Novel nucleic acids and polypeptides

2. BACKGROUND OF THE INVENTION

[0002] 2.1 Technical Field

[0003] The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.

[0004] 2.2 Background

[0005] Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.

[0006] Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.

3. SUMMARY OF THE INVENTION

[0007] The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.

[0008] The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.

[0009] The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases. The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins. These nucleic acid sequences are designated as SEQ ID NO: 1-441 and are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A is adenine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases. In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.

[0010] The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-441 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-441. A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-441 or a degenerate variant or fragment thereof. The identifying sequence can be 100 base pairs in length.

[0011] The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-441. The sequence information can be a segment of any one of SEQ ID NO: 1-441 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-441.

[0012] A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.

[0013] This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors. Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.

[0014] In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-441 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art. In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-441 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.

[0015] The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in SEQ ID NO: 1-441; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-441; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-441. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in SEQ ID NO: 1-441; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g. orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.

[0016] The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-441; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.

[0017] The invention also provides compositions comprising a polypeptide of the invention. Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

[0018] The invention also provides host cells transformed or transfected with a polynucleotide of the invention.

[0019] The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.

[0020] Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.

[0021] In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.

[0022] The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.

[0023] Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0024] In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.

[0025] The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions. The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected. The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.

[0026] The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.

[0027] The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound the binds to a polypeptide of the invention is identified.

[0028] The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies. In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.

[0029] The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have homology (set forth in Table 2); for which they have a signature region (as set forth in Table 3); or for which they have homology to a gene family (as set forth in Table 4). If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.

4. DETAILED DESCRIPTION OF THE INVENTION

4.1 DEFINITIONS

[0030] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0031] The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide. According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule. Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

[0032] The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.

[0033] The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing. For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′. Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules. The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.

[0034] The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells. The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes. The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells. PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal. Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.

[0035] The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.

[0036] As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.

[0037] The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonculeotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material. In the sequences herein A is adenine, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U). It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil). Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.

[0038] The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides. The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides. Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides. Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules. A fragment or segment may uniquely identify each polynucleotide sequence of the present invention. Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NOs: 1-441.

[0039] Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P.S. et al., 1992, PCR Methods Appl 1:241-250). They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.

[0040] The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NOs: 1-441. The sequence information can be a segment of any one of SEQ ID NOs: 1-441 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-441. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosomes. Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression studies, fifteen-mer segments can be used. The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.

[0041] Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer. The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.

[0042] The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.

[0043] The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences. For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence. While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.

[0044] The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.

[0045] The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules. A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids. The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids. Preferably the peptide is from about 5 to about 200 amino acids. To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.

[0046] The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.

[0047] The term “translated protein coding portion” means a sequence which encodes for the fill length protein which may include any leader sequence or any processing sequence.

[0048] The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence. The “mature protein portion” means that portion of the protein which does not include a signal or leader sequence. The peptide may have been produced by processing in the cell which removes any leader/signal sequence. The mature protein portion may or may not include the initial methionine residue. The methionine residue may be removed from the protein during processing in the cell. The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.

[0049] The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.

[0050] The term “variant” (or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e g., recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.

[0051] Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.

[0052] Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. “Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

[0053] Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.

[0054] The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).

[0055] The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.

[0056] The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems. “Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.

[0057] The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0058] The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers. Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cells can be prokaryotic or eukaryotic.

[0059] The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell. “Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. “Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum. “Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al. (1998) Annu. Rev. Inmunol. 16:27-55)

[0060] Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.

[0061] The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1× SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2× SSC/0.1% SDS at 42° C.). Other exemplary hybridization conditions are described herein in the examples.

[0062] In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6× SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

[0063] As used herein, “substantially equivalent” or “substantially similar” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to have 65% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity). Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. Substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. Preferably, the nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded. Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645). Identity between sequences can also be determined by other methods known in the art, e.g. by varying hybridization conditions.

[0064] The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.

[0065] The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration. The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed. The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.

[0066] As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.

[0067] Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.

4.2 NUCLEIC ACIDS OF THE INVENTION

[0068] Nucleotide sequences of the invention are set forth in the Sequence Listing.

[0069] The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-441; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 1-441; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polynucleotides of any one of SEQ ID NO: 1-441. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of SEQ ID NO: 1-441; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 1-441. Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.

[0070] The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.

[0071] The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genoric materials. Further 5′ and 3′ sequence can be obtained using methods known in the art. For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-441 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-441 or a portion thereof as a probe. Alternatively, the polynucleotides of SEQ ID NO: 1-441 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.

[0072] The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene. The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.

[0073] The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99% sequence identity to a polynucleotide recited above.

[0074] Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of SEQ ID NO: 1-441, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e. specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.

[0075] The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-441, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NOs: 1-441 with a sequence from another isolate of the same species. Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.

[0076] The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NOs: 1-441, can be obtained by searching a database using an algorithm or a program. Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J Mol. Evol. 36 290-300 (1993) and Altschul S. F. et al. J. Mol. Biol. 21:403-410 (1990)). Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.

[0077] Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.

[0078] The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.

[0079] The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation. Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature. These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues. Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.

[0080] In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis. This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983). A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.

[0081] A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.

[0082] Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.

[0083] The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.

[0084] In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-441, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. Also included are the cDNA inserts of any of the clones identified herein.

[0085] A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.

[0086] The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NOs: 1-441 or a fragment thereof or any other polynucleotides of the invention. In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NOs: 1-441 or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

[0087] The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufinan et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufinan, Methods in Enzymology 185, 537-566 (1990). As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

[0088] Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

[0089] As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0090] Polynucleotides of the invention can also be used to induce immune responses. For example, as described in Fan et al., Nat. Biotech. 17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA. The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.

4.3 ANTISENSE

[0091] 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: 1-441, 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 coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 1-441 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-441 are additionally provided.

[0092] In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention. 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 of the invention. The term “noncoding region” refers to 5′ and 3′ sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0093] Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-441, 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 an mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of an mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of an 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.

[0094] 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-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxynracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-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 subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0095] 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 protein according to the invention 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 intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0096] 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 α-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).

4.4 RIBOZYMES AND PNA MOIETIES

[0097] In still another 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 (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of an mRNA. A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-441). For example, a derivative of 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 SECX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, SECX mRNA can 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.

[0098] Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.

[0099] In various embodiments, the nucleic acids of the invention 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 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 nucleobases 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 oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS93: 14670-675.

[0100] PNAs of the invention 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 the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S 1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).

[0101] In another embodiment, PNAs of the invention can be modified, e.g., to enhance their 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 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 nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 540 -(4-methoxytrityl)amino-540 -deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

[0102] 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. W088/098 10) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, 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, etc.

4.5 HOSTS

[0103] The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.

[0104] Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences. See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO92/20808, and PCT International Publication No. WO91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.

[0105] The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)). The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.

[0106] Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.

[0107] Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981). Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0108] Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

[0109] In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

[0110] The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

[0111] The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

4.6 POLYPEPTIDES OF THE INVENTION

[0112] The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 1-441 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NOs: 1-441 or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NOs: 1-441 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 1-441 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions. The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 1-441 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 1-441.

[0113] Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.

[0114] The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins. The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences. The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form. Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which they are expressed.

[0115] Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

[0116] The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.

[0117] A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. This technique is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

[0118] The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.

[0119] The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown. For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide. The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified. Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.

[0120] In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology. Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.

[0121] The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.

[0122] In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 1-441.

[0123] The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.

[0124] The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications, in the peptide or DNA sequence, can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity. This type of analysis determines the importance of the substituted amino acid(s) in biological activity. Regions of the protein that are important for protein function may be determined by the eMATRIX program.

[0125] Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are encompassed by the present invention.

[0126] The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.”

[0127] The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

[0128] Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag. Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (“FLAG)”) is commercially available from Kodak (New Haven, Conn.).

[0129] Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.”

[0130] The polypeptides of the invention include analogs (variants). This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted. Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability. Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells. Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.

4.6.1 DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE IDENTITY AND SIMILARITY

[0131] Preferred identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp. Biol., Vol. 6, pp. 219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol. 4, pp. 202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol. 26(1), pp. 320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference). The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).

4.7 CHIMERIC AND FUSION PROTEINS

[0132] The invention also provides chimeric or fusion proteins. As used herein, a ∫chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide. Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention. In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention. In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention. Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other. The polypeptide can be fused to the N-terminus or C-terminus, or to the middle.

[0133] For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.

[0134] In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.

[0135] In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprise one or more domains fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo. The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand. Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e.g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.

[0136] A 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, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fuision moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.

4.8 GENE THERAPY

[0137] Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein. The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention. Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.

[0138] Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art. Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.

[0139] The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.

[0140] Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.

[0141] In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

[0142] The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

[0143] The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

4.9 TRANSGENIC ANIMALS

[0144] In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No 5,489,743 and PCT Publication No. WO94/28122, incorporated herein by reference.

[0145] Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

[0146] The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide. Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.

[0147] In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No 5,489,743 and PCT Publication No. WO94/28122, incorporated herein by reference.

[0148] Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

4.10 USES AND BIOLOGICAL ACTIVITY

[0149] The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment. Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity. Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.

[0150] The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.

4.10.1 RESEARCH USES AND UTILITIES

[0151] The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

[0152] The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

[0153] Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

[0154] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

4.10.2 NUTRITIONAL USES

[0155] Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

4.10.3 CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION ACTIVITY

[0156] A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the invention can be used in the following:

[0157] Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-lnterscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761, 1994.

[0158] Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

[0159] Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

[0160] Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500,1986; Takai et al., J. Immunol. 140:508-512, 1988.

4.10.4 STEM CELL GROWTH FACTOR ACTIVITY

[0161] A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells. Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors. The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.

[0162] It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).

[0163] Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells. Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations. This can be accomplished by direct administration of the polypeptide of the invention to the culture medium. Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo. Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat. No. 5,690,926).

[0164] Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments. These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.

[0165] Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions. For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders. The polypeptide of the invention may be usefull for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue. In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.

[0166] Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types. A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker. The selectable marker allows only cells of the desired type to survive. For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin. Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds. Lanza et al., Academic Press (1997)). Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.

[0167] In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity. Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines. The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g. as described by Bernstein et al., Blood, 77: 2316-2321 (1991).

4.10.5 HEMATOPOIESIS REGULATING ACTIVITY

[0168] A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.

[0169] Therapeutic compositions of the invention can be used in the following:

[0170] Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

[0171] Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

[0172] Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

4.10.6 TISSUE GROWTH ACTIVITY

[0173] A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of bums, incisions and ulcers.

[0174] A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.

[0175] A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells. Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes may also be possible using the composition of the invention.

[0176] Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation. Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

[0177] The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.

[0178] Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

[0179] Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate. A polypeptide of the present invention may also exhibit angiogenic activity.

[0180] A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

[0181] A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.

[0182] Therapeutic compositions of the invention can be used in the following:

[0183] Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium).

[0184] Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

4.10.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY

[0185] A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A polynucleotide of the invention can encode a polypeptide exhibiting such activities. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.

[0186] Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention. The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).

[0187] Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.

[0188] Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

[0189] The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.

[0190] Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

[0191] Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.

[0192] Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.

[0193] A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and β2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

[0194] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0195] Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Inmunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

[0196] Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

[0197] Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

[0198] Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.

[0199] Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.

[0200] Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

4.10.8 ACTIVIN/INHIBIN ACTIVITY

[0201] A polypeptide of the present invention may also exhibit activin- or inhibin-related activities. A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885. A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.

[0202] The activity of a polypeptide of the invention may, among other means, be measured by the following methods.

[0203] Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.

4.10.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY

[0204] A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic compositions (e.g. proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

[0205] A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.

[0206] Therapeutic compositions of the invention can be used in the following:

[0207] Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.

4.10.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY

[0208] A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).

[0209] Therapeutic compositions of the invention can be used in the following:

[0210] Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.

4.10.11 CANCER DIAGNOSIS AND THERAPY

[0211] Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer. For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.

[0212] Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness. Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.

[0213] Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer. Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.

[0214] The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine. Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

[0215] In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer. There are hereditary conditions and/or environmental situations (e.g. exposure to carcinogens) known in the art that predispose an individual to developing cancers. Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.

[0216] In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment. These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999), respectively. Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.

4.10.12 RECEPTOR/LIGAND ACTIVITY

[0217] A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions. A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selecting, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses. Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.

[0218] The activity of a polypeptide of the invention may, among other means, be measured by the following methods:

[0219] Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

[0220] By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s). Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.

[0221] Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands. The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods. (“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990) Academic Press, Inc. San Diego). Examples of radioisotopes include, but are not limited to, tritium and carbon-14 . Examples of calorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamnine, or rhodamine or other colorimetric molecules. Examples of toxins include, but are not limited, to ricin.

4.10.13 DRUG SCREENING

[0222] This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.

[0223] Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.

[0224] Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.

[0225] The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves. Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998).

[0226] Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1):1 14-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).

[0227] Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.

[0228] The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention. Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.

4.10.14 ASSAY FOR RECEPTOR ACTIVITY

[0229] The invention also provides methods to detect specific binding of a polypeptide e.g. a ligand or a receptor. The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention. For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention. There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention. Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not. The response of the two cell populations to the addition of ligands(s) are then compared. Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s). As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.

[0230] The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined. For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell. The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor. Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e. phosphorylation. Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.

4.10.15 ANTI-INFLAMMATORY ACTIVITY

[0231] Compositions of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material. Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflammation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.

4.10.16 LEUKEMIAS

[0232] Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention. Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

4.10.17 NERVOUS SYSTEM DISORDERS

[0233] Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:

[0234] (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;

[0235] (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;

[0236] (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;

[0237] (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;

[0238] (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B 12 deficiency, folic acid deficiency, Wemicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;

[0239] (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;

[0240] (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and

[0241] (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

[0242] Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention:

[0243] (i) increased survival time of neurons in culture;

[0244] (ii) increased sprouting of neurons in culture or in vivo;

[0245] (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or

[0246] (iv) decreased symptoms of neuron dysfunction in vivo.

[0247] Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

[0248] In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

4.10.18 OTHER ACTIVITIES

[0249] A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.

4.10.19 IDENTIFICATION OF POLYMORPHISMS

[0250] The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment. Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately. For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.

[0251] Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced. Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymnorphism is extended with one or more labeled nucleotides). In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed. Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms. The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention. In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.

[0252] Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.

4.10.20 ARTHRITIS AND INFLAMMATION

[0253] The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system. The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int. Arch. Allergy Appl. Immunol., 23:129. Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture. The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. The control consists of administering PBS only.

[0254] The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24. At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.

4.11 THERAPEUTIC METHODS

[0255] The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods. Examples of therapeutic applications include, but are not limited to, those exemplified herein.

4.11.1 EXAMPLE

[0256] One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention. While the mode of administration is not particularly important, parenteral administration is preferred. An exemplary mode of administration is to deliver an intravenous bolus. The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient. Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight. For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin. The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.

4.12 PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

[0257] A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.

[0258] The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects. Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents). A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.

[0259] As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site). Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0260] In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated. Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

4.12.1 ROUTES OF ADMINISTRATION

[0261] Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.

[0262] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation. In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue.

[0263] The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action. The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art. Preferably for wound treatment, one administers the therapeutic compound directly to the site. Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models. Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.

4.12.2 COMPOSITIONS/FORMULATIONS

[0264] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.

[0265] When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0266] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0267] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0268] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0269] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0270] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0271] A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.

[0272] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.

[0273] The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.

[0274] The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.

[0275] The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response. Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.

[0276] The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.

[0277] A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

[0278] The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formned, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

[0279] Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

4.12.3 EFFECTIVE DOSAGE

[0280] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.

[0281] A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g. Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0282] Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0283] An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.

[0284] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

4.12.4 PACKAGING

[0285] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

4.13 ANTIBODIES

[0286] Also included in the invention are antibodies to proteins, or fragments of proteins of the invention. The term “antibody” as used herein refers to immnunoglobulin 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, an antibody molecule 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.

[0287] An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally 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 shown in SEQ ID NO: 1-441, 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.

[0288] In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of alpha-2-macroglobulin-like protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein 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 of which is incorporated herein by reference in its 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.

[0289] 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.

[0290] The term “specific for” indicates that the variable regions of the antibodies of the invention recognize and bind polypeptides of the invention exclusively (i.e., able to distinguish the polypeptide of the invention from other similar polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, full-length polypeptides of the invention. As with antibodies that are specific for full length polypeptides of the invention, antibodies of the invention that recognize fragments are those which can distinguish polypeptides from the same family of polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.

[0291] Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of a polypeptide of the invention), diagnostic purposes to detect or quantitate a polypeptide of the invention, as well as purification of a polypeptide of the invention. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific. The invention further provides a hybridoma that produces an antibody according to the invention. Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention.

[0292] Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.

[0293] The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed. The antibodies may also be used directly in therapies or other diagnostics. The present invention further provides the above-described antibodies immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose®, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immuno-affinity purification of the proteins of the present invention.

[0294] 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.

4.13.1 POLYCLONAL ANTIBODIES

[0295] 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 that can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

[0296] 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).

4.13.2 MONOCLONAL ANTIBODIES

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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).

[0301] 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). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.

[0302] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. 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 as cites in a mammal.

[0303] The monoclonal antibodies secreted by the subcloned can be isolated or purified from the culture medium or as cites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0304] 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.

4.13.3 HUMANIZED ANTIBODIES

[0305] 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); Riechinann 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 that 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 immnunoglobulin 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)).

4.13.4 HUMAN ANTIBODIES

[0306] Fully human antibodies relate to antibody molecules in which essentially the entire sequences 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).

[0307] 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)).

[0308] Human antibodies may additionally be produced using transgenic nonhuman animals that 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 that 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.

[0309] 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.

[0310] 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.

[0311] 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.

4
.13.5 FAB FRAGMENTS AND SINGLE CHAIN ANTIBODIES

[0312] 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.

4.13.6 BISPECIFIC ANTIBODIES

[0313] 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.

[0314] 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 May 13, 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.

[0315] 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).

[0316] 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 that 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.

[0317] 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 (TN1B) 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.

[0318] 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.

[0319] 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).

[0320] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0321] 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 FcγRf (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).

4.13.7 HETEROCONJUGATE ANTIBODIES

[0322] 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.

4
.13.8 EFFECTOR FUNCTION ENGINEERING

[0323] 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).

4.13.9 IMMUNOCONJUGATES

[0324] 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).

[0325] 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.

[0326] 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.

[0327] 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.

4.14 COMPUTER READABLE SEQUENCES

[0328] In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention. As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.

[0329] A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[0330] By providing any of the nucleotide sequences SEQ ID NOs: 1-441 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NOs: 1-441 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.

[0331] As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.

[0332] As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues. However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[0333] As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

4.15 TRIPLE HELIX FORMATION

[0334] In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.

4.16 DIAGNOSTIC ASSAYS AND KITS

[0335] The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.

[0336] In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample. Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.

[0337] In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.

[0338] In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.

[0339] Conditions for incubating a nucleic acid probe or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.

[0340] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention. Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.

[0341] In detail, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe. Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

4.17 MEDICAL IMAGING

[0342] The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection). See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778. Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.

4.18 SCREENING ASSAYS

[0343] Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in SEQ ID NOs: 1-441, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of:

[0344] (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and

[0345] (b) determining whether the agent binds to said protein or said nucleic acid.

[0346] In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

[0347] Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

[0348] Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.

[0349] Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound). Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound). Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.

[0350] The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.

[0351] For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W. H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.

[0352] In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control. One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.

[0353] Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.

[0354] Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.

4.19 USE OF NUCLEIC ACIDS AS PROBES

[0355] Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences. The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NOs: 1-441. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NOs: 1-441 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.

[0356] Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described in U.S. Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences. Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both. The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.

[0357] Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes. Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences. The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.

[0358] Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.

4.20 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES

[0359] Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.

[0360] Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon. One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers. Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin. Microbiol. 28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, (1989) Mol. Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.

[0361] Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker. For example, Broude et al. (1994) Proc. Natl. Acad. Sci. USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Of course, this same linking chemistry is applicable to coating any surface with streptavidin. Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).

[0362] Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used. Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH. CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling. CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA rasmussen et al., (1991) Anal. Biochem. 198(1) 138-42).

[0363] The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991). In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res. 11(8) 6513-29). This is beneficial as immobilization using only a single covalent bond is preferred. The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm. To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group. It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.

[0364] More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7. A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.

[0365] Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well. The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).

[0366] It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference. This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support. The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support. Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.

[0367] An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed. For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991) Science 251(4995) 767-73, incorporated herein by reference. Probes may also be immobilized on nylon supports as described by Van Ness et al. (1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal. Biochem. 169(1) 104-8; all references being specifically incorporated herein.

[0368] To link an oligonucleotide to a nylon support, as described by Van Ness et al. (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.

[0369] One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference). These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips). These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.

4.21 PREPARATION OF NUCLEIC ACID FRAGMENTS

[0370] The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps. For example, Sambrook et al. (1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).

[0371] DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods. Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.

[0372] The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.

[0373] Low pressure shearing is also appropriate, as described by Schriefer et al. (1990) Nucleic Acids Res. 18(24) 7455-6, incorporated herein by reference). In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures. A lever device allows controlled application of low to intermediate pressures to the cell. The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.

[0374] One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al. (1992) Nucleic Acids Res. 20(14) 3753-62. These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.

[0375] The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.

[0376] As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed

[0377] Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization. This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip. Phosphate groups must also be removed from genomic DNA by methods known in the art.

4.22 PREPARATION OF DNA ARRAYS

[0378] Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm2, depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones. Each of the subarrays may represent replica spotting of the same samples. In one example, a selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples maybe spotted on one 8×12 cm membrane. Subarrays may contain 64 samples, one from each patient. Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.

[0379] Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g. a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips. A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.

[0380] The present invention is illustrated in the following examples. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples. The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.

[0381] All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.

5.0 EXAMPLES

5.1 EXAMPLE 1

[0382] Novel Nucleic Acid Sequences Obtained from Various Libraries

[0383] A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques. The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts. Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences. The clones were clustered into groups of similar or identical sequences. Representative clones were selected for sequencing.

[0384] In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol. PCR products were purified and subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences. In some cases RACE (Random Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.

5.2 EXAMPLE 2

[0385] Novel Nucleic Acids

[0386] The novel nucleic acids of the present invention of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases. The nucleic acids were assembled using an EST sequence as a seed. Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 119, gb pri 119, and UniGene version 119) that belong to this assemblage. The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage. Inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.

[0387] Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full length gene cDNA sequence and its corresponding protein sequence were generated from the assemblage. Any frame shifts and incorrect stop codons were corrected by hand editing. During editing, the sequence was checked using FASTY and/or BLAST against Genbank (i.e., dbEST version 120, gb pri 120, UniGene version 120, Genpept release 120). Other computer programs which may have been used in the editing process were phredphrap and Consed (University of Washington) and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). The full-length nucleotide and amino acid sequences, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NOS: 1-441.

[0388] Table 1 shows the various tissue sources of SEQ ID NO: 1-441.

[0389] The homology for SEQ ID NO: 1-441 were obtained by a BLASTP version 2.0al 19MP-WashU search against Genpept release 120 and the amino acid version of Geneseq released on Oct. 26, 2000, using BLAST algorithm. The results showed homologues for SEQ ID NO: 1-441 from Genpept. The homologues with identifiable functions for SEQ ID NO: 1-441 are shown in Table 2 below.

[0390] Using eMatrix software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp. Biol., Vol. 6 pp. 219-235 (1999) herein incorporated by reference), all the sequences were examined to determine whether they had identifiable signature regions. Table 3 shows the signature region found in the indicated polypeptide sequences, the description of the signature, the eMatrix p-value(s) and the position(s) of the signature within the polypeptide sequence.

[0391] Using the pFam software program (Sonnhammer et al., Nucleic Acids Res., Vol. 26(1) pp. 320-322 (1998) herein incorporated by reference) all the polypeptide sequences were examined for domains with homology to certain peptide domains. Table 4 shows the name of the domain found, the description, the p-value and the pFam score for the identified domain within the sequence.

[0392] The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determined from using Neural Network SignalP V1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication “Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites” Protein Engineering, Vol. 10, no.1, pp. 1-6 (1997), incorporated herein by reference. A maximum S score and a mean S score, as described in the Nielson et as reference, was obtained for the polypeptide sequences. Table 5 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide.

1TABLE 1HYSEQLIBRARY/LIBRARYTISSUE ORIGINRNA SOURCENAMESEQ ID NOS:adult brainGIBCOAB300176-77 91 106-107 115 134 163-164 178203 232 255 265 276 279 322-323adult brainGIBCOABD00316 19 24 77 80-81 85 89-90 92 96 98105 110 116 121-123 125 130-132 134-136138 142-143 151 153 158-159 163-164184 191 193 196 198 200 208-209213-214 216 219-220 223 229 232-234236 239 241 243 257-259 262 265 267274-276 278 284 292 302 317 321 324-325327 337-338 340 348 359 371 391-392400adult brainClontechABR0011 18-19 35 80 98 125 136 153 185 200209 221 228-229 239 243 274-275 302399-400adult brainClontechABR0065 7-8 18 32 35 52 57 85 91 96 111 113126 131 135 138-139 142 148 153-154181 188 192 199 209-211 217 221 224226 229 233 235 238 243 248 273 283-284286 292 316 322 348 357 361 367376 378 399 407 409 417 428adult brainClontechABR0082 4 6-11 19-21 23-25 31 35-37 39-4145-46 72-73 76 80-81 85 88-90 94-95 97102-105 109 111-112 114-119 121-122126-131 134-135 138-139 144 146-150152-153 156-157 159 168-172 174-175178 180 182 185-186 189-190 194 196198-201 203 205-210 217 219 221-222224 229-230 232-233 236-239 243-244248 253-256 260-261 263-265 273 276281-282 286-289 291-292 299-300 302304 315-317 319 321-322 324 326 329331-332 341 352-357 360 362 365 367-368370 376-377 379-380 383-384 387-389391-392 394 396-402 407-410 412-413419 425-426 433adult brainClontechABR01185 90adult brainBioChainABR012148 213adult brainInvitrogenABR01385 322adult brainInvitrogenABR0149 23 85 146 200 233 282 321 330adult brainInvitrogenABR01514 31 69 121 124 163 209 216 224 291377adult brainInvitrogenABR01692 136 219 279adult brainInvitrogenABT0042 7-8 20-21 33 85 90-91 95 97 102-103108 121 123 129-131 138-139 143 146151 153 157-158 172 178 180 209-210213 219 229-230 232 234 239 308 321330 360 365 370-373 375 401 412culturedStrategeneADP0013-4 23 36 79 81 106-107 116 129 133-134preadipocytes147 151 154 158 179 181 192 196222 230 256-257 287 292 297 313 329359adrenal glandClontechADR0022 25 27 33 57 76 85-86 88 96 98 105-108114 121-122 125 129-130 134 147164 178 180 182 198-199 201 205 207-208240-241 244 246 253-254 257 261276 280 292 320 329 336 352 403adult heartGIBCOAHR0013 17-21 27 32 74 76 85 89-91 95-96102-103 105-110 117 121 124-125 128131 134-136 139 141 148 151-153 155-156161 163 181-182 186 190 193 198200-201 205 207 211-213 215 222 225229-230 234 251-254 257-259 263 274-277280 292-297 301 303-304 315-316319 329-331 345 359 384 417 423-424adult kidneyGIBCOAKD0013-5 14 16 19-29 34-35 76 79 85 89-9094-95 97 101-103 106-107 115 117-118121-122 124-125 128-130 133-136 138144 146 158-159 163 166 171 177-178182 185-186 189 194 196-198 200-201205 207 211-213 216-217 223 225 228-232243 247-248 253-255 257 267 272277-278 283 287 291 298-301 315 326358-359 364adult kidneyInvitrogenAKT0023 6 14 20-21 25-26 76 79 85 89 94 101111 114 118 121 124 126 130-131 138146 163 170 177-178 189 196 198 201204 213 231 253-254 256-259 271 273-275277 298 315 320 329 342adult lungGIBCOALG0014 29 74 79 85 90 96 105 111 119 132134 136 142 144 149 159 181 189 198200 205-207 226 255 257 263 283 294300 302-303 328 358-359 365 426lymph nodeClontechALN0016 16 31 105 120 215 257 295 306 309359young liverGIBCOALV00110-11 25-26 29 31 33 76 85 95 115 121-122124 126 130 143 146 156 158 164178 182 187 189 229 248 253-254 261278 283 304 342 375adult liverInvitrogenALV00210-12 23 26 31 33-34 38 53 56 90-9294-95 118 121 124 128-129 138 141 146148 153 156 161 171 178 198 216 232248 253-254 256-257 264 302 306 365375 383 396adult liverClontechALV00310-11 156 171 188adult ovaryInvitrogenAOV0013-8 10-11 14 16 19-22 24 27-31 34 3657 73 75-76 81-82 85 89-91 94-98 104-109111 115-116 121-128 130-131 134136 138-139 141 143-144 146 149-150152 155 157-160 163-166 170-173 175177-178 180 182 184-187 189-190 193-194196-197 200-201 212-213 215 217222 225-226 228 230-233 235 241-243245 248 253-259 261 266-267 270 272-273276-278 283-285 287 289 292 297-299305-306 315-317 319 323-325 329-331341 343-344 352 358-359 363-366382-383 386 389-390 412adult placentaClontechAPL00173 92 117 135 182 194 232 246 261 272282 359placentaInvitrogenAPL00216 28 92 121 135 144 157 178 210 394adult spleenGIBCOASP0013-4 16 32-33 35 90 96 99-100 123-125128 131 134 136 139 151 178 181 189194 200 210 218 229 251 253-255 257276 283 307-309 315 329 354-355 357392 400testisGIBCOATS00122 73 82 91 96-97 104-105 117 124 130134 164 173 200 209 222 233 241 253-254257 285 287-288 305 325 329 351-353359adult bladderInvitrogenBLD0014 108 130 150 212 226 236 240 242 257276 287 305 395-396 415bone marrowClontechBMD0011 4-5 22 29-30 34 72 85 88 90 92 94 98104-107 109 111 113 117 120 123-125128-129 132 135 140 142 144 146 152163 165-166 170-173 177 180 182 186189-190 198-209 215 222 225 232 240-246251-252 260-261 273-275 277-280283-285 300 316 318 346-347 359bone marrowClontechBMD0021 4 7-8 10-11 16 19 25 31 49 61-62 7274 76 80 85 88 90 93-95 97-101 109-110112 114 116-117 121 126 129 132 135141 144 146 149-150 154 157 160 162-163165-166 170-172 175 178-180 182-183186-190 192-194 198-200 203 208210-213 215 223 225 234 242 245 247251-254 256-257 265 270 273 276-278280 285 287 289 291 293-294 299 302307 309 315 322 324 337-338 353 356-357359 367 369 388 407 414 419 426434bone marrowClontechBMD007144adult colonInvitrogenCLN0014 25 33 85 138 146 148 158-159 198 210229 301 360 384 397Mixture of 16VariousCTL02123 359tissues - mRNAs*Vendors*Mixture of 16VariousCTL028172tissues - mRNAs*Vendors*adult cervixBioChainCVX0013 5 10-11 18 20-21 24-25 29 36 41 4757 63 72 74 76 86 90 94 104 108-109111 125 127 130 134 138 144 147 162174 178-179 182 186 189 193 197 211222 225-226 228 232 241 243 257 261267 270 273-275 278-281 288-289 298301-302 305 315 319 324-325 329 331337-338 359 391-392 395 420endothelialStrategeneEDT0013-6 18-19 24 27-29 35 72 76 79-80 85cells89 96 98 104-107 111 117 119-121 124-134134 136 138-139 141 144 146-147149 152 158-159 166-167 170-173 178-179182-183 186-187 191 193-194 196-197200 210-211 222-224 226 231-232236 241 243 246 248 253-256 258-259276 279 282 287 292 300 302-303 315329 337-338 358-362 382-383 385-388Genomic clonesGenomic DNAEPM001435from the shortfrom Geneticarm ofResearchchromosome 8esophagusBioChainESO002257fetal brainClontechFBR00134fetal brainClontechFBR0043 139 144 271 284 337-338fetal brainClontechFBR0064 6-11 14 18-21 24 28 31 37-38 40 6376 85 87 89-90 94-95 97 105 108-109112-113 115 117-120 127-130 133 138140 144-146 148 170 172 175 180 182186-188 190 192 194 199 201 203 209-210215 219 222 229-230 232-233 240243 245 253-255 270 273 276 281 288-289292 295 304 315 317 319 324 330-331356-357 359-360 364 367-368 379-380383 389 397 399-401 408-409 411413 419 421 423fetal brainInvitrogenFBT0022 14 19 23 28 31 90 94 105 121 124 126131 135 139 142 149 158 186 193 198210 214-215 232 239 242 248 255 267326 332 365 369 371 376-383 394 399fetal heartInvitrogenFHR0014 7-8 10-11 14 17-21 28-29 31-32 6064-65 73 85 87 92 95 102-103 105 108111 113 117 119 121 125 128-129 134-135141 152 154 156-157 160-161 172176 178 194 196 198-200 203 208 212215 218 222 226 229 233-234 253-257261 265 272 276 281 292-293 295 303305 319 325 327 337-338 341 345 349354-355 367-368 389 395-396 398 412417 436fetal kidneyClontechFKD0011 14 22 94 110 115 132 134-135 146 178189 199 235-236 242 247 257 267 292295 359fetal kidneyClontechFKD00222 31 38 40 46 94 122 127 131 156 160194 198 229 253-254 270 292 303 319354-355 389 396fetal kidneyInvitrogenFKD007303fetal lungClontechFLG00185 89 98-100 111 175 271 281 369fetal lungInvitrogenFLG00384 88 106-107 122 135 140 146 160 181246 272 284 292 328 330 396 404 416426fetal liver-ColumbiaFLS0011-3 6-12 14 19 23 28-31 33 57 59-60spleenUniversity72-76 78 80 83 85-138 140-141 143-144146-155 157-161 163-197 200 204 208210-211 223 225 230 232-233 235 241-243245-266 268-273 277 281 285-287292 297 303 314 329 343 346-347 357-359369 397 399 407 415fetal liver-ColumbiaFLS0021 3-4 6 10-12 23-24 29 31-33 35-37 53-54spleenUniversity74-76 79 81-82 86-89 91 94-95 99-104106-109 111-112 115 117-120 122125-126 128-129 132 134 136-138 141146 149 153 157-159 162-166 170 172175 178-180 183 185-191 194 196-197205 207-212 222-225 228 232-233 239-241248 251-252 255-256 258-259 261-262264 266-267 270-271 273-275 277-278283 285 287 298 305 315 317-318322 330-332 337-338 341 343 349 357-360365 388 390-391 399 402 418 424fetal liver-ColumbiaFLS00312 29 91 98 111 119 156 163 165 178spleenUniversity186 193 210-211 276 286 315 322 346-347357 365 424fetal liverInvitrogenFLV0017-8 14 35 118 122-123 129 146 182 211230 232 248 251-252 264 287 304 337-338344 346-347 352 365 367-369fetal liverClontechFLV002102-103 147 149 300fetal liverClontechFLV00473 85 105 108 118 122 126 141 156-157161 165 170 178 180 182 194 215 218225 240 242 247 251-252 292 330 337-338369 407 411 440fetal muscleInvitrogenFMS00117 80 85 90 139 147 153 173 178 198201 220 229 236 293 296 303 323 334341 345 359 372fetal muscleInvitrogenFMS0025 9 17-18 20-21 29 38 85 88 97 106-107129 131 136 150-152 155 165 170 179182 192-193 212-213 229 234 242 258-259270 282 286 289 300 316 319 345351 354-355 360 389 396 408 410 437439fetal skinInvitrogenFSK0012 4 7-8 29 33 42-43 49 51-52 58 74 8285 90 94 110-111 116 118 121 133 136138-139 145 151 154 156-157 161-162172 181 184 186 193 198 200 205 207209-211 222 227-230 232 235 240 246253-257 266 270 276 292 295 299 316318 323 330 332 337-340 343 357 369389 394-395 412 422 427fetal skinInvitrogenFSK0024 9 42 44 51 66 72 81 85 89-90 95 98105 112-114 119 121 129 133 135 162172 179-182 197 200 208 210 231 243-244272 304 316 330 339 354-355 357360 389 395 410 417 437fetal spleenBioChainFSP001157 223umbilical cordBioChainFUC0014-6 20-21 25 29 73-74 83 87 89-91 94101 109 120 123 125 128 130-131 133141 143-144 147 149 154 161 165 173175 179 184 188 210-212 217 226 235240 248 251-252 257 262 267 270 277293 305 307 316 319 323 327 331 341356 359 389 392 407 416fetal brainGIBCOHFB0012-4 16 20-21 74 77 85 89-91 96-98 104-105111 114 118 121-122 124-125 127-128131 134 137-140 142 144 146-148151 153 158-159 163-164 166 173 178180 182 191 194 196 200 203 209-214216-232 234-236 238-239 243 253-255263 270 272-273 276 281 292 310 316319-321 332 348 357 359 365 399macrophageInvitrogenHMP0012 247infant brainColumbiaIB20022-4 7-8 19-22 26-27 31-32 35 73-74 80University85 89 91 96-98 106-107 110 112 118-119121-122 125 128-131 134-144 148 153164 166 172-173 177 180 186-187 191-194196 202-203 208-210 217 219 223-224227 229 232-234 236-237 239 241-243245 248 253-259 273-275 278-279282 287 294 298 309 314 317 322 327330 333-334 341 348-350 360 368 376379-380 382 396 406 424infant brainColumbiaIB20033-4 20-21 26 28 31 35 73 85 95-96 110University113 119 122-123 130-131 135 138 140142-143 146 153 155 170 172-173 186191-193 196 209 219 223 226 229 233-234236 239 245 248 253-254 256-257273 279 291-292 304 314 337-338 343359 367 371 376 397 413infant brainColumbiaIBM002129 138 140 193 209 251-252 256 317University373 397infant brainColumbiaIBS00131 48 85 138 151 180 193 229 239 265University292 326 337-338 371lung, fibroblastStrategeneLFB0013 6 31 72-73 90 92 105-107 124 126-127133 136 139 144 146 172 189 198 204233 235 246 258-259 268 272 276 282310 335 359 434lung tumorInvitrogenLGT0024 19-21 28 33 35-36 49 72 79 81 85 8890-91 94-95 101 106-107 109 118 120-125127 130-131 133 135-138 141-142144 147 149 157 159-161 163 166 170-173193-194 196-197 212 216 218 221223 226 228-229 231 233 241 247-248253-255 257 261 266-267 270-275 277-278282-283 292 298 301 303 315 318324 331 335 354-355 359 367 369 381392-393 398lymphocytesATCCLPC0014 6 10-11 16 42 76 88 91 95-98 104 109114 120 122 133-134 144 146 148 157-158161 164 171 180 186-187 203 209212 223 231 242-243 248 251-252 255258-259 271-273 281-282 299 334 336362 374 418leukocyteGIBCOLUC0011-5 15 19-21 28 30-33 37 72 74 91 94-9597-100 108-109 113 115 117 119-122124-125 127-128 134-138 141 144 146-148150-151 157-158 160 162-167 170-173175-178 180-181 187 189 192 194197 200 212-213 215-216 218-219 223225 228-232 241-242 245-246 251-254261 272-276 278-282 284 287-290 297-298305 307 310-314 325 331 336 340358-359 372 399 414leukocyteClontechLUC0031 5 124 171 176 204 225 248 253-254283 285 307 315melanoma fromClontechMEL0044-5 24 37 72-74 81 85 106-107 113 136cell line ATCC177 203 205-207 209 231 243 284-285#CRL 1424315-316 320 326 359 374 428mammary glandInvitrogenMMG0012 4-5 7-8 10-12 29 31 34-35 38 50 80-8185 89-90 92 94-97 105 108-109 119-124126 128-130 135 138-139 141-142144 146-147 153 155 157-159 163 178-179181-182 198 200 209-210 219 223228 230 232-233 235-236 239 242 248253-255 257 260-261 265-267 270 272281 287 292 294 315-316 318 324 327330 337-340 354-355 357 369 372 383392-395 401 404induced neuronStrategeneNTD00135 47 89-90 111 118 164 232 253-254cells276 324 331 382retinoid acidStrategeneNTR00120-21 37 122 147-149 170 179 181 186induced neuronal212 226 258-259 265 276 369 436 438cellsneuronal cellsStrategeneNTU0017-8 37 55 80 85 112 118 126-127 133138 140-141 151 170 181 210 214 225-226236 243 287 328 330-331 357 383400 436pituitary glandClontechPIT00492 124 159 231placentaClontechPLA00334 46 88 126 128 159 182 186 197 201267 278 281-282 305 330 356 361 365418prostateClontechPRT00118 36 72 74 86 95 106-107 111 118 122144 161 179 211 218 233 286 297rectumInvitrogenREC0019 31 85 121 128 147 171 200 219 257292 340 394 398 407 412salivary glandClontechSAL0013 24 38 80 122 136 147 189 241 282 296310 351 392 395 415salivary glandClontechSALs03118skin fibroblastATCCSFB003257small intestineClontechSIN00112 16 25 82-83 89-90 93 95 98 105-109111 122-123 125-128 133-134 137 139142 161 167 171 184 197 201 204 212218 236 242-243 248-249 253-254 257267 276 284-285 292 297 300 303 310313 317-318 325 340 343 352 354-355359 383 391 416skeletal muscleClontechSKM0013 85 90 121 136 154 200 211-212 224234-235 272 288 293 303 353 426spinal cordClontechSPC0013 39 84 86 94 96 105 115 117 130-131134 136 141 143 148 155 176 190-191203 213 224 233-234 236 239 279 283298 320-321 332 336-338 356 359 365404-406adult spleenClontechSPLc017-8 35 80 94 106-107 120 133 144 152155 241 253-254 263 329 407 428stomachClontechSTO00110-11 23 30 77 98 106-107 117 131 135139 144 257 285 343 367 382 394thalamusClontechTHA0022 20-21 23 74 81 85 105-106 116 121131 146 171 185 188 200 209 219 233239 256 258-259 273 276 362 399thymusClontechTHM00116 29 33 57 80 82 85 90 93-94 106-107120 126 128 134 141 161 176 194 223228 235 253-254 261 274-275 278 285298 319 332 336 343 353 359 425thymusClontechTHMc021-2 7-9 14 26 34 44 73 75 82 85 87 9498 106-107 109-111 117 119-120 125-126128-129 139 141 144 147-148 151 154-155162 165 170-172 175-176 179 182186 193-194 199-200 208-209 213 218233 235 240 242 247 253-254 257 265276 281 287 290 305 307 312 319 336342 354-356 359 364 367 399 408 412-413415 419 421 426 429-433thyroid glandClontechTHR0013 5 7-8 28 30-31 33 73-77 80 82 85 8890-92 94 96-98 105-107 109 113 117121-122 124-125 127-128 130 134 136141 143 146-148 152 161-163 166 175177-178 181 194 199 201 204 210 212216 218 223-226 228 230-231 234 236241 243 246 253-257 261 270 272-273276-278 281-283 287 292 295 298 303-304308 315 323 329 335 352 359 362401 416-417tracheaClontechTRC00188 138 180 226 228 279 359 411 436uterusClontechUTR0013 10-11 23 77 92 106-107 109 111 141197-198 218 241 257 270 274-275 302315 329 396 400 413*The 16 tissue-mRNAs and their vendor source, are as follows: 1) Normal adult brain mRNA (Invitrogen), 2) normal adult kidney mRNA (Invitrogen), 3) normal adult liver mRNA (Invitrogen), 4) normal fetal brain mRNA (Invitrogen), 5) normal fetal kidney mRNA (Invitrogen), 6) normal fetal liver mRNA (Invitrogen), 7) normal fetal skin mRNA (Invitrogen), 8) human adrenal gland mRNA (Clontech), 9) human bone marrow mRNA (Clontech), 10) human #leukemia lymphablastic mRNA (Clontech), 11) human thymus mRNA (Clontech), 12) human lymph node mRNA (Clontech), 13) human spinal cord mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA (BioChain), 16) human conceptional umbilical cord mRNA (BioChain).

[0393]

2

TABLE 2

SMITH-

SEQ ID
ACCESSION

WATERMAN
%

NO:
NUMBER
DESCRIPTION
SCORE
IDENTITY

1
AF255303

Homo sapiens
membrane-
2074
67

associated nucleic acid

binding protein

2
Y57911
Human transmembrane
865
94

protein HTMPN-35

3
AL121961

Homo sapiens
dJ104A17.1
1426
100

(novel protein)

4
X90872

Homo sapiens
associated
1096
98

to Golgi apparatus

5
AL161659

Homo sapiens
bA526K24.1
4348
99

(A novel protein)

6
AF047185

Homo sapiens
NADH-
505
100

ubiquinone

oxidoreductase subunit

CI-B8

7
AL162458

Homo sapiens
bA465L10.1
3765
100

(novel protein similar

to Drosophila CG11399)

8
AL162458

Homo sapiens
bA465L10.1
3366
100

(novel protein similar

to Drosophila CG11399)

9
U28928

Caenorhabditis elegans

450
30

similar to C. elegans

protein F59B2.2

10
U66411

Drosophila melanogaster

336
57

putative type III

alcohol dehydrogenase

11
U66411

Drosophila melanogaster

425
52

putative type III

alcohol dehydrogenase

13
AL096710

Homo sapiens
dJ61B2.2
733
99

(similar to MACF

cytoskeletal protein)

14
L05093

Homo sapiens
ribosomal
887
93

protein L18a

16
AF061961

Mus musculus
putative
345
33

zinc finger protein

FLIZ1

17
U76373

Mus musculus
skm-BOP1
2494
94

18
AJ272073

Torpedo marmorata
male
2307
80

sterility protein 2-like protein

19
Z82084

Caenorhabditis elegans

279
27

contains similarity to

Pfam domain: PF01769

(Divalent cation

transporter),

Score = 211.5, E-

value = 4.2e−60, N = 2

20
AF090989

Homo sapiens
high-risk
3089
68

human papilloma viruses

E6 oncoproteins targeted

protein E6TP1 alpha;

putative GAP protein

alpha

21
AF090989

Homo sapiens
high-risk
3089
68

human papilloma viruses

E6 oncoproteins targeted

protein E6TP1 alpha;

putative GAP protein

alpha

22
U20286

Rattus norvegicus
lamina
739
59

associated polypeptide

1C

23
AF207829

Homo sapiens
SCAN-
1104
100

related protein RAZ1

24
X78817

Homo sapiens
p115
1118
45

25
D86417

Bacillus subtilis
YfmJ
359
43

26
L01083
Oryctolagus cuniculus
683
36

UDP-

glucuronosyltransferase

27
AF000240

Gallus gallus

385
37

monocarboxylate

transporter 3

28
Z36948

Caenorhabditis elegans

138
42

contains 3 cysteine rich

repeats

29
D17554

Homo sapiens
TAXREB107
1207
98

30
L06505

Homo sapiens
ribosomal
845
100

protein L12

31
AL136538

Schizosaccharomyces

680
37

pombe
putative

cysteinyl-trna

synthetase

32
AB040900

Homo sapiens
KIAA1467
2232
100

protein

33
AL031685

Homo sapiens
dJ963K23.2
521
46

(novel protein)

34
AF263742

Homo sapiens
golgin-like
297
45

protein

35
AF030131

Mus musculus
Plenty of
2951
88

SH3s; POSH

36
Z22818

Canis familiaris
Rab12
1071
99

protein

37
AL110477

Caenorhabditis elegans

198
34

Y113G7B.24

38
AC002505

Arabidopsis thaliana

157
28

putative

phosphatidylinositol-4-

phosphate 5-kinase

39
AF099810

Homo sapiens
neurexin
1255
99

III-alpha

40
AL133215

Homo sapiens
bA108L7.6
4406
99

(semaphorin 4G (sema

domain, immunoglobulin

domain (Ig),

transmembrane domain

(TM) and short

cytoplasmic domain) )

41
AL109657

Homo sapiens
dJ842G6.1
247
100

(novel protein)

42
AB033744

Mus musculus
type II
2419
91

cytokeratin

43
M27685

Mus musculus
ultra-high
702
60

sulphur keratin

44
M94081

Homo sapiens
putative
110
100

46
AJ278348

Homo sapiens
pregnancy-
8966
99

associated plasma

protein-E

47
Z98762

Schizosaccharomyces

190
36

pombe
profilin.

48
AF026544
Ralstonia eutropha phbF
561
66

49
AB036882

Mus musculus
midnolin
950
67

50
Z46967

Homo sapiens
calicin
3076
99

51
AF204929

Sus scrofa
thyroxine-
1060
52

binding globulin

52
AL035694

Homo sapiens
dJ33L1.1
2634
100

(novel T-box (Brachyury)

family protein)

54
AF079864

Rattus norvegicus

508
51

putative G-protein

coupled receptor RA1c

55
AL022373

Arabidopsis thaliana

200
35

glycine-rich protein

56
AJ007767

Homo sapiens
T-cell
566
97

antigen receptor-alpha

57
AF041107

Rattus norvegicus
tulip 2
1884
94

58
L32179

Homo sapiens

1035
52

arylacetamide

deacetylase

59
AB010414

Homo sapiens
G-protein
127
56

gamma 7

60
AJ277486

Mus musculus
T-box
1463
93

transcription factor

61
AF164612

Homo sapiens
Gag protein
131
29

62
S61069

Homo sapiens
reverse
232
80

transcriptase

homolog = pol {retroviral

element }

63
AF266172
Gillichthys mirabilis
127
81

60S acidic ribosomal

protein P1

64
M18184

Mus musculus
lymphocyte
140
37

differentiation antigen

65
AL035587

Homo sapiens
dJ475N16.3
434
88

(novel protein similar

to RPL7A (60S ribosomal

protein L7A) )

66
M34225

Homo sapiens
cytokeratin 8
866
64

67
Y17166
Campylobacter jejuni
111
31

ribosomal protein S6

68
AF252290

Mus musculus
PAR6A
1492
76

69
Z46787

Caenorhabditis elegans

522
51

contains similarity to

Pfam domain: PF00097

(Zinc finger, C3HC4 type

(RING finger) ),

Score = 17.6, E-

value = 0.00013, N = 1

70
AF205718

Homo sapiens

473
76

hepatocellular

carcinoma-related

putative tumor

suppressor

71
AJ006692

Homo sapiens
ultra high
774
79

sulfer keratin

72
AK001269

Homo sapiens
unnamed
3518
100

protein product

73
AE003453

Drosophila melanogaster

756
43

CG9752 gene product

74
AK000725

Homo sapiens
unnamed
3013
100

protein product

75
AF231921

Homo sapiens
unknown
1215
100

76
AK024664

Homo sapiens
unnamed
2522
100

protein product

77
AL133661

Homo sapiens

1612
100

hypothetical protein

78
AK000765

Homo sapiens
unnamed
1450
100

protein product

79
AK023453

Homo sapiens
unnamed
774
100

protein product

80
AK000474

Homo sapiens
unnamed
737
100

protein product

81
W93948
Human regulatory
441
91

molecule HRM-4 protein

82
AK024435

Homo sapiens
FLJ00025
145
24

protein

83
AK000820

Homo sapiens
unnamed
652
100

protein product

84
AL080154

Homo sapiens

838
100

hypothetical protein

85
D45131

Homo sapiens
basigin
1302
99

86
AL365412

Homo sapiens

580
100

hypothetical protein,

similar to (NP_006139.1)

LASP-1, LIM and SH3

domain protein 1

87
L03188

Saccharomyces cerevisiae

216
26

putative

88
X74855

Mus musculus
zinc finger
362
31

protein 51

89
AF152344

Mus musculus
F2 alpha
4195
89

prostoglandin regulatory

protein

90
AF020262

Bos taurus
general
607
100

protein transport factor

p16

91
AL096882

Arabidopsis thaliana

146
24

putative protein

92
M75099

Homo sapiens
rapamycin-
740
100

and FK506-binding

protein

93
AF163151

Homo sapiens
dentin
162
22

sialophosphoprotein

precursor

94
AL078593

Homo sapiens
dJ210B1.1
745
40

(KIAA0680)

95
AJ223802

Arabidopsis thaliana
2-
1463
42

oxoglutarate

dehydrogenase, E1

subunit

96
AB020749

Arabidopsis thaliana

144
24

97
U06944

Mus musculus
PRAJA1
1087
76

98
AL096678

Homo sapiens
dJ122O8.4
10626
100

(KIAA0301)

99
AL356276

Homo sapiens
bA367J7.7
1533
100

(novel Immunoglobulin

domains containing

protein)

100
AL356276

Homo sapiens
bA367J7.7
1533
100

(novel Immunoglobulin

domains containing

protein)

101
AF200691

Drosophila melanogaster

1031
38

Dispatched

102
AF177144

Mus musculus
mammalian
1027
50

inositol

hexakisphosphate kinase 1

103
AF177144

Mus musculus
mammalian
1039
51

inositol

hexakisphosphate kinase 1

104
AL021918

Homo sapiens
b34I8.1
1624
48

(Kruppel related Zinc

Finger protein 184)

105
X69115

Homo sapiens
ZNF37A
1371
99

106
AF142406

Babesia bigemina
200 kDa
325
24

antigen p200

107
AF161358

Homo sapiens
HSPC095
419
100

108
AL163852

Arabidopsis thaliana

142
30

putative protein

109
Z99531

Schizosaccharomyces

346
31

pombe
caffeine-induced

death protein 1

110
M29580

Homo sapiens
zinc finger
510
57

protein 7 (ZFP7)

111
L00923

Mus musculus
myosin I
5389
96

112
AP001751

Homo sapiens
gene
1527
50

similar to rat protein

kinase (KID2)

113
Y12781

Homo sapiens
transducin
2431
86

(beta) like 1 protein

114
AF215703

Drosophila melanogaster

2090
66

KISMET-L long isoform

115
AL023828

Caenorhabditis elegans

445
48

cDNA EST yk167h7.3 comes

from this gene˜cDNA EST

yk167h7.5 comes from

this gene˜cDNA EST

yk289g5.3 comes from

this gene˜cDNA EST

yk332h9.3 comes from

this gene˜cDNA EST

yk289g5.5 comes from

this gene˜cDNA EST

yk332h9.5 comes from

this gene˜cDNA EST

yk391h4.5 comes from

this gene˜cDNA EST

yk653f1.5 comes from

this gene

116
AF125960

Caenorhabditis elegans

475
35

contains similarity to

dual specificity

phosphatase, catalyitic

domain (Pfam: PF00782,

Score = 16.8, E = 7.4e−05,

N = 1)

117
AF151697

Homo sapiens
sentrin-
3110
99

specific protease

118
AF022655

Homo sapiens
cep250
139
24

centrosome associated

protein

119
AK026098

Homo sapiens
unnamed
1950
99

protein product

120
X56203

Plasmodium falciparum

504
25

liver stage antigen

121
U21324

Caenorhabditis elegans

1197
52

similar to entire S.

cerevisiae
ABC1 protein

(Swiss-Prot Acc: P27697)

122
AL138641

Arabidopsis thaliana

579
33

putative protein

123
S73591

Homo sapiens
brain-
674
38

expressed HHCPA78

homolog VDUP1

124
D00761

Homo sapiens
proteasome
857
94

subunit C5

125
AF000262

Caenorhabditis elegans

343
36

similar to

CCAAT/enhancer-binding

protein

126
AF146075
Thermus thermophilus
144
31

ribosomal protein L9

127
AC006585

Arabidopsis thaliana

1129
46

putative SUDD-like

protein

128
AL031804

Arabidopsis thaliana

186
25

putative protein

129
AL035539

Arabidopsis thaliana

140
27

putative protein

130
AF151816

Homo sapiens
CGI-58
1023
54

protein

131
AC024765

Caenorhabditis elegans

223
33

contains similarity to

O-linked GlcNAc

transferases

132
M12623

Homo sapiens
high
320
95

mobility group protein

17

133
AB017614

Mus musculus
OASIS
2409
91

protein

134
AL109827

Homo sapiens
dJ309K20.4
4972
100

(KIAA0765 (HRIHFB2091,

RNA recognition motif

(RNP, RRM or RBD domain)

containing protein) )

135
AL035661

Homo sapiens
dJ568C11.3
2526
100

(novel AMP-binding

enzyme similar to

acetyl-coenzyme A

synthethase

(acetate-coAligase))

136
AL031804

Arabidopsis thaliana

237
35

putative protein

137
AL161590

Arabidopsis thaliana

232
34

putative protein

138
AB024032

Arabidopsis thaliana

372
55

gene_id: K9P8.4˜

139
AL050367

Homo sapiens

4177
94

hypothetical protein

140
AF081484

Homo sapiens
alpha-
1195
97

tubulin isoform 1

141
D14336

Mus musculus
RNA
1694
77

polymerase I associated

factor (PAF53)

142
AF023657

Rattus norvegicus
endo-
1415
67

alpha-D-mannosidase

143
AB011541

Homo sapiens
MEGF8
9785
100

144
AF112221

Homo sapiens
rap2
1189
60

interacting protein x

145
AF117382

Mus musculus

1333
91

hypermethylated in

cancer 2 protein; HIC2

146
AF151827

Homo sapiens
CGI-69
1794
97

protein

147
AC024792

Caenorhabditis elegans

424
35

contains similarity to

TR: P78316

148
AL022603

Arabidopsis thaliana

369
32

putative protein

149
AF016430

Caenorhabditis elegans

370
43

similar to Pan

Troglodytes GOR

(SP: P48778)

150
X15696

Homo sapiens
translated
129
28

region (partial)

151
AL022373

Arabidopsis thaliana

137
33

putative protein

152
AB010077

Arabidopsis thaliana

881
56

GTP-binding membrane

protein LepA homolog

153
AF062655

Mus musculus
plenty-of-
115
29

prolines-101; POP101;

SH3-philo-protein

154
AF159852

Drosophila melanogaster

243
48

RNA-binding protein

Smaug

155
AE003606

Drosophila melanogaster

465
32

CG14646 gene product

156
AE004999
Halobacterium sp. NRC-1
335
35

dihydrodipicolinate

synthase; DapA

157
AL157953
Streptomyces coelicolor
143
28

A3 (2) putative

nitroreductase

158
AF116238

Homo sapiens

1927
99

pseudouridine synthase 1

159
AF263541

Homo sapiens
protein
2844
100

kinase DYRK4

160
Z81317

Schizosaccharomyces

148
37

pombe
putative ubiquitin

carboxyl-terminal

hydrolase

161
Y18208

Rattus norvegicus

1360
89

serine-threonine

specific protein

phosphatase, glycogen-

binding (GL) subunit

162
AC005970

Arabidopsis thaliana

926
58

putative translation

initiation factor eIF-2B

alpha subunit

163
M83822

Homo sapiens
beige-like
9733
99

protein

164
AL080282

Arabidopsis thaliana

352
34

putative protein

165
U61232

Homo sapiens
cofactor E
292
27

166
AB014578

Homo sapiens
KIAA0678
5255
100

protein

167
X61123

Homo sapiens
BTG1
813
96

168
Z92954

Bacillus subtilis

570
38

product highly similar

to metabolite transport

proteins

169
AL031055

Homo sapiens
dJ28H20.1
636
44

(novel protein similar

to membrane transport

proteins)

170
AL021918

Homo sapiens
b34I8.1
1144
46

(Kruppel related Zinc

Finger protein 184)

171
U44731

Mus musculus
purine
2260
70

nucleotide binding

protein

172
AF211859

Mus musculus
cyclin
1052
92

ania-6b

173
AF181252

Mus musculus
TPR-
266
31

containing protein

involved in

spermatogenesis TPIS

174
AF096864
Pseudomonas aeruginosa
173
33

heat shock protein

175
AB042199

Homo sapiens
APC-
1783
62

stimulated guanine

nucleotide exchange

factor

176
Z11582

Saccharomyces cerevisiae

151
25

nuf1

177
AJ235270
Rickettsia prowazekii
376
37

PROBABLE O-

SIALOGLYCOPROTEIN

ENDOPEPTIDASE (gcp)

178
AK022710

Homo sapiens
unnamed
527
100

protein product

179
D50617

Saccharomyces cerevisiae

139
28

YFL027C

180
AF065389

Homo sapiens
tetraspan
867
59

NET-4

181
D21261

Homo sapiens
similar to
1048
100

human 22kDa, SM22 mRNA

(HUM22SM).

182
AL162971

Arabidopsis thaliana

529
38

putative protein

183
AL080062

Homo sapiens

1530
99

hypothetical protein

184
U93572

Homo sapiens
putative
496
53

p150

185
AF187064

Homo sapiens
p75NTR-
227
47

associated cell death

executor; NADE

186
AL035413

Homo sapiens
dJ657E11.5
1880
99

(KIAA0090 PROTEIN)

187
AC073944

Arabidopsis thaliana

755
36

ethylene-responsive RNA

helicase, putative

188
AB017615

Mus musculus
Eos protein
2750
95

189
Z99110
Bacillus subtilis
148
20

similar to hexuronate

transporter

190
AL138657

Arabidopsis thaliana

253
32

putative protein

191
AJ002397

Trichoderma harzianum

177
30

beta-1, 3 exoglucanase

192
AY004877

Mus musculus
cytoplasmic
5569
98

dynein heavy chain

193
U02313

Mus musculus
protein
6356
86

kinase

194
AE003739

Drosophila melanogaster

596
43

CG6949 gene product

195
U01317

Homo sapiens
epsilon-
397
100

globin

196
AF083116

Homo sapiens

770
46

paraneoplastic cancer-

testis-brain antigen

197
AL133156

Schizosaccharomyces

157
27

pombe
putative U4/U6

small nuclear

ribonucleoprotein

198
AF123653

Homo sapiens
FEZ1
742
42

199
Z99162

Schizosaccharomyces

167
24

pombe
putative signal

transduction pathway

protein

200
U10406

Mus musculus
capping
1440
98

protein beta-subunit,

isoform 1

201
AL365409

Homo sapiens
similar to
1653
100

(NP_034322.1 | ) sex-

determination protein

homolog Femla

202
AF116656

Homo sapiens
PRO1167
389
100

203
AL161578

Arabidopsis thaliana

142
27

putative protein

204
AF148805
Kaposi's sarcoma-
172
21

associated herpesvirus

latent nuclear antigen

205
AF102853

Rattus norvegicus

1500
62

membrane-associated

guanylate kinase-

interacting protein 1

Maguin-1

206
AF102853

Rattus norvegicus

779
69

membrane-associated

guanylate kinase-

interacting protein 1

Maguin-1

207
AF102853

Rattus norvegicus

933
71

membrane-associated

guanylate kinase-

interacting protein 1

Maguin-1

208
AF096896

Drosophila melanogaster

2089
50

Pushover

209
AF178941

Homo sapiens
ATP-binding
9833
99

cassette sub-family A

member 2

210
AC004780

Homo sapiens
F17127_1
1262
95

211
AL121845

Homo sapiens

1774
99

dJ583P15.5.1 (novel

protein (isoform 1))

212
AF170562

Homo sapiens
ubiquitin-
1248
49

specific processing

protease

213
U00060

Saccharomyces cerevisiae

637
46

Yhr088wp

214
AJ289133

Mus musculus
chondroitin
565
42

4-O-sulfotransferase

215
M63577

Saccharomyces cerevisiae

131
59

SFP1

216
AB046635

Macaca fascicularis

300
46

hypothetical protein

217
Z98884

Homo sapiens
dJ467L1.1
3640
99

(KIAA0833)

218
AB009883

Nicotiana tabacum
KED
203
22

219
D86491

Xenopus laevis
Nfr1
2416
75

220
U66707

Rattus norvegicus

3640
93

densin-180

221
AJ006701

Homo sapiens
putative
1457
84

serine/threonine protein

kinase

222
X61045
Hydra sp. mini-collagen
132
62

223
AF138883

Bos taurus
type II
170
38

collogen cyanogen

bromide fragment CB10

224
U22816

Homo sapiens
LAR-
2039
57

interacting protein 1b

225
AL109865

Homo sapiens

2298
98

bG120K12.3.1 (novel

protein similar to

archaeal, yeast and worm

N2,N2-dimethylguanosine

tRNA methyltransferase

(isoform 1))

226
AJ011654

Homo sapiens
triple LIM
557
41

domain protein

227
AB030198

Mus musculus
contains
318
50

transmembrane (TM)

region

229
AF233223

Homo sapiens
F-box
1386
87

protein FBG2

230
D88734
Equine herpesvirus 1
333
28

membrane glycoprotein

231
X94699
Mesocricetus auratus
1306
87

glucosamine-6-phosphate

isomerase

232
AL358732

Arabidopsis thaliana

385
44

putative protein

233
AB004306

Homo sapiens
osteoblast
574
100

stimulating factor-1

234
Z99281

Caenorhabditis elegans

154
22

Y57G11C.17

235
U23181

Caenorhabditis elegans

173
26

final exon in repeat

region; similar to long

tandem repeat region of

sialidase

(SP: TCNA_TRYCR,

P23253)

and neurofilament H

protein

236
X99384

Mus musculus
paladin
3563
80

237
AB020755

Arabidopsis thaliana

144
32

238
AF168122
Oryctolagus cuniculus
4053
96

hyperpolarization-

activated cyclic

nucleotide-gated channel 1

239
AF177909

Homo sapiens
TTYH1
1055
100

240
AF265296

Drosophila melanogaster

647
48

putative multipass

transmembrane

241
AC024760

Caenorhabditis elegans

212
19

contains similarity to

TR: Q10466

242
AB034991

Mus musculus
stress
614
37

protein Herp

243
AF030558

Rattus norvegicus

2090
95

phosphatidylinositol 5-

phosphate 4-kinase gamma

244
M80537

Drosophila melanogaster

1610
30

fat protein

245
U80741

Homo sapiens
CAGH44
373
56

246
L11586

Rattus norvegicus

271
25

leukocyte common

antigen-related

phosphatase

247
AF265555

Homo sapiens
ubiquitin-
330
37

conjugating BIR-domain

enzyme APOLLON

248
AC024810

Caenorhabditis elegans

450
35

contains similarity to

Pfam family PF00560

(Leucine Rich Repeat-2

copies)

249
U13831

Homo sapiens
cellular
715
99

retinol binding protein

II

250
J03799

Homo sapiens
laminin-
209
87

binding protein

251
AL109840

Homo sapiens
dJ543J19.4
1447
100

(A novel protein

(ortholog of chicken

tubulin beta-6 chain

(beta-tubulin class-

VI)))

252
AL109840

Homo sapiens
dJ543J19.4
1842
100

(A novel protein

(ortholog of chicken

tubulin beta-6 chain

(beta-tubulin class-

VI)))

253
D14710

Homo sapiens
inport
1566
99

precursor of human ATP

synthase alpha subunit

254
Y07895
Strongylocentrotus
815
78

purpuratus mitochondrial

ATP synthase alpha

subunit precursor

255
AJ243936

Homo sapiens
protein G16
592
60

256
AL034380

Homo sapiens
dJ50024.1
1262
100

(novel protein similar

to C. elegans K07B1.7

(Tr: O01886)

257
D17409

Homo sapiens
SM22 alpha
937
95

258
AL080276

Homo sapiens
dJ101K10.1
1940
99

(novel protein similar

to Prokaryotic-type

class I peptide chain

release factors)

259
AL080276

Homo sapiens
dJ101K10.1
1048
99

(novel protein similar

to Prokaryotic-type

class I peptide chain

release factors)

260
D50617

Saccharomyces cerevisiae

352
32

YFL044C

261
D63378

Rattus norvegicus
ER-60
174
28

protease

262
AJ249900

Homo sapiens
secreted
2346
99

modular calcium-binding

protein

263
U41557

Caenorhabditis elegans

281
33

proline and glycine-rich

264
AE001751
Thermotoga maritima dnaJ
172
52

protein

265
AC004144

Homo sapiens
R34001_1
1699
89

266
AF221759

Homo sapiens
Mam1
342
31

267
U33007

Saccharomyces cerevisiae

370
35

Ydr438wp; CAI: 0.11

268
X15940

Homo sapiens
ribosomal
546
92

protein L31 (AA 1-125)

269
X00955

Homo sapiens
prepro-apo
478
96

AII

270
AL035460

Homo sapiens
dJ860F19.3
3571
100

(novel protein (ortholog

of mouse

metallocarboxypeptidase

CPX-1))

271
U35730

Mus musculus
jerky
758
33

272
X16576

Homo sapiens
KUP protein
567
37

273
AK021815

Homo sapiens
unnamed
1408
97

protein product

274
AL391148

Arabidopsis thaliana

521
43

putative protein

275
AL391148

Arabidopsis thaliana

521
43

putative protein

276
U33335

Saccharomyces cerevisiae

376
29

Lpa8p

277
AB046381

Homo sapiens
testis-
2739
100

abundant finger protein

278
Z19092
Oryctolagus cuniculus
217
25

trichohyalin

279
U65079

Mus musculus
actin-
516
26

binding protein

280
M27878

Homo sapiens
DNA binding
1491
56

protein

281
U62907

Mus musculus
zinc finger
675
36

protein 95

282
AF052298
Drosophila silvestris Y
140
34

box protein

283
AB020970

Homo sapiens
up-
637
100

regulated by BCG-CWS

284
X61585

Bos taurus

2194
60

polynucleotide

adenylyltransferase

285
AJ249978

Xenopus laevis
p33 ringo
95
43

286
U96166

Streptococcus cristatus

194
17

srpA

287
AE003701

Drosophila melanogaster

286
29

CG9591 gene product

288
AL117337

Homo sapiens
bA393J16.3
2015
100

(novel KRAB box

containing zinc finger

gene)

289
AC004076

Homo sapiens
R30217_1
1793
56

290
M27337

Homo sapiens
T cell
399
91

receptor gamma chain

291
V00507

Homo sapiens
coding
886
93

sequence of DHFR (1 is

1st base in codon) (561

is 3rd base in codon)

292
AE005053
Halobacterium sp. NRC-1
1062
37

Vng1303c

293
AF061258

Homo sapiens
LIM protein
510
88

294
AE003543

Drosophila melanogaster

142
60

CG6938 gene product

295
AK024429

Homo sapiens
FLJ00018
5938
100

protein

296
AF151851

Homo sapiens
CGI-93
557
49

protein

297
AK025429

Homo sapiens
unnamed
668
63

protein product

298
Z19092

Oryctolagus cuniculus

188
21

trichohyalin

299
AF195056

Mus musculus
SorCSb
2188
47

splice variant of the

VPS10 domain receptor

SorCS

300
L32978

Mesocricetus auratus

167
22

synaptonemal complex

protein

301
AC002130

Arabidopsis thaliana

237
30

F1N21.22

302
U80736

Homo sapiens
CAGF9
290
87

303
AF178428

Bos taurus
submaxillary
183
26

mucin

304
AJ243936

Homo sapiens
protein G16
380
68

305
AF147791

Homo sapiens
mucin 11
106
22

306
AF071010
Mouse mammary tumor
177
46

virus putative integrase

307
X71997

Rattus norvegicus
myosin I
1654
62

308
AF000560

Homo sapiens
TTF-I
2081
99

interacting peptide 20;

TIP20; Transcription

Termination Factor I

Interacting Peptide 20

309
AF142780

Mus musculus

895
69

butyrophilin-like

protein

310
AF161384

Homo sapiens
HSPC266
119
44

311
A08695

Homo sapiens
rap2
204
31

312
X04956

Homo sapiens
T-cell
590
100

receptor alpha-chain

(404 is 2nd base in

codon)

313
X60489

Homo sapiens
elongation
1066
92

factor-1-beta

314
AB026190

Homo sapiens
Kelch motif
1187
44

containing protein

315
U66561

Homo sapiens
kruppel-
916
45

related zinc finger

protein

316
AF038007

Homo sapiens
FIC1
1499
54

317
AJ225124

Mus musculus

3554
96

hyperpolarization-

activated cation

channel, HAC3

318
AL031788

Schizosaccharomyces

290
40

pombe
putative

mitochondrial membrane

protease subunit 2

319
AC003682

Homo sapiens
F18547_1
814
49

320
AF188507

Mus musculus
LAB300
1347
46

isoform gamma

321
AL136125

Homo sapiens
dJ304B14.1
374
32

(novel protein)

322
U70312

Homo sapiens
integrin
447
100

binding protein Del-1

323
D13302
Megabalanus rosa lectin
155
30

BRA-3

324
U59185

Homo sapiens
putative
519
29

monocarboxylate

transporter

325
AF205599

Mus musculus

1016
35

transposase-like protein

326
Z34465

Zea mays
extensin-like
218
27

protein

327
AL049553

Homo sapiens
dJ402N21.2
1474
100

(novel protein with MAM

domain)

328
AF027505

Mus musculus
putative
1118
83

membrane-associated

guanylate kinase 1

329
U37376

Xenopus laevis
MAM
2448
63

domain protein

330
AF030430

Mus musculus
semaphorin
2958
94

VIa

331
Z30174

Mus musculus
domesticus
2087
71

zinc finger protein 30

332
U55020

Saccharomyces cerevisiae

282
33

Smp3p

333
Y07783

Rattus norvegicus
ER
285
32

transmembrane protein

334
AF291437

Rattus norvegicus

432
27

neuronal leucine-rich

repeat protein-3

335
W56097

Homo sapiens
amino acid
2470
84

sequence of the ODD4b5.3

enzyme

336
AB029333

Halocynthia roretzi

810
44

HrPET-1

337
AB030186

Mus musculus
unnamed
1634
86

protein product

338
AB030186

Mus musculus
unnamed
1671
89

protein product

339
AF128113

Mus musculus
prominin-
1021
77

like protein

340
AB007407

Mus musculus
myeloid
209
43

zinc finger protein-2

341
AF176665

Xenopus laevis
F-box
249
29

protein 27

342
AL079335

Homo sapiens
dJ132F21.2
352
56

(Contains a novel

protein similar to the

L82E from Drosophila)

343
AF177377

Homo sapiens
cytoplasmic
628
62

protein

344
AF002109

Arabidopsis thaliana

371
31

putative ABC transporter

345
AJ010482

Homo sapiens
Myopodin
579
29

protein

346
X01831

Cairina moschata
alpha
398
53

D-globin

347
X01831

Cairina moschata
alpha
314
54

D-globin

348
AL163852

Arabidopsis thaliana

184
53

putative protein

349
AF176532

Mus musculus
F-box
706
59

protein FBX17

350
D83674

Mus musculus
MesP1
564
65

351
AC006539

Homo sapiens
BC39498_3
387
100

352
AF229643

Mus musculus
ubiquitin
2025
73

specific protease

353
X17025

Homo sapiens
homologue
808
64

of yeast IPP isomerase

354
D79983

Homo sapiens
There is a
659
52

C3HC4 zinc-finger in the

C-terminal region.

355
D79983

Homo sapiens
There is a
434
44

C3HC4 zinc-finger in the

C-terminal region.

356
AF109674

Rattus norvegicus
late
968
86

gestation lung protein 1

357
AC004780

Homo sapiens
F17127_1
1258
87

358
X76116

Caenorhabditis elegans

567
48

carrier protein (c2)

359
X77953

Rattus norvegicus

671
100

ribosomal protein S15a

360
U67594

Methanococcus jannaschii

171
27

Yeast KTI12 Protein

361
AF229643

Mus musculus
ubiquitin
2711
95

specific protease

362
AF083340

Homo sapiens
double-
270
54

stranded RNA-binding

zinc finger protein JAZ

363
AE000161

Escherichia coli
K12
296
59

bacteriophage lambda

lysozyme homolog

364
AL023705

Schizosaccharomyces

241
33

pombe
phenylalanyl-trna

synthetase,

mitochondrial precursor

365
J02870

Mus musculus
laminin
321
64

receptor

366
AL137391

Homo sapiens

1620
100

hypothetical protein

367
AB012247

Arabidopsis thaliana

732
42

contains similarity to

zinc finger

proteins˜gene_id: MSL1.13

368
AF167441

Mus musculus
E-cadherin
2768
97

binding protein E7

369
AF196576

Chlamydomonas

260
34

reinhardtii
flagellar

protofilament ribbon

protein

370
AF075050

Homo sapiens
similar to
592
100

(AJ223828) small

glutamine-rich

tetratricopeptide (SGT)

371
S79410

Mus musculus
nuclear
107
51

localization signal

binding protein

372
AC015446

Arabidopsis thaliana

311
34

Similar to AIG1 protein

373
AF217227

Homo sapiens
zinc finger
1187
52

protein ZNF287

374
AB013390

Arabidopsis thaliana

605
66

ADP-ribosylation factor-

like protein

375
AB026842

Mus musculus
sialidase
732
51

376
D78174

Mus musculus
Zic4
1490
86

protein

377
X51394

Xenopus laevis
APEG
186
26

precursor protein

378
AJ001616

Mus musculus
myeloid
1106
86

associated

differentiation protein

379
AF248651

Homo sapiens
RNA-binding
869
100

protein BRUNOL4

380
U16800

Xenopus laevis

764
72

ribonucleoprotein

381
AE003503

Drosophila melanogaster

249
32

CG4768 gene product

383
AB026192

Xenopus laevis
Kielin
413
36

384
Y16430

Mus musculus
ribosomal
201
54

protein L35a

385
Z46259

Saccharomyces cerevisiae

169
40

NO381

386
AF136010
Solanum chacoense SPP30
437
51

387
AK024432

Homo sapiens
FLJ00022
1346
93

protein

388
AB004665

Mus musculus
Rab33B
1138
94

389
AF053768

Rattus norvegicus
brain
589
36

specific cortactin-

binding protein CBP90

390
M20030

Homo sapiens
small
434
94

proline rich protein

391
AF182443

Rattus norvegicus
F-box
1449
99

protein FBL2

392
U53449

Rattus norvegicus
jun
800
96

dimerization protein 2

393
AB046381

Homo sapiens
testis-
270
36

abundant finger protein

394
AL031588

Homo sapiens
dJ1163J1.2
863
100

(novel protein similar

to C. elegans B0035.16

and bacterial tRNA (5-

Methylaminomethyl-2-

thiouridylate)-

Methyltransferases)

395
AF117814

Mus musculus
odd-skipped
1413
97

related 1 protein

396
AJ010228

Homo sapiens
RFPL1L
889
58

397
U65079

Mus musculus
actin-
2402
74

binding protein

398
M64488

Rattus norvegicus

2128
95

synaptotagmin II

399
AB017026

Mus musculus
oxysterol-
548
99

binding protein

400
U42580
Paramecium bursaria
213
33

Chlorella virus 1

contains 10 ankyrin-like

repeats; similar to

human ankyrin,

corresponds to Swiss-

Prot Accession Number

P16157

401
Y00204

Xenopus laevis

464
49

nucleoplasmin

402
U80741

Homo sapiens
CAGH44
446
65

403
D16100

Rattus norvegicus
90 kDa-
4055
95

diacylglycerol kinase

404
AF132611

Homo sapiens

268
49

monocarboxylate

transporter MCT3

405
AB030505

Mus musculus
UBE-1c2
661
50

406
AK024422

Homo sapiens
FLJ00011
1450
97

protein

407
AC007228

Homo sapiens
BC37295_1
576
49

408
AB017059

Arabidopsis thaliana
FH
183
43

protein interacting

protein FIP2

409
L06434
Rattus sp.
3709
96

neurotransmitter

transporter

410
AB017139

Rattus norvegicus
Kilon
667
94

411
AF088982

Homo sapiens
heat shock
478
49

protein hsp40-3

412
L04948

Saccharomyces cerevisiae

467
37

mitochondrial

transporter protein

413
U09413

Homo sapiens
zinc finger
1241
52

protein ZNF135

414
X58079

Homo sapiens
S100 alpha
274
57

protein

415
X61617

Drosophila melanogaster

183
38

neuralized protein

416
AF176694

Mus musculus
neighbor of
344
41

Punc ell protein

417
AC002086

Homo sapiens
similar to
346
41

zinc finger 5 protein

from Gallus gallus,

U51640 (PID: g1399185)

418
AB046381

Homo sapiens
testis-
564
43

abundant finger protein

419
AF086824

Mus musculus
rho/rac-
1506
89

interacting citron

kinase

420
AE000663

Mus musculus
trypsinogen 1
898
65

421
L20468

Rattus norvegicus

1132
87

cerebroglycan

422
X73462
Ovis aries hair keratin
198
39

cysteine rich protein

423
AC002394

Homo sapiens
Gene
3043
100

product with similarity

to dynein beta subunit

424
AF118566

Mus musculus

722
48

hematopoietic zinc

finger protein

425
AJ010100

Homo sapiens
NKp44RG2
163
30

426
AF059576

Mus musculus
myosin
264
26

binding protein-C

427
M37760

Mus musculus
serine 2
461
41

ultra high sulfur

protein

428
AL359916

Homo sapiens
bA550O8.2
1734
99

(A novel protein similar

to cell division protein

kinase 4 (CDK4))

429
X68527

Homo sapiens
TCR Vbeta
613
94

5.5

430
AJ006078

Homo sapiens
beta-1,3-
373
34

galactosyltransferase

431
AF059493

Sus scrofa
proteasome
618
53

subunit LMP7

432
AL096770

Homo sapiens
bA150A6.2
816
52

(novel 7 transmembrane

receptor (rhodopsin

family) (olfactory

receptor like) protein

(hs6M1-21))

433
U38896

Homo sapiens
zinc finger
501
62

protein C2H2-171

434
AF093680

Homo sapiens

157
26

transcription factor IIB

435
AJ237660
Bacteriophage 21 NinG
392
43

protein

436
L10912

Oryctolagus cuniculus

1317
51

cytochrome P-450 2B-Bx

437
AL031055

Homo sapiens
dJ28H20.1
2624
99

(novel protein similar

to membrane transport

proteins)

438
AJ007421

Homo sapiens
spalt-like
6070
99

zinc finger protein

439
X94744

Gallus gallus
olfactory
443
45

receptor 4

440
AB037973

Homo sapiens
FGF-23
1360
100

441
X13986

Mus musculus
minopontin
1521
100

precursor (AA-66 to

272)

[0394]

3

TABLE 3

SEQ ID
ACCESSION

NO:
NO.
DESCRIPTION
RESULTS*

1
PF00642
Zinc finger C-x8-C-
PF00642 11.59 9.700e−12 426-437

x5-C-x3-H type (and

similar).

2
BL00291
Prion protein.
BL00291A 4.49 8.759e−09 185-220

3
PR00405
HIV REV INTERACTING
PR00405B 11.83 7.000e−21 49-67

PROTEIN SIGNATURE
PR00405A 17.71 6.943e−13 30-50

PR00405C 19.41 4.906e−12 70-92

4
PF01105
emp24/gp25L/p24
PF01105B 25.12 1.000e−40 178-230

family.

5
BL00307
Legume lectins beta-
BL00307G 9.91 8.531e−10 678-689

chain proteins.

7
BL01159
WW/rsp5/WWP domain
BL01159 13.85 6.073e−09 61-76

proteins.

8
BL01159
WW/rsp5/WWP domain
BL01159 13.85 6.073e−09 61-76

proteins.

10
BL00913
Iron-containing
BL00913D 24.20 8.981e−17 170-204

alcohol
BL00913C 7.62 4.375e−11 136-146

dehydrogenases
BL00913B 10.94 7.706e−11 86-102

proteins.

11
BL00913
Iron-containing
BL00913D 24.20 8.981e−17 218-252

alcohol
BL00913C 7.62 4.375e−11 184-194

dehydrogenases
BL00913B 10.94 7.706e−11 134-150

proteins.

12
BL50062
BCL2-like apoptosis
BL50062C 6.66 8.500e−11 349-358

inhibitors (spans

part of BH3, BH1 and

BH

15
BL01144
Ribosomal protein
BL01144 25.07 9.069e−26 78-130

L31e proteins.

16
PF00642
Zinc finger C-x8-C-
PF00642 11.59 6.694e−10 355-366

x5-C-x3-H type (and

similar).

18
PR00773
GRPE PROTEIN
PR00773D 16.14 5.922e−09 215-235

SIGNATURE

24
PD00930
PROTEIN GTPASE
PD00930B 33.72 7.300e−26 600-641

DOMAIN ACTIVATION.
PD00930A 25.62 1.514e−16 497-523

26
BL00375
UDP-
BL00375F 16.99 7.061e−35 291-336

glycosyltransferases
BL00375C 18.27 2.615e−19 126-150

proteins.
BL00375D 14.56 9.000e−17 192-220

BL00375B 21.22 8.627e−16 67-108

BL00375G 13.01 4.577e−13 390-430

29
BL01170
Ribosomal protein
BL01170A 12.34 9.143e−40 139-175

L6e proteins.

30
BL00359
Ribosomal protein
BL00359B 23.07 4.231e−24 56-97

L11 proteins.
BL00359C 22.18 6.586e−22 111-145

BL00359A 20.66 4.000e−21 20-56

31
PR00983
CYSTEINYL-TRNA
PR00983D 14.16 3.647e−23 270-292

SYNTHETASE SIGNATURE
PR00983C 11.27 3.415e−21 239-258

PR00983A 11.10 1.878e−12 75-87

32
PR00718
PHOSPHOLIPASE D
PR00718E 8.61 1.000e−08 327-351

SIGNATURE

33
BL00518
Zinc finger, C3HC4
BL00518 12.23 6.571e−10 49-58

type (RING finger),

proteins.

34
PF00992
Troponin.
PF00992A 16.67 7.972e−10 10-45

PF00992A 16.67 5.145e−09 17-52

PF00992A 16.67 6.684e−09 56-91

35
PR00452
SH3 DOMAIN SIGNATURE
PR00452D 17.02 8.000e−11 252-265

36
BL01019
ADP-ribosylation
BL01019A 13.20 8.000e−11 68-108

factors family

proteins.

39
PR00764
COMPLEMENT C9
PR00764F 16.89 7.783e−11 204-225

SIGNATURE

40
PR00832
PAXILLIN SIGNATURE
PR00832B 9.87 6.284e−10 768-792

42
BL00226
Intermediate
BL00226D 19.10 3.172e−34 397-444

filaments proteins.
BL00226B 23.86 5.929e−23 230-278

BL00226C 13.23 4.808e−21 296-327

BL00226A 12.77 5.065e−13 129-144

BL00226B 23.86 6.400e−10 181-229

43
BL00243
Integrins beta chain
BL00243I 31.77 2.014e−09 156-199

cysteine-rich domain
BL00243I 31.77 5.437e−09 159-202

proteins.
BL00243I 31.77 5.690e−09 30-73

45
BL00291
Prion protein.
BL00291A 4.49 4.414e−09 47-82

46
PF00084
Sushi domain
PF00084B 9.45 7.188e−10 1549-1561

proteins (SCR repeat

proteins.

47
BL00414
Profilin proteins.
BL00414D 15.59 9.182e−10 81-108

50
PR00837
ALLERGEN V5/TPX-1
PR00837D 11.12 6.023e−09 22-36

FAMILY SIGNATURE

51
BL00284
Serpins proteins.
BL00284A 15.64 2.350e−20 85-109

BL00284D 16.34 4.240e−19 323-350

BL00284C 28.56 5.600e−17 216-258

BL00284E 19.15 7.500e−14 408-433

BL00284B 17.99 9.379e−13 189-210

52
BL01283
T-box domain
BL01283A 24.15 2.125e−39 148-196

proteins.
BL01283B 23.17 9.438e−34 208-250

BL01283D 11.70 7.868e−31 298-331

BL01283C 13.05 8.448e−16 260-274

53
PD01270
RECEPTOR FC
PD01270D 24.66 8.054e−09 50-86

IMMUNOGLOBULIN

AFFIN.

54
BL00237
G-protein coupled
BL00237A 27.68 2.543e−13 181-221

receptors proteins.

55
PR00050
COLD SHOCK PROTEIN
PR00050A 11.28 3.143e−12 42-58

SIGNATURE
PR00050C 9.82 9.151e−11 85−104

58
BL01173
Lipolytic enzymes G-
BL01173B 13.27 4.462e−17 140-167

D-X-G family,
BL01173C 8.98 4.349e−14 182-196

histidine.
BL01173A 9.41 1.818e−13 454-467

BL01173C 8.98 6.553e−13 495-509

BL01173A 9.41 8.364e−13 107-120

59
PR00321
GAMMA G-PROTEIN
PR00321C 15.39 2.473e−12 123-141

(TRANSDUCIN)

SIGNATURE

60
PR00937
T-BOX DOMAIN
PR00937A 15.25 1.000e−24 117-142

SIGNATURE
PR00937D 13.41 5.500e−18 220-235

PR00937B 14.58 5.235e−13 184-198

PR00937F 12.53 1.450e−12 293-302

PR00937E 11.86 1.918e−12 259-273

PR00937C 10.51 3.571e−11 201-211

61
PD02059
CORE POLYPROTEIN
PD02059A 28.10 2.694e−09 116-157

PROTEIN GAG

CONTAINS: P.

65
BL00634
Ribosomal protein
BL00634 34.38 3.250e−15 46-97

L30 proteins.

66
BL00226
Intermediate
BL00226B 23.86 1.643e−31 264-312

filaments proteins.

70
DM00892
3 RETROVIRAL
DM00892C 23.55 6.727e−13 33-67

PROTEINASE.

71
PR00874
FUNGI-IV
PR00874C 4.37 7.652e−10 68-83

METALLOTHIONEIN

SIGNATURE

72
PR00102
ORNITHINE
PR00102C 15.77 1.988e−07 42-57

CARBAMOYLTRANSFERASE

SIGNATURE

73
PR00359
B-CLASS P450
PR00359D 16.13 6.824e−08 291-314

SIGNATURE

74
PR00066
XERODERMA
PR00066A 10.61 6.418e−08 77-95

PIGMENTOSUM GROUP G

PROTEIN SIGNATURE

75
PR00873
ECHINOIDEA (SEA
PR00873D 8.43 8.237e−08 13-32

URCHIN)

METALLOTHIONEIN

SIGNATURE

76
PR00623
HISTONE H4 SIGNATURE
PR00623B 13.68 8.888e−09 364-384

77
BL00873
Sodium: alanine
BL00873E 24.91 2.800e−07 216-257

symporter family

proteins.

78
PR00478
PHOSPHORIBULOKINASE
PR00478C 12.84 5.012e−07 102-120

FAMILY SIGNATURE

79
PF00878
Cation-independent
PF00878T 17.51 5.457e−08 52-79

mannose-6-phosphate

receptor repeat

proteins.

80
PR00715
CATION-DEPENDENT
PR00715I 10.58 3.603e−06 112-131

MANNOSE-6-PHOSPHATE

RECEPTOR SIGNATURE

81
PR00350
VITAMIN D RECEPTOR
PR00350E 11.55 6.780e−07 45-65

SIGNATURE

82
BL01131
Ribosomal RNA
BL01131A 26.62 2.376e−08 460-506

adenine dimethylases

proteins.

83
PR00629
SHC PHOSPHOTYROSINE
PR00629F 10.95 9.457e−07 96-123

INTERACTION DOMAIN

SIGNATURE

84
BL00111
Phosphoglycerate
BL00111A 9.65 7.783e−07 73-87

kinase proteins.

88
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 7.000e−13 200-213

METAL-BINDI

89
PD02870
RECEPTOR
PD02870D 15.74 8.468e−09 358-393

INTERLEUKIN-1

PRECURSOR.

90
BL00048
Protamine P1
BL00048 6.39 6.815e−13

proteins.
64-91 BL00048 6.39 4.750e−11

56-83 BL00048 6.39

4.316e−10 55-82 BL00048

6.39 5.500e−10

70-97 BL00048 6.39 2.350e−09 62-89

BL00048 6.39 3.700e−09 60-87

BL00048 6.39 5.050e−09

63-90 BL00048

6.39 6.288e−09 61-88 BL00048 6.39

9.438e−09 71-98

91
PR00320
G-PROTEIN BETA WD-40
PR00320C 13.01 8.920e−10 202-217

REPEAT SIGNATURE
PR00320B 12.19 9.486e−10 202-217

PR00320C 13.01 7.900e−09 292-307

PR00320A 16.74 8.902e−09 202-217

92
BL00453
FKBP-type peptidyl-
BL00453B 23.86 3.864e−28 106-140

prolyl cis-trans
BL00453A 15.57 1.000e−15 81-96

isomerase proteins.
BL00453C 9.72 1.000e−12 147-160

94
PR00299
ALPHA CRYSTALLIN
PR00299B 17.53 7.211e−09 324-337

SIGNATURE

95
PF00676
Dehydrogenase E1
PF00676D 14.40 4.857e−13 421-441

component.
PF00676C 16.88 1.931e−10 389-413

PF00676B 24.71 5.433e−10 192-230

98
BL00824
Elongation factor 1
BL00824B 9.21 3.919e−09 1472-1492

beta/beta′/delta

chain proteins.

101
PR00417
PROKARYOTIC DNA
PR00417A 12.66 5.415e−09 866-880

TOPOISOMERASE I

SIGNATURE

104
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 6.936e−29 17-56

ZINC-FINGER METAL-

BINDING NU.

105
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 9.438e−37 10-49

ZINC-FINGER METAL-

BINDING NU.

106
PD01781
PROTEASE
PD01781B 27.55 8.680e−09 325-369

IMMUNOGLOBULIN

PRECURSO.

107
PD01781
PROTEASE
PD01781B 27.55 8.680e−09 379-423

IMMUNOGLOBULIN

PRECURSO.

109
PR00939
C2HC-TYPE ZINC-
PR00939B 13.27 3.647e−09 1302-1311

FINGER SIGNATURE

110
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 2.800e−14 279-292

METAL-BINDI.
PD00066 13.92 4.600e−14 307-320

PD00066 13.92 1.000e−13 335-348

PD00066 13.92 7.500e−13 363-376

111
PR00193
MYOSIN HEAVY CHAIN
PR00193D 14.36 5.680e−33 391-420

SIGNATURE
PR00193C 12.60 4.789e−32 156-184

PR00193B 11.69 1.692e−26 110-136

PR00193E 19.47 5.500e−21 445-474

PR00193A 15.41 4.130e−20 54-74

PR00193E 19.47 5.091e−12 444-473

112
BL00239
Receptor tyrosine
BL00239B 25.15 2.985e−16 67-115

kinase class II

proteins.

113
BL00678
Trp-Asp (WD) repeat
BL00678 9.67 2.800e−10 366-377

proteins proteins.
BL00678 9.67 5.263e−09 417-428

BL00678 9.67 6.211e−09 186-197

114
DM00547
1 kw CHROMO
DM00547F 23.43 2.350e−35 384-431

BROMODOMAIN SHADOW
DM00547C 17.30 7.000e−19 23-45

GLOBAL.
DM00547E 13.94 5.592e−17 135-158

DM00547D 11.60 2.750e−13 105-119

116
PR00700
PROTEIN TYROSINE
PR00700D 12.47 8.788e−11 237-256

PHOSPHATASE

SIGNATURE

118
PR00884
RIBOSOMAL PROTEIN
PR00884E 8.32 4.750e−09 449-466

HS6 SIGNATURE

119
PD02890
ISOMERASE CHALCONE-
PD02890C 16.14 8.457e−09 200-235

FLAVONONE FLAV.

120
BL00226
Intermediate
BL00226B 23.86 6.513e−10 401-449

filaments proteins.

121
PD01823
PROTEIN INTERGENIC
PD01823C 16.13 7.000e−14 352-373

REGION ABC1
PD01823B 14.96 3.782e−13 328-348

PRECURSOR
PD01823D 16.66 6.857e−10 430-451

MITOCHONDRION T.

124
BL00854
Proteasome B-type
BL00854C 29.92 8.435e−19 114-143

subunits proteins.

126
BL00651
Ribosomal protein L9
BL00651A 23.25 8.477e−17 94-134

proteins.

127
BL01245
RIO1/ZK632.3/MJ0444
BL01245F 18.75 2.373e−23 334-371

family proteins.
BL01245A 14.04 8.342e−23 206-231

BL01245C 13.31 6.564e−15 262-282

BL01245E 15.28 1.000e−12 320-330

BL01245B 11.91 9.809e−10 245-255

130
PR00793
PROLYL
PR00793C 12.24 1.333e−09 168-183

AMINOPEPTIDASE (S33)

FAMILY SIGNATURE

131
BL01160
Kinesin light chain
BL01160D 10.17 7.077e−09 505-534

repeat proteins.

132
BL00355
HMG14 and HMG17
BL00355 5.97 8.412e−32 18-49

proteins.

133
PR00041
CAMP RESPONSE
PR00041E 7.20 2.976e−13 305-326

ELEMENT BINDING

(CREB) PROTEIN

SIGNATURE

134
PR00211
GLUTELIN SIGNATURE
PR00211B 0.86 1.750e−09 205-226

PR00211B 0.86 8.750e−09 199-220

135
BL00455
Putative AMP-binding
BL00455 13.31 5.125e−11 293-309

domain proteins.

138
PD00015
GLYCOPROTEIN
PD00015A 8.90 6.400e−09 243-251

PRECURSOR CELL SI.

140
BL00227
Tubulin subunits
BL00227B 19.29 1.000e−40 52-107

alpha, beta, and
BL00227C 25.48 1.000e−40 113-165

gamma proteins.
BL00227A 24.55 8.200e−36 1-35

142
PR00049
WILM'S TUMOR
PR00049D 0.00 8.377e−13 60-75

PROTEIN SIGNATURE
PR00049D 0.00 7.500e−10 63-78

PR00049D 0.00 8.071e−10 61-76

143
PR00049
WILM'S TUMOR
PR00049D 0.00 6.438e−12 1613-1628

PROTEIN SIGNATURE

144
DM01970
0 kw ZK632.12
DM01970B 8.60 4.750e−17 552-565

YDR313C ENDOSOMAL

III.

145
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 5.846e−15 579-592

METAL-BINDI.
PD00066 13.92 9.217e−11 551-564

PD00066 13.92 6.700e−09 523-536

146
PR00926
MITOCHONDRIAL
PR00926F 17.75 3.672e−10 262-285

CARRIER PROTEIN

SIGNATURE

149
DM01417
6 kw INDUCING XPMC2
DM01417C 12.93 3.250e−11 267-279

MUSHROOM
DM01417D 11.08 2.200e−10 306-322

SPAC22G7.04.

150
BL01160
Kinesin light chain
BL01160B 19.54 8.378e−10 349-403

repeat proteins.

153
PR00049
WILM'S TUMOR
PR00049D 0.00 7.807e−11 419-434

PROTEIN SIGNATURE
PR00049D 0.00 8.563e−11 1284-1299

PR00049D 0.00 3.929e−10 1283-1298

PR00049D 0.00 3.288e−09 417-432

156
BL00665
Dihydrodipicolinate
BL00665D 14.76 1.000e−11 109-132

synthetase proteins.
BL00665C 25.58 5.832e−11 50-101

158
PD02906
SYNTHASE I
PD02906C 24.17 9.115e−15 171-206

PSEUDOURIDYLATE
PD02906B 15.35 4.886e−13 142-155

PSEUDOURIDINE LYASE
PD02906D 12.27 1.000e−09 239-249

TR.
PD02906A 10.84 8.333e−09 92-105

159
BL00107
Protein kinases ATP-
BL00107B 13.31 2.286e−11 396-412

binding region
BL00107A 18.39 6.586e−11 332-363

proteins.

162
PF01008
Initiation factor 2
PF01008B 25.59 9.609e−36 366-409

subunit.
PF01008A 20.14 8.676e−12 315-336

PF01008C 12.25 7.382e−10 449-469

163
BL00591
Glycosyl hydrolases
BL00591D 8.33 6.167e−09 2099-2112

family 10 proteins.

165
PR00019
LEUCINE-RICH REPEAT
PR00019B 11.36 7.120e−09 99-113

SIGNATURE
PR00019B 11.36 7.840e−09 73-87

166
BL00636
Nt-dnaJ domain
BL00636A 8.07 3.000e−14 143-160

proteins.

167
PR00310
ANTI-PROLIFERATIVE
PR00310B 10.59 4.000e−39 41-71

PROTEIN BTG1 FAMILY
PR00310C 12.74 2.256e−33 71-101

SIGNATURE
PR00310D 9.10 9.820e−33 101-131

PR00310A 11.17 7.000e−27 16-41

168
BL00216
Sugar transport
BL00216B 27.64 2.688e−21 124-174

proteins.

169
BL00216
Sugar transport
BL00216B 27.64 5.636e−20 124-174

proteins.

170
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 5.929e−32 59-98

ZINC-FINGER METAL-

BINDING NU.

172
PR00456
RIBOSOMAL PROTEIN P2
PR00456E 3.06 2.820e−11 6-21

SIGNATURE
PR00456E 3.06 7.563e−10 3-18

173
PD00126
PROTEIN REPEAT
PD00126A 22.53 4.706e−14 140-161

DOMAIN TPR NUCLEA.
PD00126A 22.53 6.824e−14 289-310

175
BL00741
Guanine-nucleotide
BL00741B 14.27 3.418e−11 294-317

dissociation

stimulators CDC24

family sign.

177
BL01016
Glycoprotease family
BL01016C 22.84 5.292e−19 60-105

proteins.
BL01016H 13.71 6.595e−12 307-317

BL01016E 14.88 3.182e−11 141-169

BL01016D 8.86 6.741e−09 118-131

178
PR00850
GLYCOSYL HYDROLASE
PR00850B 6.67 5.455e−09 148-173

FAMILY 59 SIGNATURE

180
PR00259
TRANSMEMBRANE FOUR
PR00259A 9.27 8.676e−20 17-41

FAMILY SIGNATURE
PR00259C 16.40 4.750e−17 85-114

PR00259B 14.81 8.615e−12 58-85

PR00259D 13.50 2.528e−11 235-262

181
BL01052
Calponin family
BL01052C 18.51 6.806e−40 87-127

repeat proteins.
BL01052A 16.12 7.618e−32 3-35

BL01052B 15.31 8.031e−26 52-78

BL01052D 10.26 1.000e−24 174-194

182
BL00875
Bacterial type II
BL00875A 25.57 6.447e−09 367-399

secretion system

protein D proteins.

183
PD01351
PROTEIN REPEAT
PD01351B 13.72 5.355e−09 238-264

NEUROFILAMENT TRIPL.

184
DM01354
kw TRANSCRIPTASE
DM01354H 18.00 8.826e−27 109-149

REVERSE II ORF2.
DM01354G 11.57 2.143e−25 78-109

DM01354F 14.56 1.414e−15 42-78

DM01354E 18.69 8.650e−14 17-47

187
BL00039
DEAD-box subfamily
BL00039A 18.44 4.000e−25 222-261

ATP-dependent
BL00039D 21.67 4.529e−23 498-544

helicases proteins.
BL00039C 15.63 4.300e−16 347-371

BL00039B 19.19 9.379e−15 262-288

188
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 5.714e−12 152-165

METAL-BINDI.
PD00066 13.92 6.143e−12 124-137

189
BL01022
PTR2 family
BL01022B 22.19 4.240e−10 308-354

proton/oligopeptide

symporters proteins.

192
PR00830
ENDOPEPTIDASE LA
PR00830A 8.41 3.342e−09 881-901

(LON) SERINE

PROTEASE (S16)

SIGNATURE

193
PR00109
TYROSINE KINASE
PR00109B 12.27 9.234e−13 261-280

CATALYTIC DOMAIN

SIGNATURE

195
BL01033
Globins profile.
BL01033A 16.94 2.385e−18 25-47

197
PR00320
G-PROTEIN BETA WD-40
PR00320B 12.19 6.226e−11 140-155

REPEAT SIGNATURE
PR00320A 16.74 4.971e−10 140-155

PR00320C 13.01 9.280e−10 140-155

198
PR00832
PAXILLIN SIGNATURE
PR00832B 9.87 9.174e−10 309-333

199
PR00674
LIGHT HARVESTING
PR00674A 20.10 7.391e−09 134-155

PROTEIN B CHAIN

SIGNATURE

200
PR00192
F-ACTIN CAPPING
PR00192C 6.65 2.500e−36 57-84

PROTEIN BETA SUBUNIT
PR00192D 8.23 4.462e−36 97-125

SIGNATURE
PR00192E 8.85 7.000e−33 212-239

PR00192A 8.23 1.474e−27 5-26

PR00192B 6.20 3.000e−26 26-48

201
PF00023
Ank repeat proteins.
PF00023A 16.03 4.750e−10 45-61

203
PR00239
MOLLUSCAN RHODOPSIN
PR00239E 1.58 6.114e−09 183-195

C-TERMINAL TAIL

SIGNATURE

204
BL00412
Neuromodulin (GAP-
BL00412D 16.54 4.033e−10 319-370

43) proteins.

205
BL00790
Receptor tyrosine
BL00790R 16.20 7.677e−09 29-73

kinase class V

proteins.

206
BL00790
Receptor tyrosine
BL00790R 16.20 7.677e−09 29-73

kinase class V

proteins.

207
BL00790
Receptor tyrosine
BL00790R 16.20 7.677e−09 29-73

kinase class V

proteins.

209
BL00211
ABC transporters
BL00211B 13.37 3.077e−17 573-605

family proteins.
BL00211B 13.37 7.577e−17 1642-1674

BL00211A 12.23 1.900e−09 472-484

211
PR00049
WILM'S TUMOR
PR00049D 0.00 1.786e−10 288-303

PROTEIN SIGNATURE

212
BL00972
Ubiquitin carboxyl-
BL00972D 22.55 3.348e−11 388-413

terminal hydrolases
BL00972E 20.72 4.343e−09 415-437

family 2 proteins.

216
PD00469
PROTEIN PRECURSOR
PD00469A 13.95 6.400e−09 73-86

SIGNAL HYDROLA.

217
PF00023
Ank repeat proteins.
PF00023A 16.03 8.875e−10 839-855

PF00023A 16.03 2.286e−09 884-900

219
BL00982
Bacterial-type
BL00982A 18.41 8.013e−12 328-360

phytoene

dehydrogenase

proteins.

220
PF00595
PDZ domain proteins
PF00595 13.40 4.600e−09 688-699

(Also known as DHR

or GLGF).

221
BL00107
Protein kinases ATP-
BL00107A 18.39 7.000e−23 65-96

binding region
BL00107B 13.31 4.214e−10 130-146

proteins.

222
PR00239
MOLLUSCAN RHODOPSIN
PR00239E 1.58 3.045e−09 38-50

C-TERMINAL TAIL

SIGNATURE

224
BL00326
Tropomyosins
BL00326A 14.01 5.337e−10 825-859

proteins.

226
BL00478
LIM domain proteins.
BL00478B 14.79 8.527e−09 143-158

227
DM00179
w KINASE ALPHA
DM00179 13.97 9.526e−10 135-145

ADHESION T-CELL.

228
BL00048
Protamine P1
BL00048 6.39 6.063e−09 199-226

proteins.

230
BL00115
Eukaryotic RNA
BL00115Z 3.12 5.744e−10 113-162

polymerase II
BL00115Z 3.12 3.449e−09 120-169

heptapeptide repeat

proteins.

231
BL01161
Glucosamine/galactos
BL01161A 19.47 1.000e−40 37-77

amine-6-phosphate
BL01161D 28.14 1.000e−40 199-244

isomerases proteins.
BL01161B 21.37 5.091e−39 117-160

BL01161C 18.47 1.500e−23 170-199

233
PR00269
PLEIOTROPHIN/MIDKINE
PR00269A 13.91 3.571e−30 88-113

FAMILY SIGNATURE

238
BL00888
Cyclic nucleotide-
BL00888B 14.79 9.069e−13 499-523

binding domain

proteins.

240
BL01188
GNS1/SUR4 family
BL01188B 13.46 4.115e−26 120-151

proteins.
BL01188C 22.65 4.136e−26 151-202

BL01188D 8.62 1.290e−11 238-255

BL01188A 18.82 6.718e−10 55-87

241
PR00929
AT-HOOK-LIKE DOMAIN
PR00929B 4.38 8.875e−09 571-583

SIGNATURE
PR00929C 5.26 8.914e−09 571-582

244
BL00232
Cadherins
BL00232B 32.79 2.765e−25 541-589

extracellular repeat
BL00232B 32.79 8.263e−22 766-814

proteins domain
BL00232B 32.79 2.397e−21 67-115

proteins.
BL00232B 32.79 4.571e−19 1481-1529

BL00232B 32.79 1.000e−18 1371-1419

BL00232B 32.79 2.662e−18 1691-1739

BL00232B 32.79 5.292e−18 1287-1335

BL00232B 32.79 9.585e−18 1586-1634

BL00232B 32.79 1.265e−17 980-1028

BL00232B 32.79 1.529e−17 426-474

BL00232B 32.79 2.588e−17 1084-1132

BL00232B 32.79 1.386e−16 1184-1232

BL00232C 10.65 5.390e−12 1369-1387

BL00232C 10.65 1.391e−11 642-660

BL00232C 10.65 2.174e−11 1584-1602

BL00232C 10.65 4.522e−11 1689-1707

BL00232C 10.65 1.000e−10 65-83

BL00232C 10.65 4.115e−10 1285-1303

BL00232B 32.79 7.200e−10 649-697

BL00232C 10.65 9.827e−10 978-996

BL00232C 10.65 1.947e−09 170-188

BL00232B 32.79 2.137e−09 172-220

BL00232C 10.65 4.474e−09 1182-1200

BL00232C 10.65 8.737e−09 539-557

245
BL00795
Involucrin proteins.
BL00795C 17.06 4.977e−10 64-109

BL00795C 17.06 6.300e−09 55-100

246
BL00790
Receptor tyrosine
BL00790I 20.01 7.823e−15 23-54

kinase class V
BL00790I 20.01 9.400e−11 310-341

proteins.
BL00790I 20.01 1.900e−10 117-148

BL00790I 20.01 3.893e−09 215-246

247
BL00183
Ubiquitin-
BL00183 28.97 7.037e−10 140-188

conjugating enzymes

proteins.

248
PR00019
LEUCINE-RICH REPEAT
PR00019A 11.19 8.800e−12 205-219

SIGNATURE
PR00019B 11.36 2.000e−11 202-216

249
BL00214
Cytosolic fatty-acid
BL00214B 26.51 7.618e−24 206-251

binding proteins.
BL00214A 21.17 6.250e−22 165-191

250
PR00395
RIBOSOMAL PROTEIN S2
PR00395C 16.17 2.047e−13 46-64

SIGNATURE

251
BL00227
Tubulin subunits
BL00227D 18.46 1.000e−40 74-128

alpha, beta, and
BL00227F 21.16 1.529e−33 226-280

gamma proteins.
BL00227E 24.15 1.409e−26 178-213

252
BL00227
Tubulin subunits
BL00227C 25.48 1.000e−40 39-91

alpha, beta, and
BL00227D 18.46 1.000e−40 148-202

gamma proteins.
BL00227F 21.16 1.529e−33 300-354

BL00227E 24.15 1.409e−26 252-287

253
BL00152
ATP synthase alpha
BL00152B 21.40 1.900e−31 191-229

and beta subunits
BL00152A 15.38 5.154e−21 134-160

proteins.
BL00152C 11.41 6.250e−12 291-303

254
BL00152
ATP synthase alpha
BL00152E 22.68 1.000e−32 285-323

and beta subunits
BL00152A 15.38 5.154e−21 134-160

proteins.
BL00152C 11.41 6.250e−12 247-259

255
BL00518
Zinc finger, C3HC4
BL00518 12.23 2.200e−11 54-63

type (RING finger),

proteins.

256
DM00892
3 RETROVIRAL
DM00892C 23.55 9.739e−12 417-451

PROTEINASE.

257
BL01052
Calponin family
BL01052C 18.51 1.000e−40 88-128

repeat proteins.
BL01052A 16.12 2.875e−35 3-35

BL01052B 15.31 5.219e−26 52-78

258
BL00745
Prokaryotic-type
BL00745C 13.66 1.000e−40 238-285

class I peptide
BL00745B 22.56 8.683e−33 184-227

chain release
BL00745D 14.90 8.435e−23 316-339

factors signat.
BL00745A 26.45 1.818e−19 123-162

259
BL00745
Prokaryotic-type
BL00745C 13.66 1.000e−40 202-249

class I peptide
BL00745B 22.56 8.683e−33 148-191

chain release
BL00745D 14.90 8.435e−23 280-303

factors signat.

261
BL00194
Thioredoxin family
BL00194 12.16 7.429e−10 684-697

proteins.

262
BL00612
Osteonectin domain
BL00612E 13.12 3.948e−10 391-436

proteins.

264
PR00625
DNAJ PROTEIN FAMILY
PR00625A 12.84 2.375e−09 288-308

SIGNATURE

265
PR00320
G-PROTEIN BETA WD-40
PR00320B 12.19 2.125e−09 207-222

REPEAT SIGNATURE

268
BL01144
Ribosomal protein
BL01144 25.07 1.000e−40 21-73

L31e proteins.

270
DM00516
186 DISCOIDIN I N-
DM00516 30.53 8.606e−13 153-198

TERMINAL.

271
BL00622
Bacterial regulatory
BL00622 32.69 9.780e−09 11-58

proteins, luxR

family proteins.

272
PR00048
C2H2-TYPE ZINC
PR00048A 10.52 1.000e−11 447-461

FINGER SIGNATURE
PR00048A 10.52 4.316e−11 389-403

PR00048A 10.52 6.684e−11 362-376

276
DM00303
6 LEA 11-MER REPEAT
DM00303A 13.20 3.310e−09 467-517

REPEAT.

277
PF00622
Domain in SPla and
PF00622B 21.00 9.357e−14 374-396

the RYanodine
PF00622C 12.62 1.857e−12 458-472

Receptor.

279
PF00651
BTB (also known as
PF00651 15.00 9.571e−10 65-78

BR-C/Ttk) domain

proteins.

280
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 8.200e−16 295-308

METAL-BINDI.
PD00066 13.92 8.200e−16 519-532

PD00066 13.92 1.692e−15 351-364

PD00066 13.92 4.462e−15 547-560

PD00066 13.92 4.600e−14 323-336

PD00066 13.92 4.600e−14 435-448

PD00066 13.92 7.000e−14 463-476

PD00066 13.92 1.500e−13 239-252

PD00066 13.92 3.143e−12 267-280

PD00066 13.92 3.143e−12 407-420

PD00066 13.92 8.826e−11 211-224

PD00066 13.92 2.038e−10 491-504

PD00066 13.92 2.385e−10 379-392

281
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 6.400e−16 449-462

METAL-BINDI.
PD00066 13.92 6.538e−15 504-517

PD00066 13.92 9.308e−15 421-434

PD00066 13.92 7.000e−14 476-489

PD00066 13.92 6.087e−11 393-406

282
BL00291
Prion protein.
BL00291A 4.49 5.229e−10 429-464

287
BL00276
Channel forming
BL00276A 8.87 6.500e−09 257-269

colicins proteins.

288
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 2.000e−30 10-49

ZINC-FINGER METAL-

BINDING NU.

289
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 7.407e−23 3-42

ZINC-FINGER METAL-

BINDING NU.

291
PR00070
DIHYDROFOLATE
PR00070C 13.09 6.143e−16 51-63

REDUCTASE SIGNATURE
PR00070D 11.63 2.929e−15 112-127

294
PR00250
FUNGAL PHEROMONE
PR00250D 14.62 9.163e−09 254-278

MATING FACTOR STE2

GPCR SIGNATURE

296
PR00081
GLUCOSE/RIBITOL
PR00081A 10.53 2.731e−09 39-57

DEHYDROGENASE FAMILY

SIGNATURE

297
PR00806
VINCULIN SIGNATURE
PR00806B 4.28 8.920e−09 276-290

PR00806B 4.28 9.640e−09 275-289

298
PF00992
Troponin.
PF00992A 16.67 3.789e−10 553-588

300
PR00511
TEKTIN SIGNATURE
PR00511C 7.86 4.214e−09 371-388

302
BL00353
HMG1/2 proteins.
BL00353B 11.47 9.609e−19 228-278

303
PR00240
ALPHA-1A ADRENERGIC
PR00240C 8.38 3.941e−10 316-336

RECEPTOR SIGNATURE

304
BL00518
Zinc finger, C3HC4
BL00518 12.23 2.200e−11 54-63

type (RING finger),

proteins.

307
PR00193
MYOSIN HEAVY CHAIN
PR00193D 14.36 1.545e−31 390-419

SIGNATURE
PR00193C 12.60 1.209e−25 143-171

PR00193B 11.69 2.543e−24 95-121

PR00193A 15.41 6.885e−19 39-59

PR00193E 19.47 3.291e−12 444-473

308
PR00239
MOLLUSCAN RHODOPSIN
PR00239E 1.58 5.920e−11 47-59

C-TERMINAL TAIL

SIGNATURE

309
PD00015
GLYCOPROTEIN
PD00015A 8.90 6.400e−09 35-43

PRECURSOR CELL SI.

312
DM00031
IMMUNOGLOBULIN V
DM00031B 15.41 3.662e−11 80-114

REGION.

313
BL00824
Elongation factor 1
BL00824C 14.58 1.000e−40 129-167

beta/beta′/delta
BL00824D 14.04 6.192e−39 167-202

chain proteins.
BL00824B 9.21 2.080e−21 96-116

BL00824E 12.49 3.333e−19 210-226

314
DM00099
4 kw A55R REDUCTASE
DM00099B 14.73 8.364e−11 525-535

TERMINAL
DM00099B 14.73 9.438e−09 478-488

DIHYDROPTERIDINE.

315
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 6.200e−30 43-82

ZINC-FINGER METAL-

BINDING NU.

316
PR00121
SODIUM/POTASSIUM-
PR00121D 16.72 1.577e−13 210-232

TRANSPORTING ATPASE

SIGNATURE

317
BL00888
Cyclic nucleotide-
BL00888B 14.79 1.692e−10 396-420

binding domain

proteins.

318
PR00727
BACTERIAL LEADER
PR00727C 13.04 9.063e−16 108-128

PEPTIDASE 1 (S26)
PR00727B 12.51 7.848e−11 81-94

FAMILY SIGNATURE

319
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 9.471e−27 13-52

ZINC-FINGER METAL-

BINDING NU.

321
PR00004
ANAPHYLATOXIN DOMAIN
PR00004C 12.46 8.579e−09 91-103

SIGNATURE

322
DM00060
338 kw NEUREXIN
DM00060 6.92 6.500e−11 28-38

ALPHA III CYSTEINE.

327
PR00020
MAM DOMAIN SIGNATURE
PR00020A 18.17 5.776e−12 344-363

PR00020C 13.66 6.932e−10 417-429

328
BL00048
Protamine P1
BL00048 6.39 6.566e−10 167-194

proteins.

329
PR00020
MAM DOMAIN SIGNATURE
PR00020C 13.66 2.615e−11 581-593

PR00020B 15.52 5.059e−10 52-69

PR00020B 15.52 1.789e−09 553-570

331
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 9.357e−32 8-47

ZINC-FINGER METAL-

BINDING NU.

334
PD02870
RECEPTOR
PD02870B 18.83 5.871e−11 468-501

INTERLEUKIN-1

PRECURSOR.

335
BL00738
S-adenosyl-L-
BL00738J 18.61 1.000e−40 592-642

homocysteine
BL00738H 23.08 5.320e−36 468-521

hydrolase proteins.
BL00738F 12.23 7.261e−29 387-419

BL00738A 16.27 9.660e−27 216-256

BL00738C 16.53 7.923e−25 281-319

BL00738G 14.29 6.268e−23 446-468

BL00738B 12.28 8.085e−21 256-281

BL00738E 14.18 9.200e−19 361-384

BL00738I 14.57 5.135e−17 545-583

BL00738D 7.16 5.109e−13 335-350

339
PR00425
BRADYKININ RECEPTOR
PR00425C 13.23 3.586e−09 80-100

SIGNATURE

344
PD01823
PROTEIN INTERGENIC
PD01823E 9.30 6.824e−12 108-121

REGION ABC1
PD01823D 16.66 1.265e−09 46-67

PRECURSOR

MITOCHONDRION T.

345
PR00976
RIBOSOMAL PROTEIN
PR00976C 10.41 2.837e−09 396-407

S21 FAMILY

SIGNATURE.

346
PR00613
MYOGLOBIN SIGNATURE
PR00613B 9.02 2.581e−10 25-49

347
PR00814
BETA HAEMOGLOBIN
PR00814C 9.20 6.523e−10 104-122

SIGNATURE

350
BL00038
Myc-type, ‘helix-
BL00038B 16.97 7.568e−10 117-138

loop-helix’

dimerization domain

proteins.

351
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 6.571e−32 6-45

ZINC-FINGER METAL-

BINDING NU.

352
BL00972
Ubiquitin carboxyl-
BL00972A 11.93 6.318e−19 364-382

terminal hydrolases
BL00972D 22.55 7.968e−16 648-673

family 2 proteins.
BL00972B 9.45 1.600e−12 445-455

354
BL00518
Zinc finger, C3HC4
BL00518 12.23 4.429e−10 214-223

type (RING finger),

proteins.

355
BL00518
Zinc finger, C3HC4
BL00518 12.23 4.429e−10 179-188

type (RING finger),

proteins.

356
BL01009
Extracellular
BL01009D 14.19 9.341e−17 160-181

proteins SCP/Tpx-
BL01009A 13.75 3.769e−14 80-98

1/Ag5/PR-1/Sc7
BL01009E 13.50 5.333e−14 194-210

proteins.
BL01009C 10.54 2.667e−11 127-141

358
BL00215
Mitochondrial energy
BL00215A 15.82 8.500e−17 16-41

transfer proteins.
BL00215B 10.44 4.900e−09 177-190

BL00215A 15.82 6.786e−09 133-158

BL00215B 10.44 7.300e−09 278-291

359
BL00053
Ribosomal protein S8
BL00053C 16.71 5.500e−26 107-140

proteins.
BL00053B 14.56 4.789e−14 67-85

BL00053A 8.83 5.320e−12 14-27

360
PR00326
GTP1/OBG GTP-BINDING
PR00326A 8.75 7.150e−11 21-42

PROTEIN FAMILY

SIGNATURE

361
BL00972
Ubiquitin carboxyl-
BL00972A 11.93 6.318e−19 324-342

terminal hydrolases
BL00972D 22.55 3.903e−16 608-633

family 2 proteins.
BL00972B 9.45 1.600e−12 405-415

364
PR00759
BASIC PROTEASE
PR00759C 14.15 7.750e−10 123-139

(KUNITZ-TYPE)

INHIBITOR FAMILY

SIGNATURE

367
DM00215
PROLINE-RICH PROTEIN 3.
DM00215 19.43 1.482e−10 355-388

368
BL00518
Zinc finger, C3HC4
BL00518 12.23 2.800e−11 125-134

type (RING finger),

proteins.

370
PD00126
PROTEIN REPEAT
PD00126A 22.53 8.448e−09 2-23

DOMAIN TPR NUCLEA.

373
BL00028
Zinc finger, C2H2
BL00028 16.07 7.353e−14 157-174

type, domain
BL00028 16.07 1.000e−13 269-286

proteins.
BL00028 16.07 8.200e−13 493-510

BL00028 16.07 3.739e−12 213-230

BL00028 16.07 6.478e−12 381-398

BL00028 16.07 1.346e−11 185-202

BL00028 16.07 2.385e−11 129-146

BL00028 16.07 2.385e−11 325-342

BL00028 16.07 5.154e−11 241-258

BL00028 16.07 9.654e−11 437-454

BL00028 16.07 1.300e−10 297-314

BL00028 16.07 9.100e−10 409-426

BL00028 16.07 9.100e−10 465-482

374
BL01020
SAR1 family
BL01020C 15.35 8.063e−20 79-130

proteins.

376
BL00028
Zinc finger, C2H2
BL00028 16.07 4.522e−12 208-225

type, domain

proteins.

377
PR00308
TYPE I ANTIFREEZE
PR00308A 5.90 7.288e−11 533-548

PROTEIN SIGNATURE
PR00308A 5.90 8.835e−09 534-549

380
PD02784
PROTEIN NUCLEAR
PD02784B 26.46 7.538e−09 147-190

RIBONUCLEOPROTEIN.

381
PD01351
PROTEIN REPEAT
PD01351A 8.69 7.469e−09 155-166

NEUROFILAMENT TRIPL.

383
PF00094
von Willebrand
PF00094C 12.88 1.918e−09 43-53

factor type D domain

proteins.

384
BL01105
Ribosomal protein
BL01105B 12.95 7.930e−13 43-83

L35Ae proteins.

387
PR00049
WILM'S TUMOR
PR00049D 0.00 9.643e−10 10-25

PROTEIN SIGNATURE
PR00049D 0.00 1.915e−09 9-24

388
BL01115
GTP-binding nuclear
BL01115A 10.22 8.909e−13 34-78

protein ran

proteins.

389
BL00115
Eukaryotic RNA
BL00115Z 3.12 7.977e−10 397-446

polymerase II

heptapeptide repeat

proteins.

390
PR00021
SMALL PROLINE-RICH
PR00021A 4.31 2.125e−19 4-17

PROTEIN SIGNATURE
PR00021D 6.09 3.323e−13 21-30

PR00021D 6.09 3.323e−13 30-39

PR00021E 6.82 7.545e−13 57-67

PR00021B 7.29 9.333e−13 18-28

PR00021B 7.29 3.302e−12 27-37

PR00021C 5.55 1.225e−10 21-28

PR00021C 5.55 1.225e−10 30-37

PR00021D 6.09 2.770e−09 39-48

391
PF00646
F-box domain
PF00646A 14.37 9.036e−10 28-42

proteins.

392
BL00036
bZIP transcription
BL00036 9.02 6.294e−12 81-94

factors basic domain

proteins.

393
PF00622
Domain in SPla and
PF00622B 21.00 2.500e−13 85-107

the RYanodine

Receptor.

394
BL00564
Argininosuccinate
BL00564A 19.93 6.114e−09 7-44

synthase proteins.

395
PR00048
C2H2-TYPE ZINC
PR00048A 10.52 7.750e−14 230-244

FINGER SIGNATURE
PR00048A 10.52 4.316e−11 202-216

396
PF00622
Domain in SPla and
PF00622B 21.00 1.391e−16 132-154

the RYanodine

Receptor.

397
PR00501
KELCH REPEAT
PR00501A 8.25 1.409e−09 537-551

SIGNATURE

398
PR00399
SYNAPTOTAGMIN
PR00399A 9.52 3.571e−19 146-162

SIGNATURE
PR00399C 12.82 8.200e−17 222-238

PR00399B 14.27 7.750e−16 161-175

PR00399D 14.48 4.000e−14 242-253

399
BL01013
Oxysterol-binding
BL01013A 25.14 7.231e−21 558-594

protein family
BL01013B 11.33 1.000e−11 623-634

proteins.

400
PF00023
Ank repeat proteins.
PF00023A 16.03 1.750e−10 55-71

401
BL00422
Granins proteins.
BL00422C 16.18 5.787e−10 134-162

402
PR00029
OCTAMER-BINDING
PR00029C 6.10 5.708e−09 140-153

TRANSCRIPTION FACTOR

SIGNATURE

403
PR00450
RECOVERIN FAMILY
PR00450D 16.58 8.986e−11 161-181

SIGNATURE

404
PR00049
WILM'S TUMOR
PR00049D 0.00 7.407e−09 325-340

PROTEIN SIGNATURE

405
DM01117
2 kw TRANSPOSASE
DM01117A 11.17 7.750e−09 364-382

WITHIN TRANSPOSITION

VASOTOCIN.

406
DM01206
CORONAVIRUS
DM01206B 10.69 9.286e−12 724-744

NUCLEOCAPSID
DM01206B 10.69 3.466e−10 726-746

PROTEIN.
DM01206B 10.69 9.630e−10 722-742

DM01206B 10.69 7.152e−09 718-738

DM01206B 10.69 8.861e−09 728-748

407
PD01066
PROTEIN ZINC FINGER
PD01066 19.43 1.353e−27 31-70

ZINC-FINGER METAL-

BINDING NU.

408
PR00169
POTASSIUM CHANNEL
PR00169A 16.77 1.711e−09 94-114

SIGNATURE

409
BL00610
Sodium: neurotransmitter
BL00610A 17.73 1.000e−40 68-118

symporter family
BL00610B 23.65 1.000e−40 132-182

proteins.
BL00610C 12.94 1.000e−40 225-277

BL00610D 20.97 1.000e−40 291-344

BL00610F 29.02 6.143e−36 540-595

BL00610E 20.34 3.647e−35 448-491

BL00610G 12.89 2.200e−15 611-634

410
DM00179
w KINASE ALPHA
DM00179 13.97 5.304e−09 111-121

ADHESION T-CELL.

411
PR00625
DNAJ PROTEIN FAMILY
PR00625B 13.48 1.800e−16 45-66

SIGNATURE
PR00625A 12.84 6.700e−12 15-35

412
PR00927
ADENINE NUCLEOTIDE
PR00927E 14.93 4.136e−11 246-268

TRANSLOCATOR 1

SIGNATURE

413
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 6.400e−17 411-424

METAL-BINDI.
PD00066 13.92 8.200e−17 327-340

PD00066 13.92 5.154e−15 271-284

PD00066 13.92 2.800e−14 215-228

PD00066 13.92 9.000e−13 355-368

PD00066 13.92 6.143e−12 439-452

PD00066 13.92 6.478e−11 187-200

PD00066 13.92 9.217e−11 243-256

414
BL00303
S-100/ICaBP type
BL00303B 26.15 2.552e−23 51-88

calcium binding
BL00303A 21.77 5.846e−20 4-41

protein.

416
PR00014
FIBRONECTIN TYPE III
PR00014C 15.44 4.600e−10 73-92

REPEAT SIGNATURE

417
PR00806
VINCULIN SIGNATURE
PR00806A 6.63 1.493e−09 785-796

418
PF00622
Domain in SPla and
PF00622B 21.00 1.000e−13 331-353

the RYanodine
PF00622C 12.62 3.160e−11 415-429

Receptor.

419
PF00780
Domain found in
PF00780B 23.03 5.929e−33 442-485

NIK1-like kinases,

mouse citron and

yeast ROM.

420
BL01253
Type I fibronectin
BL01253H 13.15 8.452e−13 203-238

domain proteins.
BL01253D 4.84 2.016e−12 41-55

421
BL01207
Glypicans proteins.
BL01207B 23.69 9.122e−28 191-237

BL01207A 12.21 1.000e−16 62-78

426
PD02870
RECEPTOR
PD02870D 15.74 4.351e−09 693-728

INTERLEUKIN-1

PRECURSOR.

427
BL00203
Vertebrate
BL00203 13.94 5.041e−09 13-59

metallothioneins

proteins.

428
BL00107
Protein kinases ATP-
BL00107A 18.39 8.579e−18 217-248

binding region

proteins.

431
PR00141
PROTEASOME COMPONENT
PR00141C 11.15 6.333e−12 234-246

SIGNATURE
PR00141D 12.45 8.615e−12 259-271

PR00141B 11.15 9.561e−12 223-235

PR00141A 11.36 2.050e−11 102-118

432
PR00245
OLFACTORY RECEPTOR
PR00245A 18.03 9.413e−17 59-81

SIGNATURE
PR00245C 7.84 7.500e−16 238-254

PR00245E 12.40 2.500e−12 291-306

PR00245B 10.38 9.550e−11 177-192

433
PF00651
BTB (also known as
PF00651 15.00 1.000e−11 87-100

BR-C/Ttk) domain

proteins.

436
BL00086
Cytochrome P450
BL00086 20.87 3.647e−23 430-462

cysteine heme-iron

ligand proteins.

437
BL00216
Sugar transport
BL00216B 27.64 7.943e−19 101-151

proteins.

438
PR00049
WILM'S TUMOR
PR00049D 0.00 8.429e−10 10-25

PROTEIN SIGNATURE

439
PR00245
OLFACTORY RECEPTOR
PR00245A 18.03 2.667e−23 100-122

SIGNATURE
PR00245C 7.84 1.783e−14 232-248

PR00245D 10.47 7.070e−10 268-280

440
PR00262
IL1/HBGF FAMILY
PR00262A 28.26 1.000e−08 80-108

SIGNATURE

441
BL00884
Osteopontin
BL00884B 12.47 1.000e−40 50-94

proteins.
BL00884C 22.45 6.625e−39 131-173

BL00884A 11.35 5.846e−32 1-31

BL00884E 11.04 8.364e−23 273-295

BL00884D 8.79 3.323e−18 255-272

*Results include in order: accession number subtype; raw score; p-value; postion of signature in amino acid sequence.

[0395]

4

TABLE 4

SEQ ID

NO:
pFAM NAME
DESCRIPTION
p-value
pFAM SCORE

1
zf-CCCH
Zinc finger C-x8-C-x5-C-
2.5e−09
38.4

x3-H type (and similar).

3
ArfGap
Putative GTP-ase
1.3e−52
188.3

activating protein for

Arf

4
EMP24_GP25L
emp24/gp25L/p24 family
4.1e−105
362.6

7
WW
WW domain
1.2e−05
32.2

8
WW
WW domain
1.2e−05
32.2

9
Aa_trans
Transmembrane amino acid
9.6e−64
225.2

transporter protein

10
Fe-ADH
Iron-containing alcohol
9.9e−35
124.5

dehydrogenases

11
Fe-ADH
Iron-containing alcohol
9.9e−35
124.5

dehydrogenases

12
Bcl-2
Apoptosis regulator
0.016
−2.1

proteins, Bcl-2 family

13
spectrin
Spectrin repeat
1.3e−10
43.6

14
Ribosomal_L18ae
Ribosomal L18ae protein
1.9e−128
440.1

family

15
Ribosomal_L31e
Ribosomal protein L31e
2.4e−47
170.7

17
zf-MYND
MYND finger
1.4e−13
58.5

19
MgtE
Divalent cation
8.6e−39
142.3

transporter

20
Rap_GAP
Rap/ran-GAP
2e−124
426.7

21
Rap_GAP
Rap/ran-GAP
2e−124
426.7

23
SCAN
SCAN domain
1.3e−24
95.2

24
RhoGAP
RhoGAP domain
3e−58
206.9

25
adh_zinc
Zinc-binding
1.5e−05
−25.4

dehydrogenases

26
UDPGT
UDP-glucoronosyl and UDP-
1.6e−84
294.3

glucosyl transferases

27
MCT
Monocarboxylate
3.3e−49
176.9

transporter

29
Ribosomal_L6e
Ribosomal_protein L6e
9.5e−78
271.7

30
Ribosomal_L11
Ribosomal protein L11
4.9e−64
226.2

31
tRNA-synt_1e
tRNA synthetases class I
1.6e−137
470.2

(C)

33
zf-C3HC4
Zinc finger, C3HC4 type
0.00041
17.6

(RING finger)

35
SH3
SH3 domain
8.7e−32
119.0

36
ras
Ras family
2.6e−78
273.6

38
SET
SET domain
3.2e−05
10.0

39
laminin_G
Laminin G domain
1.5e−11
44.7

40
Sema
Sema domain
5e−127
435.4

42
filament
Intermediate filament
1.6e−138
473.6

proteins

43
Keratin_B2
Keratin, high sulfur B2
1.8e−18
74.8

protein

46
sushi
Sushi domain (SCR repeat)
3.8e−06
33.9

47
profilin
Profilins
4.1e−13
51.7

49
ubiquitin
Ubiquitin family
0.069
10.3

50
BTB
BTB/POZ domain
2.6e−21
84.2

51
serpin
Serpins (serine protease
2.4e−178
605.4

inhibitors)

52
T-box
T-box
3.6e−125
429.2

54
7tm_1
7 transmembrane receptor
1.2e−17
58.3

(rhodopsin family)

55
CSD
‘Cold-shock’ DNA-binding
1.8e−16
63.6

domain

56
ig
Immunoglobulin domain
2.5e−07
28.7

57
Rap_GAP
Rap/ran-GAP
5e−18
73.3

59
G-gamma
GGL domain
1.1e−11
45.4

60
T-box
T-box
8.9e−114
391.4

63
60s_ribosomal
60s Acidic ribosomal
0.0089
12.0

protein

64
UPAR_LY6
u-PAR/Ly-6 domain
5.4e−05
22.3

65
Ribosomal_L30
Ribosomal protein
0.00042
18.5

L30p/L7e

66
filament
Intermediate filament
1.1e−78
274.8

proteins

67
Ribosomal_S6
Ribosomal protein S6
0.00082
7.5

68
PDZ
PDZ domain (Also known as
5.1e−09
43.4

DHR or GLGF).

69
zf-C3HC4
Zinc finger, C3HC4 type
0.005
14.0

(RING finger)

70
G-patch
G-patch domain
0.00051
26.8

71
Keratin_B2
Keratin, high sulfur B2
0.037
−45.9

protein

85
ig
Immunoglobulin domain
8.5e−09
33.4

88
zf-C2H2
Zinc finger, C2H2 type
7.1e−71
248.9

89
ig
Immunoglobulin domain
2.7e−35
118.7

91
WD40
WD domain, G-beta repeat
7.5e−10
46.2

92
FKBP
FKBP-type peptidyl-prolyl
1.3e−53
173.3

cis-trans isomerases

95
E1_dehydrog
Dehydrogenase E1
8.7e−23
89.1

component

97
zf-C3HC4
Zinc finger, C3HC4 type
8.7e−09
32.7

(RING finger)

99
ig
Immunoglobulin domain
1.8e−20
71.0

100
ig
Immunoglobulin domain
1.8e−20
71.0

104
zf-C2H2
Zinc finger, C2H2 type
2.4e−94
326.8

105
zf-C2H2
Zinc finger, C2H2 type
1.6e−55
197.9

109
zf-CCHC
Zinc finger, CCHC class
2.6e−12
54.4

110
zf-C2H2
Zinc finger, C2H2 type
2.2e−42
154.3

111
myosin_head
Myosin head (motor
0
1267.5

domain)

112
pkinase
Eukaryotic protein kinase
1.2e−96
334.5

domain

113
WD40
WD domain, G-beta repeat
5.9e−65
229.2

114
SNF2_N
SNF2 and others N-
4.2e−78
272.9

terminal domain

115
DUF15
Domain of unknown
0.0011
−65.2

function DUF15

116
DSPc
Dual specificity
0.00056
3.7

phosphatase, catalytic

domain

119
Rhodanese
Rhodanese-like domain
1e−05
32.5

124
proteasome
Proteasome A-type and B-
7.4e−43
155.8

type

126
Ribosomal_L9
Ribosomal protein L9
3.1e−05
−3.4

127
RIO1
RIO1/ZK632.3/MJ0444
7.8e−80
278.6

family

130
abhydrolase
alpha/beta hydrolase fold
9e−20
79.1

131
TPR
TPR Domain
5.7e−27
103.0

132
HMG14_17
HMG14 and HMG17
1.9e−15
64.7

133
bZIP
bZIP transcription factor
8.3e−19
71.7

134
rrm
RNA recognition motif.
1.9e−31
117.9

(a.k.a. RRM, RBD, or RNP

domain)

135
AMP-binding
AMP-binding enzyme
6.3e−98
338.7

140
tubulin
Tubulin/FtsZ family
2.1e−151
516.4

143
laminin_EGF
Laminin EGF-like (Domains
7.6e−12
52.8

III and V)

144
FYVE
FYVE zinc finger
2.3e−29
109.1

145
zf-C2H2
Zinc finger, C2H2 type
2.4e−33
124.2

146
mito_carr
Mitochondrial carrier
3e−54
188.9

proteins

148
DAGKc
Diacylglycerol kinase
0.00015
26.0

catalytic domain

(presumed)

149
Exonuclease
Exonuclease
2.1e−17
71.3

152
GTP_EFTU
Elongation factor Tu
8.5e−09
32.3

family

153
WH2
Wiskott Aldrich syndrome
6.5e−20
79.6

homology region 2

154
SAM
SAM domain (Sterile alpha
0.032
11.4

motif)

156
DHDPS
Dihydrodipicolinate
1.3e−26
101.9

synthetase family

158
PseudoU_synth_1
tRNA pseudouridine
1e−30
115.4

synthase

159
pkinase
Eukaryotic protein kinase
2.3e−59
210.6

domain

162
IF-2B
Initiation factor 2
1.7e−98
340.7

subunit family

163
Beach
Beige/BEACH domain
1.1e−224
759.8

166
DnaJ
DnaJ domain
8.1e−11
49.4

167
Anti_proliferat
BTG1 family
7.4e−85
295.3

168
sugar_tr
Sugar (and other)
3.1e−80
280.0

transporter

169
sugar_tr
Sugar (and other)
2.9e−52
187.0

transporter

170
zf-C2H2
Zinc finger, C2H2 type
2.2e−93
323.6

171
GBP
Guanylate-binding protein
4.3e−255
860.8

173
TPR
TPR Domain
1.1e−42
155.2

175
RhoGEF
RhoGEF domain
3.3e−40
147.0

176
zf-C3HC4
Zinc finger, C3HC4 type
0.00011
19.4

(RING finger)

177
Peptidase_M22
Glycoprotease family
2.3e−73
257.2

179
TBC
TBC domain
4.7e−08
10.1

180
transmembrane4
Transmembrane 4 family
1.9e−49
161.1

181
CH
Calponin homology (CH)
1.2e−25
98.6

domain

184
AP_endonucleas1
AP endonuclease family 1
0.088
−90.2

186
Bacterial_PQQ
PQQ enzyme repeat
9.3e−05
29.2

187
DEAD
DEAD/DEAH box helicase
1.6e−60
194.3

188
zf-C2H2
Zinc finger, C2H2 type
4.2e−24
93.5

189
sugar_tr
Sugar (and other)
0.0011
−88.8

transporter

190
tRNA_int_endo
tRNA intron endonuclease
0.0012
−32.1

191
WSC
WSC domain
1e−35
132.1

193
pkinase
Eukaryotic protein kinase
5.1e−75
262.6

domain

195
globin
Globin
1.9e−26
96.6

197
WD40
WD domain, G-beta repeat
1.8e−07
38.2

200
F_actin_cap_B
F-actin capping protein,
1.7e−224
759.2

beta subunit

201
ank
Ank repeat
3.1e−59
210.2

205
PDZ
PDZ domain (Also known as
4.2e−07
37.0

DHR or GLGF).

206
SAM
SAM domain (Sterile alpha
2.2e−05
31.3

motif)

207
SAM
SAM domain (Sterile alpha
2.2e−05
31.3

motif)

208
zf-UBR1
Putative zinc finger in
3.9e−26
100.3

N-recognin

209
ABC_tran
ABC transporter
2.4e−112
386.6

211
zf-C2H2
Zinc finger, C2H2 type
0.00031
27.5

212
UCH-2
Ubiquitin carboxyl-
1.5e−19
78.4

terminal hydrolase family 2

213
IMP4
Domain of unknown
2.2e−33
124.3

function

215
zf-C2H2
Zinc finger, C2H2 type
3.5e−08
40.6

216
PG_binding_2
Putative peptidoglycan
2.1e−11
51.3

binding domain

219
pyr_redox
Pyridine nucleotide-
1.2e−70
241.7

disulphide oxidoreductase

220
PDZ
PDZ domain (Also known as
8.5e−19
75.9

DHR or GLGF).

221
pkinase
Eukaryotic protein kinase
8.1e−67
235.4

domain

222
dsrm
Double-stranded RNA
0.095
7.5

binding motif

223
PHD
PHD-finger
4.7e−05
29.9

225
TRM
N2, N2-dimethylguanosine
7.3e−22
86.1

tRNA methyltransferase

226
LIM
LIM domain containing
8.6e−08
32.7

proteins

227
ig
Immunoglobulin domain
1.1e−07
29.8

229
F-box
F-box domain.
2.2e−07
38.0

231
Glucosamine_iso
Glucosamine-6-phosphate
1.8e−146
500.0

isomerase

233
PTN_MK
PTN/MK heparin-binding
1.9e−44
161.1

protein family

238
CNG_membrane
Transmembrane region
5.8e−31
116.3

cyclic Nucleotide Gated

Channel

240
GNS1_SUR4
GNS1/SUR4 family
4.1e−46
166.6

243
PIP5K
Phosphatidylinositol-4-
6.2e−142
484.9

phosphate 5-Kinase

244
cadherin
Cadherin domain
0
1298.9

246
fn3
Fibronectin type III
1.2e−31
118.6

domain

247
UQ_con
Ubiquitin-conjugating
1.4e−16
68.5

enzyme

248
LRR
Leucine Rich Repeat
3.4e−14
60.6

249
lipocalin
Lipocalin / cytosolic
1.2e−28
102.8

fatty-acid binding

protein family

250
Ribosomal_S2
Ribosomal protein S2
2.9e−11
43.7

251
tubulin
Tubulin/FtsZ family
8.5e−163
554.2

252
tubulin
Tubulin/FtsZ family
2.4e−212
718.8

253
ATP-synt_ab
ATP synthase alpha/beta
6.3e−108
372.0

family

254
ATP-synt_A-c
ATP synthase Alpha chain,
4.3e−106
313.5

C terminal

255
zf-C3HC4
Zinc finger, C3HC4 type
5e−12
43.2

(RING finger)

256
G-patch
G-patch domain
1e−07
39.1

257
CH
Calponin homology (CH)
1.6e−11
51.7

domain

258
RF-1
Peptidyl-tRNA hydrolase
5.9e−66
232.5

domain

259
RF-1
Peptidyl-tRNA hydrolase
5.9e−66
232.5

domain

261
thiored
Thioredoxin
2e−09
35.7

262
thyroglobulin_1
Thyroglobulin type-1
3.1e−34
127.2

repeat

264
DnaJ
DnaJ domain
7.9e−13
56.0

267
DUF6
Integral membrane protein
0.083
9.1

DUF6

268
Ribosomal_L31e
Ribosomal protein L31e
1.7e−61
217.7

270
Zn_carbOpept
Zinc carboxypeptidase
3.5e−50
180.1

272
BTB
BTB/POZ domain
7.7e−18
72.7

273
Glycos_transf_1
Glycosyl transferases
0.027
12.8

group 1

277
SPRY
SPRY domain
2.5e−28
107.6

279
BTB
BTB/POZ domain
6e−27
103.0

280
zf-C2H2
Zinc finger, C2H2 type
3.7e−116
399.3

281
SCAN
SCAN domain
2.4e−52
187.3

284
NTP_transf_2
Nucleotidyltransferase
8.5e−13
55.9

domain

288
zf-C2H2
Zinc finger, C2H2 type
5.4e−93
322.4

289
zf-C2H2
Zinc finger, C2H2 type
4.5e−124
425.6

291
DiHfolate_red
Dihydrofolate reductase
1.5e−63
221.6

293
PDZ
PDZ domain (Also known as
7.4e−17
69.4

DHR or GLGF).

295
PH
PH domain
1.6e−08
35.4

296
adh_short
short chain dehydrogenase
4.5e−32
120.0

299
BNR
BNR repeat
3.1e−06
34.1

302
HMG_box
HMG (high mobility group)
5.4e−05
20.0

box

303
ig
Immunoglobulin domain
0.05
11.6

304
zf-C3HC4
Zinc finger, C3HC4 type
5e−12
43.2

(RING finger)

305
START
START domain
0.013
4.8

306
integrase
Integrase DNA binding
7.2e−06
32.9

domain

307
myosin_head
Myosin head (motor
7.6e−279
939.7

domain)

308
zf-C2H2
Zinc finger, C2H2 type
1.5e−53
191.4

309
ig
Immunoglobulin domain
0.00023
19.1

311
ras
Ras family
0.00079
−93.3

312
ig
Immunoglobulin domain
2.1e−06
25.7

313
EF1BD
EF-1 guanine nucleotide
4.3e−56
199.8

exchange domain

314
Kelch
Kelch motif
3.6e−94
326.2

315
zf-C2H2
Zinc finger, C2H2 type
6.8e−59
209.1

317
CNG_membrane
Transmembrane region
1.5e−28
108.3

cyclic Nucleotide Gated

Channel

318
Peptidase_S26
Signal peptidase I
2.8e−16
56.3

319
zf-C2H2
Zinc finger, C2H2 type
4.6e−56
199.7

322
EGF
EGF-like domain
4.7e−08
40.2

323
lectin_c
Lectin C-type domain
6.6e−13
56.3

324
MCT
Monocarboxylate
1.9e−50
181.0

transporter

327
MAM
MAM domain.
1.8e−51
184.4

329
MAM
MAM domain.
2e−179
609.5

330
Sema
Sema domain
9.9e−211
713.5

331
zf-C2H2
Zinc finger, C2H2 type
1.5e−84
294.3

333
PAP2
PAP2 superfamily
9.8e−10
45.8

334
LRR
Leucine Rich Repeat
2.8e−36
134.0

335
AdoHcyase
S-adenosyl-L-homocysteine
1.2e−238
806.2

hydrolase

336
TBC
TBC domain
9.4e−38
138.9

343
WD40
WD domain, G-beta repeat
8.7e−08
39.3

346
globin
Globin
3e−45
162.2

347
globin
Globin
7.5e−39
139.9

349
F-box
F-box domain.
8.5e−07
36.0

350
HLH
Helix-loop-helix DNA-
2e−08
41.4

binding domain

351
KRAB
KRAB box
2.7e−39
144.0

352
UCH-2
Ubiquitin carboxyl-
1.7e−19
78.2

terminal hydrolase family 2

353
IPP_isomerase
Isopentenyl-diphosphate
9.5e−94
324.9

delta-isomerase

354
IBR
IBR domain
1.6e−12
55.0

355
IBR
IBR domain
1.6e−12
55.0

356
SCP
SCP-like extracellular
1.4e−34
128.3

protein

358
mito_carr
Mitochondrial carrier
3.9e−74
257.0

proteins

359
Ribosomal_S8
Ribosomal protein S8
6e−58
192.1

361
UCH-2
Ubiquitin carboxyl-
6.7e−23
89.5

terminal hydrolase family 2

363
Phage_lysozyme
Lysozyme
9.3e−05
23.5

365
Ribosomal_S2
Ribosomal protein S2
3.3e−08
32.9

367
zf-C3HC4
Zinc finger, C3HC4 type
5.3e−09
33.4

(RING finger)

368
zf-C3HC4
Zinc finger, C3HC4 type
0.0096
13.1

(RING finger)

370
TPR
TPR Domain
0.041
20.5

373
zf-C2H2
Zinc finger, C2H2 type
5.6e−109
375.5

374
arf
ADP-ribosylation factor
4.9e−39
143.1

family

375
BNR
BNR repeat
0.033
20.8

376
zf-C2H2
Zinc finger, C2H2 type
1.5e−24
95.0

379
rrm
RNA recognition motif.
0.00019
28.2

(a.k.a. RRM, RBD, or RNP

domain)

380
rrm
RNA recognition motif.
2.2e−19
77.9

(a.k.a. RRM, RBD, or RNP

domain)

383
vwc
von Willebrand factor
1.6e−31
118.2

type C domain

384
Ribosomal_L35Ae
Ribosomal protein L35Ae
0.00013
7.0

388
ras
Ras family
3.9e−63
223.2

391
F-box
F-box domain.
2.3e−05
31.3

392
bZIP
bZIP transcription factor
6.3e−09
36.9

393
SPRY
SPRY domain
0.092
−9.4

395
zf-C2H2
Zinc finger, C2H2 type
3.1e−17
70.7

396
SCAN
SCAN domain
3.1e−39
143.8

397
Kelch
Kelch motif
1.1e−55
198.4

398
C2
C2 domain
2.2e−80
280.4

399
ank
Ank repeat
1.8e−29
111.3

400
ank
Ank repeat
2.4e−19
77.7

403
DAGKa
Diacylglycerol kinase
1.9e−124
426.8

accessory domain

(presumed)

404
MCT
Monocarboxylate
0.012
−34.1

transporter

406
PDZ
PDZ domain (Also known as
7.7e−46
165.7

DHR or GLGF).

407
zf-C2H2
Zinc finger, C2H2 type
3.9e−48
173.3

408
K_tetra
K+ channel
2.6e−23
90.9

tetramerisation domain

409
SNF
Sodium: neurotransmitter
0
1268.7

symporter family

410
ig
Immunoglobulin domain
1.1e−06
26.5

411
DnaJ
DnaJ domain
9.9e−28
105.6

412
mito_carr
Mitochondrial carrier
7.6e−66
228.6

proteins

413
zf-C2H2
Zinc finger, C2H2 type
6e−97
335.5

414
S_100
S-100/ICaBP type calcium
9.7e−13
55.8

binding domain

416
fn3
Fibronectin type III
8.6e−14
59.3

domain

417
zf-C2H2
Zinc finger, C2H2 type
3.8e−27
103.6

418
zf-C3HC4
Zinc finger, C3HC4 type
4.4e−14
49.9

(RING finger)

419
pkinase
Eukaryotic protein kinase
1.2e−54
195.0

domain

420
trypsin
Trypsin
4.6e−38
122.5

421
Glypican
Glypican
5.7e−131
448.5

422
Keratin_B2
Keratin, high sulfur B2
0.0013
−23.4

protein

424
zf-C2H2
Zinc finger, C2H2 type
0.00021
28.1

425
ig
Immunoglobulin domain
0.00074
17.5

426
ig
Immunoglobulin domain
2.9e−23
80.0

427
Keratin_B2
Keratin, high sulfur B2
0.0023
−27.1

protein

428
pkinase
Eukaryotic protein kinase
2.3e−55
197.3

domain

429
ig
Immunoglobulin domain
4.1e−09
34.4

430
Galactosyl_T
Galactosyltransferase
1.8e−36
134.6

431
proteasome
Proteasome A-type and B-
5.5e−28
106.4

type

432
7tm_1
7 transmembrane receptor
3.4e−38
123.5

(rhodopsin family)

433
BTB
BTB/POZ domain
8.1e−23
89.2

436
p450
Cytochrome P450
6.9e−175
594.4

437
sugar_tr
Sugar (and other)
5.9e−65
229.2

transporter

438
zf-C2H2
Zinc finger, C2H2 type
2.1e−52
187.5

439
7tm_1
7 transmembrane receptor
2.2e−40
130.4

(rhodopsin family)

440
FGF
Fibroblast growth factor
4.6e−14
51.6

441
Osteopontin
Osteopontin
3.7e−181
615.2

[0396]

5

TABLE 5

POSITION OF SIGNAL

IN AMINO ACID
maxS (MAXIMUM

SEQ ID NO:
SEQUENCE
SCORE)
meanS (MEAN SCORE)

2
1-32
0.995
0.681

4
1-37
0.979
0.718

22
1-31
0.947
0.775

25
1-30
0.924
0.720

27
1-28
0.982
0.649

39
1-27
0.983
0.898

40
1-17
0.991
0.955

46
1-22
0.990
0.921

56
1-18
0.925
0.822

58
1-18
0.981
0.951

62
1-28
0.939
0.749

64
1-33
0.979
0.757

72
1-41
0.989
0.690

81
1-26
0.960
0.674

85
1-18
0.979
0.963

86
1-22
0.967
0.792

89
1-25
0.980
0.867

99
1-16
0.973
0.925

100
1-24
0.978
0.760

101
1-17
0.978
0.925

115
1-18
0.887
0.579

117
1-18
0.952
0.670

122
1-42
0.977
0.587

139
1-21
0.966
0.848

142
1-25
0.993
0.954

155
1-28
0.909
0.664

158
1-18
0.954
0.747

176
1-23
0.913
0.597

177
1-20
0.986
0.936

180
1-42
0.978
0.689

182
1-32
0.929
0.583

186
1-21
0.979
0.941

194
1-21
0.930
0.662

202
1-45
0.985
0.714

214
1-37
0.992
0.855

227
1-24
0.971
0.882

230
1-20
0.979
0.911

239
1-17
0.982
0.964

253
1-13
0.918
0.692

254
1-13
0.918
0.692

258
1-20
0.912
0.693

259
1-20
0.912
0.693

262
1-26
0.974
0.824

264
1-18
0.965
0.833

269
1-25
0.956
0.765

290
1-16
0.912
0.705

291
1-18
0.896
0.634

292
1-19
0.966
0.897

296
1-18
0.991
0.973

297
1-20
0.906
0.580

301
1-27
0.957
0.652

309
1-19
0.983
0.871

312
1-22
0.968
0.844

322
1-23
0.952
0.812

326
1-27
0.982
0.911

329
1-18
0.983
0.941

330
1-18
0.932
0.884

334
1-27
0.990
0.923

337
1-45
0.983
0.793

338
1-45
0.983
0.793

348
1-29
0.991
0.729

356
1-22
0.978
0.877

366
1-26
0.939
0.709

367
1-22
0.966
0.843

378
1-29
0.961
0.842

382
1-16
0.951
0.777

404
1-44
0.975
0.876

410
1-33
0.977
0.822

420
1-17
0.989
0.969

421
1-23
0.974
0.799

425
1-18
0.981
0.952

429
1-21
0.982
0.912

431
1-30
0.936
0.679

432
1-43
0.978
0.712

436
1-28
0.993
0.948

437
1-43
0.930
0.624

440
1-24
0.993
0.810

441
1-16
0.978
0.939

[0397]

	Number	Date	Country
Parent	09774528	Jan 2001	US
Child	10120988	Apr 2002	US

Novel nucleic acids and polypeptides

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

1. CROSS REFERENCE TO RELATED APPLICATIONS

Continuations (1)