

muscle

97482_Patient-08ut_uterus
15.0
94714_Donor 2 AD - C_adipose
40.9

97483_Patient-08pl_placenta
19.5
94742_Donor 3 U - A_Mesenchymal Stem
24.0

Cells

97486_Patient-09sk_skeletal
5.6
94743_Donor 3 U - B_Mesenchymal Stem
18.8

muscle

Cells

97487_Patient-09ut_uterus
18.4
94730_Donor 3 AM - A_adipose
40.1

97488_Patient-09pl_placenta
15.5
94731_Donor 3 AM - B_adipose
22.8

97492_Patient-10ut_uterus
29.3
94732_Donor 3 AM - C_adipose
28.9

97493_Patient-10pl_placenta
79.6
94733_Donor 3 AD - A_adipose
59.0

97495_Patient-11go_adipose
13.5
94734_Donor 3 AD - B_adipose
29.1

97496_Patient-11sk_skeletal
16.8
94735_Donor 3 AD - C_adipose
35.6

muscle

97497_Patient-11ut_uterus
36.6
77138_Liver_HepG2untreated
100.0

97498_Patient-11pl_placenta
39.2
73556_Heart_Cardiac stromal cells
15.3

(primary)

97500_Patient-12go_adipose
29.5
81735_Small Intestine
34.4

97501_Patient-12sk_skeletal
47.6
72409_Kidney_Proximal Convoluted
9.0

muscle

Tubule

97502_Patient-12ut_uterus
38.4
82685_Small intestine_Duodenum
25.7

97503_Patient-12pl_placenta
21.9
90650_Adrenal_Adrenocortical adenoma
40.3

94721_Donor 2 U -
25.0
72410_Kidney_HRCE
35.4

A_Mesenchymal Stem Cells

94722_Donor 2 U -
18.6
72411_Kidney_HRE
24.3

B_Mesenchymal Stem Cells

94723_Donor 2 U -
31.9
73139_Uterus_Uterine smooth muscle
9.7

C_Mesenchymal Stem Cells

cells

[0540] CNS_neurodegeneration_v1.0 Summary: Ag5850/Ag5952 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

[0541] General_screening_panel_v1.4 Summary: Ag3883 Highest expression is seen in the adrenal gland (CT=25.7). This gene is widely expressed in this panel with high to moderate levels of expression in pituitary, adipose, adrenal gland, pancreas, thyroid, and adult and fetal skeletal muscle, heart, and liver. This widespread expression among these tissues suggests that this gene product may play a role in normal neuroendocrine and metabolic function and that disregulated expression of this gene may contribute to neuroendocrine disorders or metabolic diseases, such as obesity and diabetes.

[0542] This gene is also expressed at high to moderate levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurological disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

[0543] This gene is also widely expressed in brain, colon, gastric, lung, breast, ovarian, and melanoma cancer cell lines. This expression profile suggests a role for this gene product in cell survival and proliferation. Modulation of this gene product may be useful in the treatment of cancer.

[0544] General_screening−panel_v1.5 Summary: Ag5850 Expression is limited to the adrenal gland (CT=34.3).

[0545] Ag5952 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

[0546] Panel 4.1D Summary: Ag7342 Highest expression is seen in an ionomycin treated sample of the B cell line Ramos (CT=33). This gene is also expressed at low but significant levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members of the T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_v1.4 and also suggests a role for the gene product in cell survival and proliferation. Therefore, modulation of the gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.

[0547] Ag5850/Ag5952 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

[0548] Panel 5 Islet Summary: Ag5850 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

[0549] Panel 5D Summary: Ag3883 Highest expression is seen in a liver cell line (CT=30), with moderate to low levels of expression seen in many samples on this panel, including adipose, placenta, uterus and skeletal muscle.

[0550] D. CG163701-01: F-box Protein.

[0551] Expression of gene CG163701-01 was assessed using the primer-probe set Ag5866, described in Table DA. Results of the RTQ-PCR runs are shown in Tables DB, DC and DD.

62TABLE DAProbe Name Ag5866StartSEQ IDPrimersLengthPositionNoForward5′-agttttatttggatcggaatctactc-3′2634139ProbeTET-5′-tttcacctgcaagatcaaattggaaa-3′-TAMRA2637540Reverse15′-catagacatagctatcttctctactgaattaaa-3′3340141

[0552]

63

TABLE DB

CNS neurodegeneration v1.0

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5866,

Ag5866,

Run

Run

Tissue Name
247108840
Tissue Name
247108840

AD 1 Hippo
19.3
Control (Path) 3 Temporal Ctx
9.0

AD 2 Hippo
95.3
Control (Path) 4 Temporal Ctx
43.8

AD 3 Hippo
13.7
AD 1 Occipital Ctx
11.7

AD 4 Hippo
13.0
AD 2 Occipital Ctx (Missing)
0.0

AD 5 Hippo
59.0
AD 3 Occipital Ctx
17.1

AD 6 Hippo
52.9
AD 4 Occipital Ctx
33.0

Control 2 Hippo
55.5
AD 5 Occipital Ctx
61.6

Control 4 Hippo
52.5
AD 6 Occipital Ctx
14.9

Control (Path) 3 Hippo
14.3
Control 1 Occipital Ctx
7.0

AD 1 Temporal Ctx
25.0
Control 2 Occipital Ctx
62.0

AD 2 Temporal Ctx
58.2
Control 3 Occipital Ctx
34.9

AD 3 Temporal Ctx
14.9
Control 4 Occipital Ctx
25.3

AD 4 Temporal Ctx
42.0
Control (Path) 1 Occipital Ctx
92.0

AD 5 Inf Temporal Ctx
100.0
Control (Path) 2 Occipital Ctx
13.0

AD 5 Sup Temporal Ctx
74.2
Control (Path) 3 Occipital Ctx
3.5

AD 6 Inf Temporal Ctx
26.1
Control (Path) 4 Occipital Ctx
34.2

AD 6 Sup Temporal Ctx
54.0
Control 1 Parietal Ctx
11.7

Control 1 Temporal Ctx
12.5
Control 2 Parietal Ctx
68.8

Control 2 Temporal Ctx
27.2
Control 3 Parietal Ctx
25.7

Control 3 Temporal Ctx
29.5
Control (Path) 1 Parietal Ctx
80.7

Control 3 Temporal Ctx
24.0
Control (Path) 2 Parietal Ctx
41.5

Control (Path) 1 Temporal Ctx
59.9
Control (Path) 3 Parietal Ctx
13.4

Control (Path) 2 Temporal Ctx
45.4
Control (Path) 4 Parietal Ctx
41.2

[0553]

64

TABLE DC

General screening panel v1.5

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5866,

Ag5866,

Run

Run

Tissue Name
246284012
Tissue Name
246284012

Adipose
1.3
Renal ca. TK-10
8.5

Melanoma* Hs688(A).T
2.0
Bladder
6.1

Melanoma* Hs688(B).T
1.7
Gastric ca. (liver met.) NCI-N87
9.9

Melanoma* M14
0.0
Gastric ca. KATO III
2.5

Melanoma* LOXIMVI
0.8
Colon ca. SW-948
0.0

Melanoma* SK-MEL-5
1.2
Colon ca. SW480
12.8

Squamous cell carcinoma SCC-4
1.2
Colon ca.* (SW480 met) SW620
2.2

Testis Pool
89.5
Colon ca. HT29
0.0

Prostate ca.* (bone met) PC-3
9.0
Colon ca. HCT-116
4.5

Prostate Pool
3.1
Colon ca. CaCo-2
0.8

Placenta
0.7
Colon cancer tissue
1.6

Uterus Pool
1.3
Colon ca. SW1116
0.6

Ovarian ca. OVCAR-3
20.9
Colon ca. Colo-205
0.7

Ovarian ca. SK-OV-3
4.2
Colon ca. SW-48
0.0

Ovanan ca. OVCAR-4
0.7
Colon Pool
2.1

Ovarian ca. OVCAR-5
9.2
Small Intestine Pool
5.0

Ovarian ca. IGROV-1
4.7
Stomach Pool
3.6

Ovarian ca. OVCAR-8
7.5
Bone Marrow Pool
0.0

Ovary
14.1
Fetal Heart
0.9

Breast ca. MCF-7
11.3
Heart Pool
0.5

Breast ca. MDA-MB-231
0.7
Lymph Node Pool
2.3

Breast ca. BT 549
16.0
Fetal Skeletal Muscle
1.5

Breast ca. T47D
2.5
Skeletal Muscle Pool
0.0

Breast ca. MDA-N
0.0
Spleen Pool
6.3

Breast Pool
3.7
Thymus Pool
8.7

Trachea
53.6
CNS cancer (glio/astro) U87-MG
13.4

Lung
1.8
CNS cancer (glio/astro) U-118-MG
11.7

Fetal Lung
41.2
CNS cancer (neuro; met) SK-N-AS
3.6

Lung ca. NCI-N417
0.0
CNS cancer (astro) SF-539
5.0

Lung ca. LX-1
0.0
CNS cancer (astro) SNB-75
2.5

Lung ca. NCI-H146
0.9
CNS cancer (glio) SNB-19
3.9

Lung ca. SHP-77
22.8
CNS cancer (glio) SF-295
12.2

Lung ca. A549
7.7
Brain (Amygdala) Pool
19.5

Lung ca. NCI-H526
0.0
Brain (cerebellum)
38.4

Lung ca. NCI-H23
39.5
Brain (fetal)
33.4

Lung ca. NCI-H460
3.3
Brain (Hippocampus) Pool
22.4

Lung ca. HOP-62
4.1
Cerebral Cortex Pool
17.2

Lung ca. NCI-H522
100.0
Brain (Substantia nigra) Pool
18.7

Liver
0.9
Brain (Thalamus) Pool
39.5

Fetal Liver
5.7
Brain (whole)
15.0

Liver ca. HepG2
1.0
Spinal Cord Pool
24.8

Kidney Pool
3.5
Adrenal Gland
9.3

Fetal Kidney
38.4
Pituitary gland Pool
19.5

Renal ca. 786-0
3.2
Salivary Gland
1.5

Renal ca. A498
1.6
Thyroid (female)
12.2

Renal ca. ACHN
10.6
Pancreatic ca. CAPAN2
3.5

Renal ca. UO-31
7.4
Pancreas Pool
11.4

[0554]

65

TABLE DD

Panel 4.1D

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5866,

Ag5866,

Run

Run

Tissue Name
247283916
Tissue Name
247283916

Secondary Th1 act
4.7
HUVEC IL-1beta
0.0

Secondary Th2 act
10.8
HUVEC IFN gamma
3.1

Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN gamma
0.0

Secondary Th1 rest
3.6
HUVEC TNF alpha + IL4
0.0

Secondary Th2 rest
0.0
HUVEC IL-11
0.0

Secondary Tr1 rest
0.0
Lung Microvascular EC none
6.3

Primary Th1 act
0.0
Lung Microvascular EC TNFalpha + IL-beta
0.0

Primary Th2 act
23.3
Microvascular Dermal EC none
0.0

Primary Tr1 act
13.4
Microsvasular Dermal EC TNFalpha + IL-1beta
9.0

Primary Th1 rest
0.0
Bronchial epithelium TNFalpha + IL1beta
7.6

Primary Th2 rest
0.0
Small airway epithelium none
2.9

Primary Tr1 rest
4.6
Small airway epithelium TNFalpha + IL-1beta
4.9

CD45RA CD4 lymphocyte act
6.4
Coronary artery SMC rest
0.0

CD45RO CD4 lymphocyte act
58.6
Coronary artery SMC TNFalpha + IL-1beta
0.0

CD8 lymphocyte act
0.0
Astrocytes rest
5.0

Secondary CD8 lymphocyte rest
6.6
Astrocytes TNFalpha + IL-1beta
0.0

Secondary CD8 lymphocyte act
2.7
KU-812 (Basophil) rest
0.0

CD4 lymphocyte none
0.0
KU-812 (Basophil) PMA/ionomycin
17.4

2ry Th1/Th2/Tr1_anti-CD95 CH11
0.0
CCD1106 (Keratinocytes) none
4.9

LAK cells rest
21.0
CCD1106 (Keratinocytes) TNFalpha + IL-1beta
0.0

LAK cells IL-2
5.3
Liver cirrhosis
0.0

LAK cells IL-2 + IL-12
0.0
NCI-H292 none
20.9

LAK cells IL-2 + IFN gamma
0.0
NCI-H292 IL-4
8.8

LAK cells IL-2 + IL-18
2.7
NCI-H292 IL-9
25.0

LAK cells PMA/ionomycin
20.6
NCI-H292 IL-13
21.6

NK Cells IL-2 rest
8.9
NCI-H292 IFN gamma
5.7

Two Way MLR 3 day
14.1
HPAEC none
0.0

Two Way MLR 5 day
0.0
HPAEC TNF alpha + IL-1beta
0.0

Two Way MLR 7 day
7.0
Lung fibroblast none
3.6

PBMC rest
0.0
Lung fibroblast TNF alpha + IL-1
3.1

beta

PBMC PWM
2.9
Lung fibroblast IL-4
4.9

PBMC PHA-L
3.7
Lung fibroblast IL-9
0.0

Ramos (B cell) none
0.0
Lung fibroblast IL-13
6.2

Ramos (B cell) ionomycin
0.0
Lung fibroblast IFN gamma
3.0

B lymphocytes PWM
13.7
Dermal fibroblast CCD1070 rest
4.7

B lymphocytes CD40L and IL-4
6.5
Dermal fibroblast CCD1070 TNF
21.2

alpha

EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070 IL-1
3.5

beta

EOL-1 dbcAMP PMA/ionomycin
0.0
Dermal fibroblast IFN gamma
0.0

Dendritic cells none
19.1
Dermal fibroblast IL-4
0.0

Dendritic cells LPS
0.0
Dermal Fibroblasts rest
8.6

Dendritic cells anti-CD40
7.5
Neutrophils TNFa + LPS
0.0

Monocytes rest
0.0
Neutrophils rest
0.0

Monocytes LPS
2.3
Colon
0.0

Macrophages rest
13.7
Lung
0.0

Macrophages LPS
5.4
Thymus
0.0

HUVEC none
61.1
Kidney
100.0

HUVEC starved
0.0

[0555] CNS_neurodegeneration_v1.0 Summary: Ag5866 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at low levels in the brain. Please see Panel 1.5 for discussion of this gene in the central nervous system.

[0556] General_screening_panel_v1.5 Summary: Ag5886 Highest expression of this gene is seen in a lung cancer cell line (CT=32.3). Low levels of expression are also seen in testis, fetal lung, fetal kidney and trachea.

[0557] This gene is also expressed at low levels in the CNS, including the hippocampus, thalamus, substantia nigra, amygdala, cerebellum and cerebral cortex. Therefore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of neurological disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, stroke and epilepsy.

[0558] Panel 4.1D Summary: Ag5886 This gene is only expressed at detectable levels in the kidney (CT=34.3). Thus, expression of this gene could be used to differentiate the kidney derived sample from other samples on this panel and as a marker of kidney tissue. In addition, therapeutic targeting of the expression or function of this gene may modulate kidney function and be important in the treatment of inflammatory or autoimmune diseases that affect the kidney, including lupus and glomerulonephritis.

[0559] E. CG164211-01: Colonic and Hepatic Tumor Over-expressed Protein.

[0560] Expression of gene CG 164211-01 was assessed using the primer-probe sets Ag5879 and Ag6106, described in Tables EA and LB. Results of the RTQ-PCR runs are shown in Tables EC, ED and EL.

66TABLE EAProbe Name Ag5879StartSEQPrimersLengthPositionID NoForward5′-ggcatgatacaactaatttaagagtcttt-3′29662442ProbeTET-5′-tgcattaactatacctgactcccaagatgc-3′-TAMRA30666643Reverse5′-gcccaacactccatgatatg-3′20669844

[0561]

67

TABLE EB

Probe Name Ag6106

SEQ

Start
ID

Primers

Length
Position
No

Forward
5′-aacatgttacgcaagggacc-3′
20
4420
45

Probe
TET-5′-tgaggacatgtcttccaaactcaaaattatgtat-3′TAMRA
34
4443
46

Reverse
5′-cgggcttggatcctataagtg-3′
21
4479
47

[0562]

68

TABLE EC

CNS neurodegeneration v1.0

Rel.
Rel.

Rel.
Rel.

Exp. (%)
Exp. (%)

Exp. (%)
Exp. (%)

Ag5879,
Ag6106,

Ag5879,
Ag6106,

Run
Run

Run
Run

Tissue Name
247854376
249008722
Tissue Name
247854376
249008722

AD 1 Hippo
7.1
1.1
Control (Path) 3
0.0
2.1

Temporal Ctx

AD 2 Hippo
73.2
9.2
Control (Path) 4
53.6
11.4

Temporal Ctx

AD 3 Hippo
7.9
1.0
AD 1 Occipital Ctx
21.0
4.6

AD 4 Hippo
4.7
5.3
AD 2 Occipital Ctx
0.0
0.0

(Missing)

AD 5 Hippo
83.5
69.3
AD 3 Occipital Ctx
8.5
2.2

AD 6 Hippo
55.1
22.4
AD 4 Occipital Ctx
29.9
12.1

Control 2 Hippo
21.8
24.3
AD 5 Occipital Ctx
29.1
70.7

Control 4 Hippo
7.0
0.0
AD 6 Occipital Ctx
6.5
16.7

Control (Path) 3 Hippo
24.8
0.8
Control 1 Occipital Ctx
27.9
1.4

AD 1 Temporal Ctx
18.9
3.2
Control 2 Occipital Ctx
55.5
100.0

AD 2 Temporal Ctx
24.3
9.8
Control 3 Occipital Ctx
46.0
10.8

AD 3 Temporal Ctx
23.5
0.0
Control 4 Occipital Ctx
12.5
0.0

AD 4 Temporal Ctx
43.5
5.1
Control (Path) 1
78.5
51.8

Occipital Ctx

AD 5 Inf Temporal Ctx
100.0
43.5
Control (Path) 2
4.0
8.4

Occipital Ctx

AD 5 Sup Temporal
31.6
12.7
Control (Path) 3
0.0
0.8

Ctx

Occipital Ctx

AD 6 Inf Temporal Ctx
19.1
17.9
Control (Path) 4
40.6
12.4

Occipital Ctx

AD 6 Sup Temporal
45.4
26.1
Control 1 Parietal Ctx
5.4
0.9

Ctx

Control 1 Temporal
7.3
0.6
Control 2 Parietal Ctx
67.8
13.9

Ctx

Control 2 Temporal
7.9
66.4
Control 3 Parietal Ctx
10.4
15.8

Ctx

Control 3 Temporal
16.5
10.2
Control (Path) 1
85.3
41.8

Ctx

Parietal Ctx

Control 3 Temporal
17.2
1.4
Control (Path) 2
23.3
8.5

Ctx

Parietal Ctx

Control (Path) 1
31.4
20.4
Control (Path) 3
10.4
1.1

Temporal Ctx

Parietal Ctx

Control (Path) 2
27.0
11.9
Control (Path) 4
13.8
54.0

Temporal Ctx

Parietal Ctx

[0563]

69

TABLE ED

General screening panel v1.5

Rel.
Rel.

Rel.
Rel.

Exp. (%)
Exp. (%)

Exp. (%)
Exp. (%)

Ag5879,
Ag6106,

Ag5879,
Ag6106,

Run
Run

Run
Run

Tissue Name
247834837
248650760
Tissue Name
247834837
248650760

Adipose
1.8
0.0
Renal ca. TK-10
33.2
0.0

Melanoma*
3.3
0.0
Bladder
1.4
0.3

Hs688(A).T

NCI-N87

Melanoma*
2.6
0.0
Gastric ca. (liver met.)
2.7
0.0

Hs688(B).T

NCI-N87

Melanoma* M14
6.6
0.0
Gastric ca. KATO III
13.7
0.0

Melanoma*
3.6
0.0
Colon ca. SW-948
2.2
0.0

LOXIMVI

Melanoma*
8.8
0.0
Colon ca. SW480
13.2
1.0

SK-MEL-5

Squamous cell
2.6
0.0
Colon ca.* (SW480 met)
11.0
2.1

carcinoma SCC-4

SW620

Testis Pool
1.0
0.0
Colon ca. HT29
4.5
0.0

Prostate ca.* (bone
4.3
0.0
Colon ca. HCT-116
15.1
0.0

met) PC-3

Prostate Pool
0.6
0.0
Colon ca. CaCo-2
27.2
0.0

Placenta
0.4
0.0
Colon cancer tissue
1.9
0.0

Uterus Pool
0.1
0.0
Colon ca. SW1116
1.0
0.0

Ovarian ca.
7.9
0.0
Colon ca. Colo-205
1.2
0.0

OVCAR-3

Ovarian ca. SK-OV-3
20.0
0.0
Colon ca. SW-48
0.6
0.0

Ovarian ca.
1.7
0.0
Colon Pool
3.7
0.0

OVCAR-4

Ovarian ca.
15.3
0.0
Small Intestine Pool
3.1
1.5

OVCAR-5

Ovarian ca. IGROV-1
12.1
0.0
Stomach Pool
2.0
0.2

Ovarian ca.
2.6
0.0
Bone Marrow Pool
1.1
0.0

OVCAR-8

Ovary
3.6
0.0
Fetal Heart
1.4
0.0

Breast ca. MCF-7
8.4
0.0
Heart Pool
1.0
0.0

Breast ca.
9.1
0.0
Lymph Node Pool
4.7
0.0

MDA-MB-231

Breast ca. BT 549
3.6
0.0
Fetal Skeletal Muscle
0.8
0.0

Breast ca. T47D
2.8
0.0
Skeletal Muscle Pool
3.8
0.0

Breast ca. MDA-N
4.1
0.0
Spleen Pool
0.2
0.2

Breast Pool
4.0
0.0
Thymus Pool
1.9
0.0

Trachea
0.3
0.0
CNS cancer (glio/astro)
5.2
0.0

U87-MG

Lung
2.4
0.0
CNS cancer (glio/astro)
5.7
0.0

U-118-MG

Fetal Lung
2.5
0.0
CNS cancer (neuro;met)
15.2
0.4

SK-N-AS

Lung ca. NCI-N417
2.1
15.5
CNS cancer (astro)
3.9
0.0

SF-539

Lung ca. LX-1
9.1
0.2
CNS cancer (astro)
9.9
0.0

SNB-75

Lung ca. NCI-H146
3.1
64.6
CNS cancer (glio)
3.8
0.0

SNB-19

Lung ca. SHP-77
11.3
9.6
CNS cancer (glio)
15.1
0.0

SF-295

Lung ca. A549
8.6
0.0
Brain (Amygdala) Pool
0.7
3.7

Lung ca. NCI-H526
2.5
3.6
Brain (cerebellum)
7.8
100.0

Lung ca. NCI-H23
9.7
0.0
Brain (fetal)
4.4
59.9

Lung ca. NCI-H460
5.5
0.0
Brain (Hippocampus)
0.9
3.7

Pool

Lung ca. HOP-62
4.4
0.0
Cerebral Cortex Pool
1.2
8.1

Lung ca. NCI-H522
10.4
0.0
Brain (Substantia nigra)
1.1
4.2

Pool

Liver
4.5
0.0
Brain (Thalamus) Pool
1.2
7.9

Fetal Liver
100.0
0.0
Brain (whole)
3.7
8.1

Liver ca. HepG2
49.7
0.0
Spinal Cord Pool
1.2
10.1

Kidney Pool
6.1
0.0
Adrenal Gland
1.6
0.2

Fetal Kidney
1.2
0.0
Pituitary gland Pool
0.1
0.5

Renal ca. 786-0
4.6
0.0
Salivary Gland
0.6
0.3

Renal ca. A498
1.0
0.0
Thyroid (female)
0.1
0.0

Renal ca. ACHN
3.8
0.0
Pancreatic ca. CAPAN2
5.0
0.0

Renal ca. UO-31
4.0
0.0
Pancreas Pool
3.2
0.2

[0564]

70

TABLE EE

Panel 4.1D

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5879,

Ag5879,

Run

Run

Tissue Name
247683783
Tissue Name
247683783

Secondary Th1 act
61.1
HUVEC IL-1beta
22.7

Secondary Th2 act
51.8
HUVEC IFN gamma
8.6

Secondary Tr1 act
23.7
HUVEC TNF alpha + IFN gamma
8.5

Secondary Th1 rest
8.7
HUVEC TNF alpha + IL4
23.7

Secondary Th2 rest
7.2
HUVEC IL-11
8.5

Secondary Tr1 rest
11.4
Lung Microvascular EC none
36.9

Primary Th1 act
9.9
Lung Microvascular EC TNFalpha + IL-1beta
7.2

Primary Th2 act
29.7
Microsvascular Dermal EC none
12.2

Primary Tr1 act
38.2
Microsvasular Dermal EC TNFalpha + IL-1beta
2.6

Primary Th1 rest
2.0
Bronchial epithelium TNFalpha + IL1beta
0.0

Primary Th2 rest
9.2
Small airway epithelium none
0.0

Primary Tr1 rest
0.0
Small airway epithelium TNFalpha + IL-1beta
16.3

CD45RA CD4 lymphocyte act
36.6
Coronary artery SMC rest
12.7

CD45RO CD4 lymphocyte act
48.6
Coronary artery SMC TNFalpha + IL-1beta
2.1

CD8 lymphocyte act
12.4
Astrocytes rest
12.5

Secondary CD8 lymphocyte rest
66.0
Astrocytes TNFalpha + IL-1beta
9.5

Secondary CD8 lymphocyte act
0.0
KU-812 (Basophil) rest
45.7

CD4 lymphocyte none
9.0
KU-812 (Basophil) PMA/ionomycin
43.2

2ry Th1/Th2/Tr1_anti-CD95 CH11
7.7
CCD1106 (Keratinocytes) none
18.3

LAK cells rest
10.7
CCD1106 (Keratinocytes) TNFalpha + IL-1beta
5.7

LAK cells IL-2
15.4
Liver cirrhosis
17.1

LAK cells IL-2 + IL-12
17.2
NCI-H292 none
24.7

LAK cells IL-2 + IFN gamma
3.6
NCI-H292 IL-4
47.0

LAK cells IL-2 + IL-18
11.0
NCI-H292 IL-9
94.0

LAK cells PMA/ionomycin
9.9
NCI-H292 IL-13
59.9

NK Cells IL-2 rest
62.0
NCI-H292 IFN gamma
19.6

Two Way MLR 3 day
6.9
HPAEC none
8.3

Two Way MLR 5 day
2.7
HPAEC TNF alpha + IL-1beta
33.2

Two way MLR 7 day
4.5
Lung fibroblast none
11.7

PBMC rest
6.9
Lung fibroblast TNF alpha + IL-
3.0

1beta

PBMC PWM
12.6
Lung fibroblast IL-4
0.0

PBMC PHA-L
20.6
Lung fibroblast IL-9
5.3

Ramos (B cell) none
31.2
Lung fibroblast IL-13
6.3

Ramos (B cell) ionomycin
31.6
Lung fibroblast IFN gamma
7.0

B lymphocytes PWM
23.2
Dermal fibroblast CCD1070 rest
32.1

B lymphocytes CD40L and IL-4
46.0
Dermal fibroblast CCD1070 TNF
100.0

alpha

EOL-1 dbcAMP
54.3
Dermal fibroblast CCD1070 IL-
40.1

1beta

EOL-1 dbcAMP PMA/ionomycin
31.6
Dermal fibroblast IFN gamma
27.7

Dendritic cells none
16.2
Dermal fibroblast IL-4
18.4

Dendritic cells LPS
6.1
Dermal Fibroblasts rest
13.5

Dendritic cells anti-CD40
12.0
Neutrophils TNFa + LPS
3.1

Monocytes rest
4.6
Neutrophils rest
3.0

Monocytes LPS
7.7
Colon
0.0

Macrophages rest
10.8
Lung
0.0

Macrophages LPS
0.0
Thymus
3.2

HUVEC none
8.4
Kidney
5.0

HUVEC starved
27.9

[0565] CNS_neurodegeneration_v1.0 Summary: Ag5879/Ag6106 This panel does not show differential expression of this gene in Alzheimer's disease. However, this profile confirms the expression of this gene at low levels in the brain. Please see Panel 1.5 for discussion of this gene in the central nervous system.

[0566] General_screening_panel_v1.5 Summary: Ag5879 Highest expression is seen in fetal liver (CT=29.7). Prominent expression is also seen in a liver cell line. Thus, expression of this gene could be used to differentiate between these liver derived samples and other samples on this panel and as a marker of liver tissue. Furthermore, therapeutic modulation of the expression or function of this gene may be useful in the treatment of liver related disease.

[0567] Ag6106 Highest expression is seen in cerebellum, with prominent expression also seen in fetal brain and two lung cancer cell lines. Thus, this gene product may be a useful and specific target of drugs for the treatment of CNS disorders that have this brain region as the site of pathology, such as autism and the ataxias.

[0568] Panel 4.1D Summary: Ag5879 Highest expression of this gene is seen in TNF-a treated dermal fibroblasts (CT=34). This expression suggests that this gene product may be involved in skin disorders, including psoriasis.

[0569] Ag6106 Expression of this gene is low/undetectable in all samples on this panel (CTs>35).

[0570] F. CG164330-01: Splice Variant of Dishevelled-3.

[0571] Expression of gene CG164330-01 was assessed using the primer-probe set Ag5973, described in Table FA. Results of the RTQ-PCR runs are shown in Tables FB and FC.

71TABLE FAProbe Name Ag5973StartSEQ IDPrimersLengthPositionNoForward5′-atggacaagtggaagtcgtctag-3′23172548ProbeTET-5′-atcaccagttccatccctgacacaga-3′-TAMRA26175249Reverse5′-aaatataacttcttgagcaccatcac-3′26179450

[0572]

72

TABLE FB

CNS neurodegeneration v1.0

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5973,

Ag5973,

Run

Run

Tissue Name
248162716
Tissue Name
248162716

AD 1 Hippo
22.8
Control (Path) 3 Temporal Ctx
11.4

AD 2 Hippo
45.1
Control (Path) 4 Temporal Ctx
36.9

AD 3 Hippo
15.8
AD 1 Occipital Ctx
25.9

AD 4 Hippo
12.5
AD 2 Occipital Ctx (Missing)
0.0

AD 5 Hippo
92.0
AD 3 Occipital Ctx
18.9

AD 6 Hippo
95.3
AD 4 Occipital Ctx
38.4

Control 2 Hippo
47.3
AD 5 Occipital Ctx
64.2

Control 4 Hippo
23.5
AD 6 Occipital Ctx
25.0

Control (Path) 3 Hippo
15.1
Control 1 Occipital Ctx
6.5

AD 1 Temporal Ctx
32.8
Control 2 Occipital Ctx
90.1

AD 2 Temporal Ctx
55.5
Control 3 Occipital Ctx
18.2

AD 3 Temporal Ctx
14.1
Control 4 Occipital Ctx
14.4

AD 4 Temporal Ctx
39.8
Control (Path) 1 Occipital Ctx
95.9

AD 5 Inf Temporal Ctx
92.7
Control (Path) 2 Occipital Ctx
16.6

AD 5 Sup Temporal Ctx
66.0
Control (Path) 3 Occipital Ctx
7.2

AD 6 Inf Temporal Ctx
87.7
Control (Path) 4 Occipital Ctx
24.5

AD 6 Sup Temporal Ctx
80.7
Control 1 Parietal Ctx
13.3

Control 1 Temporal Ctx
14.4
Control 2 Parietal Ctx
60.3

Control 2 Temporal Ctx
70.2
Control 3 Parietal Ctx
21.3

Control 3 Temporal Ctx
39.0
Control (Path) 1 Parietal Ctx
100.0

Control 3 Temporal Ctx
19.2
Control (Path) 2 Parietal Ctx
22.7

Control (Path) 1 Temporal Ctx
84.1
Control (Path) 3 Parietal Ctx
8.8

Control (Path) 2 Temporal Ctx
45.4
Control (Path) 4 Parietal Ctx
74.2

[0573]

73

TABLE FC

General screening panel v1.5

Rel.

Rel.

Exp. (%)

Exp. (%)

Ag5973,

Ag5973,

Run

Run

Tissue Name
248102148
Tissue Name
248102148

Adipose
6.0
Renal ca. TK-10
48.6

Melanoma* Hs688(A).T
46.0
Bladder
30.1

Melanoma* Hs688(B).T
55.9
Gastric ca. (liver met.) NCI-N87
63.3

Melanoma* M14
42.0
Gastric ca. KATO III
100.0

Melanoma* LOXIMVI
23.5
Colon ca. SW-948
14.0

Melanoma* SK-MEL-5
35.4
Colon ca. SW480
70.7

Squamous cell carcinoma SCC-4
50.3
Colon ca.* (SW480 met) SW620
27.9

Testis Pool
10.7
Colon ca. HT29
21.6

Prostate ca.* (bone met) PC-3
67.4
Colon ca. HCT-116
28.3

Prostate Pool
12.2
Colon ca. CaCo-2
36.1

Placenta
61.6
Colon cancer tissue
17.2

Uterus Pool
6.2
Colon ca. SW1116
9.1

Ovarian ca. OVCAR-3
52.5
Colon ca. Colo-205
7.2

Ovarian ca. SK-OV-3
55.9
Colon ca. SW-48
16.2

Ovarian ca. OVCAR-4
30.1
Colon Pool
29.3

Ovarian ca. OVCAR-5
68.8
Small Intestine Pool
13.4

Ovarian ca. IGROV-1
27.4
Stomach Pool
11.3

Ovarian ca. OVCAR-8
22.7
Bone Marrow Pool
8.5

Ovary
20.9
Fetal Heart
24.5

Breast ca. MCF-7
39.0
Heart Pool
12.3

Breast ca. MDA-MB-231
35.8
Lymph Node Pool
30.1

Breast ca. BT 549
54.3
Fetal Skeletal Muscle
18.9

Breast ca. T47D
40.1
Skeletal Muscle Pool
26.8

Breast ca. MDA-N
22.1
Spleen Pool
9.5

Breast Pool
26.2
Thymus Pool
15.4

Trachea
16.7
CNS cancer (glio/astro) U87-MG
34.4

Lung
2.7
CNS cancer (glio/astro)
60.7

U-118-MG

Fetal Lung
70.7
CNS cancer (neuro; met) SK-N-AS
60.3

Lung ca. NCI-N417
8.7
CNS cancer (astro) SF-539
55.1

Lung ca. LX-1
30.4
CNS cancer (astro) SNB-75
84.7

Lung ca. NCI-H146
33.9
CNS cancer (glio) SNB-19
30.4

Lung ca. SHP-77
33.0
CNS cancer (glio) SF-295
64.6

Lung ca. A549
15.3
Brain (Amygdala) Pool
10.9

Lung ca. NCI-H526
13.4
Brain (cerebellum)
44.1

Lung ca. NCI-H23
47.6
Brain (fetal)
39.0

Lung ca. NCI-H460
14.9
Brain (Hippocampus) Pool
12.2

Lung ca. HOP-62
16.3
Cerebral Cortex Pool
15.6

Lung ca. NCI-H522
76.8
Brain (Substantia nigra) Pool
13.4

Liver
2.9
Brain (Thalamus) Pool
17.1

Fetal Liver
28.3
Brain (whole)
19.8

Liver ca. HepG2
32.5
Spinal Cord Pool
13.1

Kidney Pool
28.5
Adrenal Gland
29.1

Fetal Kidney
21.6
Pituitary gland Pool
4.4

Renal ca. 786-0
32.1
Salivary Gland
13.3

Renal ca. A498
18.9
Thyroid (female)
7.2

Renal ca. ACHN
18.2
Pancreatic ca. CAPAN2
23.2

Renal ca. UO-31
47.6
Pancreas Pool
17.9

[0574] CNS_neurodegeneration_v1.0 Summary: Ag5973 This panel confirms the expression of this gene at low levels in the brains of an independent group of individuals. However, no differential expression of this gene was detected between Alzheimer's diseased postmortem brains and those of non-demented controls in this experiment. Please see Panel 1.5 for a discussion of the potential role of this gene in treatment of central nervous system disorders.

[0575] General_screening_panel_v1.5 Summary: Ag5973 Highest expression of this gene is seen in gastric cancer KATO III cell line (CT=25.5). High expression of this gene is also seen in cluster of cancer cell lines derived from pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers. Thus, expression of this gene could be used as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of pancreatic, gastric, colon, lung, liver, renal, breast, ovarian, prostate, squamous cell carcinoma, melanoma and brain cancers.

[0576] Among tissues with metabolic or endocrine function, this gene is expressed at moderate to high levels in pancreas, adipose, adrenal gland, thyroid, pituitary gland, skeletal muscle, heart, liver and the gastrointestinal tract. Therefore, therapeutic modulation of the activity of this gene may prove useful in the treatment of endocrine/metabolically related diseases, such as obesity and diabetes.

[0577] In addition, this gene is expressed at moderate to high levels in all regions of the central nervous system examined, including amygdala, hippocampus, substantia nigra, thalamus, cerebellum, cerebral cortex, and spinal cord. Therefore, therapeutic modulation of this gene product may be useful in the treatment of central nervous system disorders such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, schizophrenia and depression.

[0578] Interestingly, this gene is expressed at much higher levels in fetal (CTs=26-27) when compared to adult liver and lung(CTs=30.7). This observation suggests that expression of this gene can be used to distinguish fetal from adult liver and lung. In addition, the relative overexpression of this gene in fetal tissue suggests that the protein product may enhance liver and lung growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of liver and lung related diseases.

[0579] G. CG164521-01: IPAS.

[0580] Expression of gene CG164521-01 was assessed using the primer-probe set Ag5897, described in Table GA.

74TABLE GAProbe Name Ag5897StartSEQ IDPrimersLengthPositionNoForward5′-agtcagttggtatctgttccttttct-3′2636751ProbeTET-5′-tcgttaccccactcctggtaccaatt-3′-TAMRA2639852Reverse5′-aaatcaaagatgctgtgtccaat-3′2343853

[0581] AI_comprehensive panel_v1.0 Summary: Ag5897 Expression of this gene is low/undetectable (CTs>35) across all of the samples on this panel.

[0582] General_screeningpanel_v1.5 Summary: Ag5897 Expression of this gene is low/undetectable (CTs>35) across all of the samples on this panel.

[0583] Panel 4.1D Summary: Ag5897 Expression of this gene is low/undetectable (CTs>35) across all of the samples on this panel.

Example D: Identification of Single Nucleotide Polymorphisms in NOVX Nucleic Acid Sequences

[0584] Variant sequences are also included in this application. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a “cSNP” to denote that the nucleotide sequence containing the SNP originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymorphic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymorphic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration of the amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may also be silent, when a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern. Examples include alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, and stability of transcribed message.

[0585] SeqCalling assemblies produced by the exon linking process were selected and extended using the following criteria. Genomic clones having regions with 98% identity to all or part of the initial or extended sequence were identified by BLASTN searches using the relevant sequence to query human genomic databases. The genomic clones that resulted were selected for further analysis because this identity indicates that these clones contain the genomic locus for these SeqCalling assemblies. These sequences were analyzed for putative coding regions as well as for similarity to the known DNA and protein sequences. Programs used for these analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and other relevant programs.

[0586] Some additional genomic regions may have also been identified because selected SeqCalling assemblies map to those regions. Such SeqCalling sequences may have overlapped with regions defined by homology or exon prediction. They may also be included because the location of the fragment was in the vicinity of genomic regions identified by similarity or exon prediction that had been included in the original predicted sequence. The sequence so identified was manually assembled and then may have been extending using one or more additional sequences taken from CuraGen Corporation's human SeqCalling database. SeqCalling fragments suitable for inclusion were identified by the CuraTools™ program SeqExtend or by identifying SeqCalling fragments mapping to the appropriate regions of the genomic clones analyzed.

[0587] The regions defined by the procedures described above were then manually integrated and corrected for apparent inconsistencies that may have arisen, for example, from miscalled bases in the original fragments or from discrepancies between predicted exon junctions, EST locations and regions of sequence similarity, to derive the final sequence disclosed herein. When necessary, the process to identify and analyze SeqCalling assemblies and genomic clones was reiterated to derive the full length sequence (Alderborn et al., Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. Genome Research. 10 (8) 1249-1265, 2000).

[0588] Variants are reported individually but any combination of all or a select subset of variants are also included as contemplated NOVX embodiments of the invention.

[0589] NOV5a SNP Data:

[0590] V5a has four SNP variants, whose variant positions for its nucleotide and amino acids sequences are numbered according to SEQ ID NOs: 11 and 12, respectively. The nucleotide sequence of the NOV5a variant differs as shown in Table SNP1.

75TABLE SNP1cSNP and Coding Variants for CG 163438-01.NucleotidesAmino AcidsVariantPositionInitialModifiedPositionInitialModified13381773132CT32ProSer13381774384AG116ThrAla13381775494AG152ValVal13381776503GA155LeuLeu

[0591] NOV7a SNP Data:

[0592] NOV7a has three SNP variants, whose variant positions for its nucleotide and amino acid sequences are numbered according to SEQ ID NOs: 15 and 16, respectively. The nucleotide sequence of the NOV7a variant differs as shown in Table SNP2.

76TABLE SNP2cSNP and Coding Variants for CG 164211-01.NucleotidesAmino AcidsVariantPositionInitialModifiedPositionInitialModified133817772161GA697AspAsn133817783792TG1240GlyGly133817794232TC1387LeuPro

OTHER EMBODIMENTS

[0593] Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims. The claims presented are representative of the inventions disclosed herein. Other, unclaimed inventions are also contemplated. Applicants reserve the right to pursue such inventions in later claims.

Number	Date	Country
60341477	Dec 2001	US
60341540	Dec 2001	US
60342592	Dec 2001	US
60344903	Dec 2001	US
60373288	Apr 2002	US
60380981	May 2002	US
60381495	May 2002	US
60383744	May 2002	US
60384024	May 2002	US
60401788	Aug 2002	US
60406353	Aug 2002	US

Novel human proteins, polynucleotides encoding them and methods of using the same

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