Novel nucleic acids and polypeptides

1. BACKGROUND OF THE INVENTION

[0001] 1.1 Technical Field

[0002] 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.

[0003] 1.2 Background

[0004] 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.

[0005] 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.

2. SUMMARY OF THE INVENTION

[0006] 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.

[0007] 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.

[0008] 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: 1-362 and are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A is adenosine; 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.

[0009] The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-362 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: 1-362. A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-362 or a degenerate variant or fragment thereof. The identifying sequence can be 100 base pairs in length.

[0010] The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-362. The sequence information can be a segment of any one of SEQ ID NO: 1-362 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-362.

[0011] 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.

[0012] 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.

[0013] In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-362 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: 1-362 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.

[0014] 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: 1-362; a polynucleotide comprising any of the full length protein coding sequences of SEQ ID NO: 1-362; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ ID NO: 1-362. 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: 1-362; (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.

[0015] 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: 1-362; 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.

[0016] 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.

[0017] The invention also provides host cells transformed or transfected with a polynucleotide of the invention.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 1); 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.

3. DETAILED DESCRIPTION OF THE INVENTION

[0029] 3.1 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:1-362.

[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: 1-362. The sequence information can be a segment of any one of SEQ ID NOs: 1-362 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-362. 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 full 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 omithine, 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. Immunol. 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” 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 90% sequence identity. Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, and most preferably at least about 95% 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.

[0068] 3.2 Nucleic Acids of the Invention

[0069] Nucleotide sequences of the invention are set forth in the Sequence Listing.

[0070] The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-362; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO:1-362; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polynucleotides of any one of SEQ ID NO: 1-362. 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: 1-362; (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: 1-362. 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.

[0071] 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.

[0072] 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 genomic 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: 1-362 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-362 or a portion thereof as a probe. Alternatively, the polynucleotides of SEQ ID NO: 1-362 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.

[0073] 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.

[0074] 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%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide recited above.

[0075] 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: 1-362, 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.

[0076] 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: 1-362, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NOs: 1-362 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.

[0077] The nearest neighbor or homology result for the nucleic acids of the present invention, including SEQ ID NOs: 1-362, 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-362, 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.

[0086] 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.

[0087] The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NOs: 1-362 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: 1-362 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).

[0088] 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 Kaufman 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. Kaufman, 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.

[0089] 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 lac, 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.

[0090] 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.

[0091] 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.

[0092] 3.3 Hosts

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 3.4 Polypeptides of the Invention

[0103] 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: 1-362 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NOs: 1-362 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: 1-362 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 1-362 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: 1-362 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%, at least about 90%, typically at least about 95%, 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: 1-362.

[0104] 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.

[0105] 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.

[0106] Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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: 1-362.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.”

[0118] 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.

[0119] 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.).

[0120] 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.”

[0121] 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.

[0122] 3.4.1 Determining Polypeptide and Polynucleotide Identity and Similarity

[0123] 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).

[0124] 3.5 Gene Therapy

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 3.6 Transgenic Animals

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 3.7 Uses and Biological Activity

[0139] 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.

[0140] The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.

[0141] 3.7.1 Research Uses and Utilities

[0142] 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.

[0143] 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.

[0144] Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

[0145] 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.

[0146] 3.7.2 Nutritional Uses

[0147] 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.

[0148] 3.7.3 Cytokine and Cell Proliferation/Differentiation Activity

[0149] 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:

[0150] 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-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; 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 3.7.4 Stem Cell Growth Factor Activity

[0155] 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.

[0156] 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).

[0157] 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).

[0158] 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.

[0159] 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 useful 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.

[0160] 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.

[0161] 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).

[0162] 3.7.5 Rematopoiesis Regulating Activity

[0163] 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.

[0164] Therapeutic compositions of the invention can be used in the following:

[0165] Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

[0166] 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.

[0167] 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.

[0168] 3.7.6 Tissue Growth Activity

[0169] 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 burns, incisions and ulcers.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] Therapeutic compositions of the invention can be used in the following:

[0179] 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).

[0180] 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).

[0181] 3.7.7 Immune Stimulating or Suppressing Activity

[0182] 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.

[0183] 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).

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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).

[0188] 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.

[0189] 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.

[0190] 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 1 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.

[0191] The activity of a protein of the invention may, among other means, be measured by the following methods:

[0192] 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. Immunol. 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 3.7.8 Activin/Inhibin Activity

[0199] 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.

[0200] The activity of a polypeptide of the invention may, among other means, be measured by the following methods.

[0201] 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.

[0202] 3.7.9 Chemotactic/Chemokinetic Activity

[0203] 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.

[0204] 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.

[0205] Therapeutic compositions of the invention can be used in the following:

[0206] 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.

[0207] 3.7.10 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.

[0211] 3.7.11 Cancer Diagnosis and Therapy

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 3.7.12 Receptor/Ligand Activity

[0219] 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 selectins, 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.

[0220] The activity of a polypeptide of the invention may, among other means, be measured by the following methods:

[0221] 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.

[0222] 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.

[0223] 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, calorimetric 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 fluorescamine, or rhodamine or other colorimetric molecules. Examples of toxins include, but are not limited, to ricin.

[0224] 3.7.13 Drug Screening

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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).

[0229] 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):114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).

[0230] 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.

[0231] 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.

[0232] 3.7.14 Assay for Receptor Activity

[0233] 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.

[0234] 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.

[0235] 3.7.15 Anti-Inflammatory Activity

[0236] 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, inflamation 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.

[0237] 3.7.16 Leukemias

[0238] 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).

[0239] 3.7.17 Nervous System Disorders

[0240] 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:

[0241] (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;

[0242] (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;

[0243] (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;

[0244] (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;

[0245] (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, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;

[0246] (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;

[0247] (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and

[0248] (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.

[0249] 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:

[0250] (i) increased survival time of neurons in culture;

[0251] (ii) increased sprouting of neurons in culture or in vivo;

[0252] (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

[0253] (iv) decreased symptoms of neuron dysfunction in vivo.

[0254] 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.

[0255] 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).

[0256] 3.7.18 Other Activities

[0257] 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.

[0258] 3.7.19 Identification of Polymorphisms

[0259] 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.

[0260] 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 polymorphism 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.

[0261] 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.

[0262] 3.7.20 Arthritis and Inflammation

[0263] 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.

[0264] 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.

[0265] 3.8 Therapeutic Methods

[0266] 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.

3.8.1 EXAMPLE

[0267] 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.

[0268] 3.9 Pharmaceutical Formulations and Routes of Administration

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 3.9.1 Routes of Administration

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 3.9.2 Compositions/Formulations

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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 br 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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).

[0292] 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 formed, 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.

[0293] 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.

[0294] 3.9.3 Effective Dosage

[0295] 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.

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 3.9.4 Packaging

[0301] 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.

[0302] 3.10 Antibodies

[0303] Another aspect of the invention is an antibody that specifically binds the polypeptide of the invention. Such antibodies include monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR and/or antigen-binding sequences, which specifically recognize a polypeptide of the invention. Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fab′, F(ab′)2, and Fv, are also provided by the invention. 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. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.

[0304] Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[0305] 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.

[0306] Polypeptides of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).

[0307] 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. In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, A. M., Monoclonal Antibodies Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. 35:1-21 (1990); Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).

[0308] Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with a peptide or polypeptide of the invention. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of the protein encoded by the ORF of the present invention used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection. The protein that is used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity. Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or β-galactosidase) or through the inclusion of an adjuvant during immunization.

[0309] For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, Western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124 (1988)). Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)). Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention.

[0310] For polyclonal antibodies, antibody-containing antiserum is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures. The present invention further provides the above-described antibodies in delectably labeled form. Antibodies can be delectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art, for example, see (Sternberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

[0311] 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.

[0312] 3.11 Computer Readable Sequences

[0313] 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.

[0314] 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.

[0315] By providing any of the nucleotide sequences SEQ ID NOs: 1-362 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of SEQ ID NOs: 1-362 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.

[0316] 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.

[0317] 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.

[0318] 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).

[0319] 3.12 Triple Helix Formation

[0320] 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.

[0321] 3.13 Diagnostic Assays and Kits

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] 3.14 Medical Imaging

[0330] 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.

[0331] 3.15 Screening Assays

[0332] 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: 1-362, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of:

[0333] (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and

[0334] (b) determining whether the agent binds to said protein or said nucleic acid.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 3.16 Use of Nucleic Acids as Probes

[0345] 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: 1-362. 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: 1-362 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 3.17 Preparation of Support Bound Oligonucleotides

[0350] 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.

[0351] 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.

[0352] 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.).

[0353] 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).

[0354] 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.

[0355] 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.

[0356] 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.).

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] 3.18 Preparation of Nucleic Acid Fragments

[0362] 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).

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. A typical 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.

[0368] 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

[0369] 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.

[0370] 3.19 Preparation of DNA Arrays

[0371] 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 may be 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.

[0372] 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.

[0373] 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.

[0374] All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.

4.0 EXAMPLES

4.1 Example 1

[0375] Novel Nucleic Acid Sequences Obtained from Various Libraries

[0376] 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.

[0377] 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.

4.2 Example 2

[0378] Novel Nucleic Acids

[0379] 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 114, gb pri 114, and UniGene version 101) 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%.

[0380] 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, Genepet 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 gc-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: 1-362.

[0381] Table 1 shows the various tissue sources of SEQ ID NO: 1-362.

[0382] The homology for SEQ ID NO: 1-362 were obtained by a BLASTP version 2.0al 19 MP-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: 1-362 from Genpept. The homologues with identifiable functions for SEQ ID NO: 1-362 are shown in Table 2 below.

[0383] 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.

[0384] 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.

[0385] The nucleotide sequence within the sequences that codes for signal peptide sequences and their cleavage sites can be determine 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 1LIBRARY/HYSEQ LIBRARYTISSUE ORIGINRNA SOURCENAMESEQ ID NOS:adult brainGIBCOAB30014 18 39-40 83 88 98 110 112-113136 168-169 201-203adult brainGIBCOABD0037 15-16 31-32 39-41 45 54 5863 70 73-75 82-84 92 98 106110 114 116-117 126 128 130139 144 155 164 168-169 191-192195 198 204-215 239-240249 252 258 272-274adult brainClontechABR00110-11 15 19 39-40 88 106 120144 168 215-216 258adult brainClontechABR00613 17 20 23 33 39-40 50 58 6275 82 84 88 100 104 121-122129 149 168 208 216 223 232-233239 256 269 277 287-288353 360adult brainClontechABR0084 10-11 13 17 20 23 25 28-3032 34-35 39-41 48 50 53-54 5861 63 68-69 74 76 78 80 84-8991 98 104 107 112-114 118 121-122130 134-136 143 153-155158-160 163-166 168 172-173184-188 199-200 203 212-213215-216 219-220 226-227 234239 242 244 251-252 255-257263 268 271-272 277-280 287291 300-301 305-306 316 322338 346-347 360adult brainClontechABR011157 306adult brainBioChainABR01236 247adult brainInvitrogenABR013176adult brainInvitrogenABR01450 53 100 269adult brainInvitrogenABR01519 38 74 161-162brainInvitrogenABR01653 74 137 139 239adult brainInvitrogenABT0048 15 19-20 28 30 35 75 78 100106-107 113 134 160 179 181184 198-199 210 216 224 227252 254-255 288 340adipocytesStratageneADP0019 13 19 45 74 98 121-122 131164 187 189-190 217 239adrenal glandClontechADR0029 15 18-19 24-25 31-32 46 5677-78 112 114-115 117-119 121-122124 139 170 182 192 209213 218 220 225 249 276 306adult heartGIBCOAHR0012 4 7 17 19-22 26-27 34 38 45-4650 53-54 58 60-61 63 74 76-7786-87 91 96 98 108 112 114121-122 131 133 136-140 144155 160 165-168 184 188 217226 239 241-242 251 259 265277-278 290 306adult kidneyGIBCOAKD0014 6-11 13 15-17 19-20 24 30-3234 36-38 47 53-54 60-63 66 6973-75 78 82-85 87 89-92 96 98100 103 106 108 110 112-113116 121-123 126 129 131 134136 139-142 144 153 155 158-159169-170 176 181 207 237239 266-267 271-272 306adult kidneyInvitrogenAKT0027-8 10-11 13 15 19 25-27 3237-38 53 55-56 66 75 86 90 92108 123 144 165-166 172 182199 218 225 233 236 238 260266-267 332adult lungGIBCOALG0018 22 26-28 38-40 47 54 78 9198 104 110 112 117 139 148 168189 196 225 239 248 351-352lymph nodeClontechALN0017 26-27 32 35 38-40 79 82 120127 152 158-159 169 171 219239 244young liverGIBCOALV0017 14 16-17 19 33 37 53 72 77107 113 116 118 134 152 168212 249adult liverInvitrogenALV00212 14 17 24 28 32-33 36 58 7375-76 84 101 116 131 138 140158-160 182 194 212 238 275284 323 342-343adult liverClontechALV003271 284 358ovaryInvitrogenAOV0014 6-11 13 15-16 18-21 25-2731-32 34 36 38-40 46 48 50 53-5456 58 60 65 70 73-78 80 83-8486 91-92 95 98 100-101 103-106108 110-112 115 117-118124 126-127 129-131 136 139-142144 148 155 157-161 163-167169 173-174 178 180-186188-189 191-193 196 199-200204-208 210-211 220-223 233236 239 249-252 260-263 266-270287-288 306 315 351-352placentaClontechAPL00130 50 74 82 230placentaInvitrogenAPL00245 50 59 70 75 103 163 223adult spleenGIBCOASP0017 19 30 38 45 54 58 62 74 8183 91 106 110 112-113 116 131144 151 155 162 165-166 172176 189 191 215 230 236 239249 329testisGIBCOATS0014 15 19-20 30 48 53 74 89 94110 126 140 158-159 173 214-215220 239 245 306bladderInvitrogenBLD00130 35 59 61 74-75 123 164 221241 318bone marrowClontechBMD0013 6-7 9 13 17 20 26-27 30-3134 38-40 42 46 53-54 63-79 82-8385 91 93-98 101 105 110 115121-122 126 128-129 133-134143 145 154 161-162 176 192205-206 234 236 239 243 264289 306 322bone marrowClontechBMD0023-4 7 9 13 16-17 19-20 23 3032 34 36 38-40 47-48 54-56 5861 68-69 74-75 79 84 108 118-119121-122 125 128-129 131133 140 144 147 149 153-154158-159 161 163 167 171 174176 185-187 200 211 218 232239 241 247 252 277-278 285296 303 310 320 324 329 339341 353 356 359bone marrowClontechBMD00464colonInvitrogenCLN00118 32 100 106 110 143 153 163-164178 213 247 266-267 284cervixBioChainCVX0014 6 8-9 19 22 24-25 28 32 45-4653 55-56 63 74-75 77-78 8387 91-92 95 102 105 108 110123 127 136-137 140 169 172182 184-186 189-191 199 211238 249 266-267 274 283 306-308317 354endothelialStratageneEDT0012 4 6-7 9 15 17-21 25-28 30 32cells36 39-40 45 47-48 53 55-57 6062-63 69-70 74-76 78 83 85-8798 101-104 106 108 112-113 119121-123 130-131 136-137 139-142155-156 158-159 161 174189-192 204 208 218 220 223230 239 251 280 306Genomic clonesGenomicEPM001223from short armData fromof chromosome 8GeneticResearchGenomic clonesGenomicEPM003223from short armData fromof chromosome 8GeneticResearchGenomic clonesGenomicEPM004223from short armData fromof chromosome 8GeneticResearchfetal brainClontechFBR00132 227fetal brainClontechFBR004319fetal brainClontechFBR0067 10-11 13 17 20 23-25 28-2932 35 41-42 48 50 53 63 75 8089 91 104 112 121-122 125 130154 163 165-166 168 171 173191 199 210 215-216 218 226232 239 256 272 277 290 300306 309 319-320 333 353 360fetal brainInvitrogenFBT00215 17 19 35 69 75 87 104 109140 163 174 192-193 198-199207 220 228 239 252 256-258fetal heartInvitrogenFHR0013 8 19 32 41 48 77-79 91 114119 126 163 165-166 172 174176 200 218 232 244 263 331351-352 360-361fetal kidneyClontechFKD00116-17 36 46 53 74 82 95 104111 117 169 189fetal kidneyClontechFKD00226-27 165-166 218 220 232 238263 306fetal kidneyInvitrogenFKD00738 74fetal lungClontechFLG00132 48 139 173 217fetal lungInvitrogenFLG00310-11 19 36 58 61 69 74 134163 168 178 194 249 263 266-267351-352fetal liver-ColumbiaFLS0011-19 21-38 41-62 68 70 72 74-78spleenUniversity87 90-91 93 100-104 106-121123-125 127 130-131 133-134141-142 144 149 155-156 161163 165-167 169 176 194-196200 207 210 221 224-225 227231-233 236 238 263 303 306313 324 336 342fetal liver-ColumbiaFLS0022 5 7-9 12 14 16-18 22-24 30-33spleenUniversity35-40 43-46 48-50 52-53 5770 72 76-78 84-85 87 90 92101-102 106-108 110 112 114116-120 124 127-128 130-131134-135 140-142 144 155 163172 174 187 189-190 192 195-196199 205-207 210 220-221223-224 230-234 244 251 258260-261 263 265 275 296 313-315331 337-338 345 362fetal liver-ColumbiaFLS00319 30 33 139 174 265 313 339spleenUniversity355fetal liverInvitrogenFLV00110-11 14 17 19 21 37 46 50 6163 156 163 165-166 172 200 210238 253fetal liverClontechFLV00219 32 74 163 356fetal liverClontechFLV0043 14 19 32-33 37 42 47-48 5058 60 82 85 121-122 129 131152 171 193 272 353fetal muscleInvitrogenFMS00128 32 39-40 45 48 50 57 74 107121-122 131 137 139-140 147173 204 230 281fetal muscleInvitrogenFMS00219 23 32 34 55-56 80-81 98121-122 124 131-132 158-159199 212 230 280-281 353 357360fetal skinInvitrogenFSK0012 4 14-15 17-19 22 41 46 50 5359 72 75-76 81-82 84 94 103106 113 128 135 140 144 156164 167 170 174 188 209-210220 227 230 238-239 254 306321-322 333-335fetal skinInvitrogenFSK0024 34 47 54 79 84 113 126-127129 134 156 192-193 208 223230 241 277 285 333fetal spleenBioChainFSP00132 104umbilical cordBioChainFUC0014 19 22-23 32 38-40 46 55-5658 61 73-75 91 98-99 103 106110 112 116 120 123 129-130139 160 165-166 175 182 230234 249 251 302fetal brainGIBCOHFB0016 9 16 19-20 25 32 35-36 39-4145 48 53-54 56 60 73 80-81 83-9298 107 112 114 157-159 163165-166 172 191 197-198 211226-227 239 350infant brainColumbiaIB20026-8 13 15-17 19 21 32 35 41-42University48 50 60-61 77 81 84-85 88 92104-106 112-113 116 119 134139 144 160 165-166 168-169173 176 191 196 199-201 215223 225 227-228 239 261 285290 329 339-340 348infant brainColumbiaIB20037-9 13 32 39-41 58 92 103 105-106University144 160 162 199 205-206219 227-228 271 357infant brainColumbiaIBM00232 88 340Universityinfant brainColumbiaIBS0016 26-27 32 164 199 340Universitylung,StratageneIFB0012 4 18-19 25 39-40 46 53 55-56fibroblast106 112 124 129 136 139 146150 164 169 189-190 215 230239 260 349adult lungInvitrogenLGT0022 6 8-11 15-16 19 26-28 30 3239-40 46 48 50 53-56 60-61 6672 74-75 85 87 92 94 96 98103-104 108 110 112-113 117119-120 124 130-131 139-140149 152-153 155 158-159 167169 174 176 178 184 189-190195-196 217 220 229-230 234-239248-250 263 265-267 280286 310 329-330 351-352lymphocyteATCCLPC0017 13 16 19 32 39-40 54 63 7482 96 113 120 126 130-131 133144 150 178 184-186 223 239241 260 262 294 305 339leukocytesGIBCOLUC0011 3-4 7-9 13 16-20 26-27 30 3234-35 38-40 46 48 51 53-56 6366 70 72-76 78 82 84-85 87 8991-92 95-96 101 106 108 110-112114 116 120-122 126-127129-133 136 139 144 146-152164 175-179 187 192 232 236239 241 266-267 292-294 306325-327 329 339 359leukocytesClontechLUC0037-8 17 55-56 76 84 112 129 131161-162 176 180 185-186 329melanomaClontechMEL0044 13 17 28 30-31 39-40 83 8592 113 126 129 139 160 162 182198 232 239 303 324mammary glandInvitrogenMMG0018-11 16-21 28 30 32 35 41 4558-59 61 72 74-75 78 84 87 92103-104 106-107 110 113 115-116123 128 131 134-135 144152 163 176 181 183 210 212220-221 230 234 236 238-239248 251 260 272-273 275-276306 331 351-352 360neuronStratageneNTD00118-19 39-40 45 74 78 85 91neuronStratageneNTR00119 21 57 246 265neuronal cellsStratageneNTU0018-9 18-19 21 32 81 85 87 128164 174 184pituitaryClontechPIT00413 47 82 87 98 112 288 354glandplacentaClontechPLA00313 48 50 58 77 100 106 112 126129 152 178 232prostateClontechPRT00116 19 22 26-27 32 34 46-47 76-7792 98 106 112 124 172 214239 260 280 294rectumInvitrogenREC0018 10-11 18 30 54 74-76 106 113123-124 143 163 172 213 220232 237 260 322-323 340salivary glandClontechSAL0018 19 36 74 83 104 118 124 150176 260 295 304skinATCCSFB002239fibroblastsmallClontechSIN0019 17 19 22 32 34 54 57 59-60intestine73 75 84-85 96 99 107 113 118134 139 144 149 151 185-187189 197 199 217 219 221 230-231248 250 253-254 260 266-267295 304 356skeletalClontechSKM00117 19 39-40 48 89 104 116 131muscle281spinal cordClontechSPC0018 19 32 34 38-40 47 58 61 7480 83-84 89 104 108 131 139-140168 187 213 226 236 239300 350adult spleenClontechSPLc011 46 54 134 236stomachClontechSTO0017 32 38-40 51 66 74 76 89 117124 128 169 229 239 253 280294 296thalamusClontechTHA00224 30 50 87 124 127 143 163201 207 220 223 230 266-267269 279thymusClontechTHM0017 13 19 25 32 36 39-40 54-5672 74 82 96 108 113 119 127137 139 141-142 146 169 184192 260 276 296thymusClontechTHMc029 17 28 30 32 39-40 48 53 6172 74-75 77 79 82 91 107 112119-122 125-126 131 139-142153 171 175-176 178 184 187205-206 222-223 227 235-236269 278 289 297 305 310-311325 327-329 336thyroid glandClontechTHR0017-11 15 17 19-20 25-27 32 3436 46 48 53 59 72 82-87 89 9196 98-99 104 106 110 118-119121-122 127 130 136 139 144151-152 158-159 165-167 179187 204 208 220 239 249 281283 295 298-299 312 316 344tracheaClontechTRC00162-63 73 75 86-87 89 101 147192 239 266-267 282-283uterusClontechUTR0014 8 17 19 22 26-27 32 39-40 4663 82 98 110 130 151

[0386]

2

TABLE 2

SMITH-

SEQ ID
ACCESSION

WATERMAN
%

NO:
NUMBER
DESCRIPTION
SCORE
IDENTITY

1
L29075

Dictyostelium discoideum
G-box
173
21

binding factor

2
AL359215

Streptomyces coelicolor
A3(2)
133
28

putative phosphoglycerate mutase.

3
AF228713

Homo sapiens
EDAG-1
1671
100

4
AC007130

Homo sapiens
similar to 3-
1557
100

hydroxyisobutyrate dehydrogenase;

similar to P29266 (PTD: g416873)

5
AB040926

Homo sapiens
KIAA1493 protein
1973
98

6
AF193016

Homo sapiens
methyltransferase COQ3
1609
99

7
U95825

Homo sapiens
androgen-induced
2968
63

prostate proliferative shutoff

associated protein

8
AL390081

Homo sapiens
SEMA4B, Semaphorin 4B
3560
99

9
AC002130

Arabidopsis thaliana
F1N21.9
258
50

10
Z38061

Saccharomyces cerevisiae
mal5,
323
27

stal, len: 1367, CAI: 0.3,

AMYH_YEAST P08640 GLUCOAMYLASE S1

(EC 3.2.1.3)

11
D88733
Equine herpesvirus 1 membrane
284
24

glycoprotein

13
M80783

Homo sapiens
B12 protein
1144
70

14
U72678

Mus musculus
EF-9
792
92

15
AK026486

Homo sapiens
unnamed protein
427
83

product

16
AK025813

Homo sapiens
unnamed protein
1010
100

product

17
AF151036

Homo sapiens
HSPC202
722
84

18
AY007148

Homo sapiens
similar to Homo
984
100

sapiens
HSPC197 mRNA with GenBank

Accession Number AF151031.1

19
X57432

Rattus rattus
ribosomal protein S2
956
97

20
AF164793

Homo sapiens
protein x 013
386
100

21
J02642

Homo sapiens
glyceraldehyde 3-
1639
95

phosphate dehydrogenase (EC

1.2.1.12)

22
M34573

Homo sapiens
alpha-2 collagen type
515
100

VI-a

23
AL109928

Homo sapiens
dJ551D2.5 (novel
1999
100

protein)

24
AF111858

Homo sapiens
dimethylglycine
3918
99

dehydrogenase precursor

25
U64854

Caenorhabditis elegans
partial CDS
184
25

26
AF151072

Homo sapiens
HSPC238
838
99

27
AF151072

Homo sapiens
HSPC238
393
96

28
AK024825

Homo sapiens
unnamed protein
1794
99

product

29
AF285631

Rattus norvegicus
secretory carrier
894
75

membrane protein 4

30
AK024113

Homo sapiens
unnamed protein
3672
99

product

31
AL161515

Arabidopsis thaliana
putative
146
52

protein

32
AJ007798

Homo sapiens
stromal antigen 3,
6320
99

(STAG3)

33
D31856

Bacillus subtilis
HutI protein,
667
39

imidazolone-5-propionate hydrolase

34
AL391145

Arabidopsis thaliana
putative
423
24

protein

35
AF134726

Homo sapiens
G7A
1591
46

36
AJ276485

Homo sapiens
integral membrane
1502
100

transporter protein

37
J05158

Homo sapiens
carboxypeptidase N (EC
2274
88

3.4.17.3)

38
X57351

Homo sapiens
1-8D
673
97

39
AF230904

Homo sapiens
c-Cbl-interacting
3437
100

protein

40
AF230904

Homo sapiens
c-Cbl-interacting
2615
99

protein

41
AF276893

Homo sapiens
p21-activated protein
3550
100

kinase 6

42
AF269255

Homo sapiens
lysosomal apyrase-like
3198
100

protein 1

43
S85655

Homo sapiens
prohibitin
742
84

44
AB040926

Homo sapiens
KIAA1493 protein
1973
98

45
AF151063

Homo sapiens
HSPC229
1012
100

46
X68277

Homo sapiens
protein-tyrosine
1886
100

phosphatase

47
Z98745

Homo sapiens
dJ29K1.2
889
51

48
AF032668

Rattus norvegicus
rsec15
3738
92

50
AF195534

Rattus norvegicus
GERp95
4513
99

51
AF161368

Homo sapiens
HSPC105
513
98

52
W73147
Amino acid sequence of the soluble
651
81

complement receptor 1

53
AF271212

Homo sapiens
disrupter of silencing
2431
100

SAS10

54
AF116646

Homo sapiens
PRO0082
598
100

55
AF145613

Drosophila melanogaster

817
46

BcDNA.GH03108

56
AF145613

Drosophila melanogaster

884
38

BcDNA.GH03108

57
AL023803

Homo sapiens
dJ616B8.3 (novel gene)
2287
100

59
AC024877

Caenorhabditis elegans
contains
296
31

similarity to Pfam families PF00621

(Guanine nucleotide exchange factor

for Rho/Rac/Cdc42-like GTPases,

score = 58.2, E = 1.7e−13, N = 10 and

PF00169 (PH (pleckstrin homology)

domain, score = 17.0, E = 0.00071, N = 1)

60
AL390114

Leishmania major
probable
154
30

proteophosphoglycan

61
AL031427

Homo sapiens
dJ167A19.1 (novel
732
51

protein)

62
AL390935

Leishmania major
possible CG17807
151
43

protein

63
J04067

Canis familiaris
microsomal signal
930
99

peptidase

64
AF062378

Mus musculus
calmodulin-binding
1782
60

protein SHA1

65
AE001002

Archaeoglobus fulgidus
ATP-
195
29

dependent RNA helicase, putative

66
X69065
Erythroid ankyrin [Mus musculus]
181
30

68
AF017807

Homo sapiens
Arp2/3 complex 16 kDa
371
100

subunit

69
AC007660

Arabidopsis thaliana
putative
173
29

translation initiation factor

70
AJ243177

Xenopus laevis
Xenopus RPA
447
42

interacting protein alpha

71
AF226055

Homo sapiens
HTGN29
1367
100

72
AF090930

Homo sapiens
PRO0478
180
89

73
AF118084

Homo sapiens
PRO1914
350
98

74
AB028893

Homo sapiens
ribosomal protein S11
824
100

75
AK024500

Homo sapiens
FLJ00109 protein
1514
100

76
AF238866

Mus musculus
LNR42
1041
99

77
AC026875

Arabidopsis thaliana
T6D22.6
129
30

78
U42436

Caenorhabditis elegans
Hypothetical
130
32

protein C49H3.3

79
M80902

Homo sapiens
AHNAK nucleoprotein
8529
99

80
W90962
Human CSGP-2 protein [homo sapiens]
2346
99

81
AF206661

Gallus gallus
neuronal tetraspanin
1066
81

82
S73591

Homo sapiens
brain-expressed
800
42

HHCPA78 homolog VDUP1

83
AF116650

Homo sapiens
PRO0786
302
100

84
L26335

Cavia porcellus
zinc finger protein
1493
99

85
AF209198

Homo sapiens
zinc finger protein
2357
100

277

86
AE001399

Plasmodium falciparum
GAF domain
178
35

protein (cyclic nt signal

transduct.)

87
Y48226
Human prostate cancer-associated
1204
96

protein 12 [Homo sapiens]

88
M94389

Loligo pealei
neurofilament protein
165
23

89
AF121775

Homo sapiens
nasopharyngeal
903
58

carcinoma susceptibility protein

LZ16

90
AF116675

Homo sapiens
PRO1942
257
100

91
AE002760

Drosophila melanogaster
CG14464
195
43

gene product

92
AK00100

Homo sapiens
unnamed protein
841
100

product

93
AB020236

Homo sapiens
ribosomal protein L27A
754
99

94
AF119865

Homo sapiens
PRO2176
470
97

96
AF138863

Homo sapiens
PRO1677
868
99

97
X14361

Homo sapiens
CR-1 receptor SCR9 (or
135
100

16) C-term. (21 is 3rd base in

codon) (106 is 1st base in codon)

98
Z24725

Homo sapiens
mitogen inducible gene
3576
99

mig-2

99
U64598

Caenorhabditis elegans
weakly
398
45

similar to S. cervisiae PTM1

precursor (SP: P32857)

100
AC004770

Homo sapiens
BC269730 4
1527
84

101
AL139075

Campylobacter jejuni
NOL1\NOP2\sun
312
35

family protein

102
AF113694

Homo sapiens
PRO1359
416
100

103
U15158

Homo sapiens
ESP-2
564
41

104
AL020996

Homo sapiens
dJ317E23.3 (novel
1818
99

protein)

105
AF161370

Homo sapiens
HSPC107
824
100

106
AK000161

Homo sapiens
unnamed protein
284
100

product

107
AK001784

Homo sapiens
unnamed protein
684
100

product

108
AE000913

Methanobacterium

221
25

thermoautotrophicum
conserved

protein

109
AF165527

Homo sapiens
DGCR8
859
100

110
AF230200

Homo sapiens
OVN6-2
358
95

111
Z72516

Caenorhabditis elegans
T25G3.1
180
36

112
AF201940

Homo sapiens
DC6
505
100

113
AK001301

Homo sapiens
unnamed protein
2040
98

product

114
U23515

Caenorhabditis elegans
weakly
205
47

similar to gastrula zinc finger

protein

115
AF228021

Bos taurus
cyclophilin I
345
91

116
AF166124

Homo sapiens
selenoprotein X
527
100

117
AF079445

Dictyostelium discoideum
TipC
529
30

118
AB032179

Homo sapiens
similar to mouse Ehm2
2255
100

119
U89867

Homo sapiens
nuclear matrix protein
2449
98

55

120
U29056

Mus musculus
Src-like adapter
352
47

protein

121
U22015

Mus musculus
retinoid X receptor
2190
73

interacting protein

122
AF113538

Homo sapiens
retinoid x receptor
1800
100

interacting protein

123
AK000158

Homo sapiens
unnamed protein
740
100

product

124
AF260924

Mus musculus
UFD2/D4COLE1E fusion
1222
82

protein

125
U12465

Homo sapiens
ribosomal protein L35
591
97

126
AJ277591

Homo sapiens
p15-2a protein
749
100

127
AF205599

Mus musculus
transposase-like
2406
74

protein

128
U58975

Homo sapiens
proto-oncogene
659
90

129
X98374

Rattus norvegicus
KIS
2193
99

130
AF151049

Homo sapiens
HSPC215
627
100

131
M59807

Homo sapiens
putative
907
99

132
U12979

Homo sapiens
PC4
563
99

133
AF076642

Homo sapiens
regulator of G-protein
1218
100

signaling 13

134
AF116718

Homo sapiens
PRO2900
396
100

135
AC018758

Homo sapiens
GPI-anchored
213
31

metastasis-associated protein

homolog

136
AC025416

Arabidopsis thaliana
F5O11.12
135
36

137
M83186

Homo sapiens
cytochrome c oxidase
247
100

subunit VIIa

138
AF232937

Mus musculus
thymic stroma derived
247
41

lymphopoietin

139
M15841

Homo sapiens
U2 small nuclear
638
100

ribonucleoprotein B″

140
AK026916

Homo sapiens
unnamed protein
2612
99

product

141
Y05317
Human secreted protein bn97_1 [Homo
1508
100

sapiens
]

142
Y05317
Human secreted protein bn97_1 [Homo
851
99

sapiens
]

143
AF041083

Rattus norvegicus
RoBo-1
139
25

144
AC024260

Arabidopsis thaliana
cell division
194
25

control protein, putative; 15914-18846

146
AL022398

Homo sapiens
dJ434O14.3.2 (putative
575
100

protein) (isoform 2)

147
AF212842

Homo sapiens
immunoglobulin-like
1280
99

transcript 11 protein

148
AB042827

Rattus norvegicus
Nadrin
477
66

149
AK001841

Homo sapiens
unnamed protein
1916
100

product

150
AJ278120

Homo sapiens
putative ankyrin-
540
98

repeat containing protein

151
AL135959

Homo sapiens
dJ233G16.1 (novel
770
100

protein)

152
Y58196
[Homo sapiens] Human STRAP-3
671
100

protein, encoded by testis EST

AI139607

153
U41060

Homo sapiens
LIV-1 protein
373
50

154
AJ007590

Homo sapiens
XRP2 protein
1766
100

155
AB046868

Xenopus laevis
beta-catenin-
125
46

interacting protein

156
AB027258

Homo sapiens
basal transcriptional
1408
100

activator hABT1

157
AF039656

Homo sapiens
neuronal tissue-
1109
96

enriched acidic protein

158
AK001425

Homo sapiens
unnamed protein
1695
99

product

159
AK001425

Homo sapiens
unnamed protein
858
98

product

160
AK002030

Homo sapiens
unnamed protein
1029
100

product

161
X79417

Sus scrofa
40S ribosomal protein
510
83

S12

162
X12597

Homo sapiens
HMG-1 protein (AA 1-215)
1140
99

163
AK001159

Homo sapiens
unnamed protein
764
100

product

164
AK000020

Homo sapiens
unnamed protein
1613
100

product

165
AK001322

Homo sapiens
unnamed protein
1207
100

product

166
AK001322

Homo sapiens
unnamed protein
892
98

product

167
AE003822

Drosophila melanogaster
CG8493 gene
357
36

product

168
AF023451

Bos taurus
guanine nucleotide-
187
21

exchange protein

169
AK000154

Homo sapiens
unnamed protein
673
100

product

170
AJ132702

Mus musculus
ATFa-associated factor
435
64

172
AL022311

Homo sapiens
dJ1014D13.3 (novel
405
38

protein)

174
AB017634

Mus musculus
ENP
770
65

175
U40407
synthetic construct T cell receptor
1119
80

alpha chain

176
AF043179

Homo sapiens
T cell receptor beta
681
73

chain

177
AF116678

Homo sapiens
PRO1995
587
100

178
AF217522

Homo sapiens
uncharacterized bone
262
42

marrow protein BM046

179
AB046074

Macaca fascicularis
unnamed protein
515
83

product

180
X79417

Sus scrofa
40S ribosomal protein
429
84

S12

181
AF002668

Homo sapiens
MLD
1235
65

182
AB036422

Bos taurus
molybdopterin cofactor
3509
79

sulfurase

184
AF036696

Caenorhabditis elegans
contains
662
42

similarity to Brassica oleracea

non-green plastid phosphate/triose-

phosphate translocator precursor

(GB: U13632)

185
AJ277276

Homo sapiens
rapa-2
5155
99

186
AJ277275

Homo sapiens
rapa-1
5086
100

187
U22296

Rattus norvegicus
casein kinase 1
1444
93

gamma 1 isoform

188
AE003750

Drosophila melanogaster
CG9996 gene
468
44

product

189
Z97056

Homo sapiens
dJ434P1.2 (KDEL (Lys-
1103
100

Asp-Glu-Leu) endoplasmic reticulum

protein retention receptor 3)

190
AF081126

Drosophila melanogaster
ER lumen
409
75

protein retaining receptor

192
AF226047

Homo sapiens
GL002
863
100

193
AF269167

Homo sapiens
arsenite related gene 1
906
60

195
U41805

Mus musculus
putative T1/ST2
162
26

receptor binding protein precursor

197
AL357374

Homo sapiens
bA353C18.2 (novel
404
97

protein)

199
M34551

Homo sapiens
52-kD Ro/SSA
964
42

ribonucleoprotein

202
AF230201

Homo sapiens
OVC10-2
396
100

203
AK001984

Homo sapiens
unnamed protein
658
100

product

204
AK000530

Homo sapiens
unnamed protein
691
100

product

205
U37134

Drosophila melanogaster
inturned
248
23

protein

206
U37134

Drosophila melanogaster
inturned
244
23

protein

208
AB033130

Mus musculus
testis-specific gene
871
85

209
AK000464

Homo sapiens
unnamed protein
221
100

product

210
AJ277557

Homo sapiens
mitochondrial 5′ (3′) -
617
100

deoxyribonucleotidase (dNT-2)

211
AF127564

Arabidopsis thaliana
ubiquitin-
854
42

protein ligase 1

213
Y17108

Homo sapiens
rhomboid-related
485
39

protein

214
AL132776

Homo sapiens
dJ393D12.2 (novel LIM
1660
99

domain protein)

215
U73819

Mus musculus
polypeptide GalNAc
1039
42

transferase-T4

216
AL035406

Homo sapiens
dJ233K16.1 (KIAA0444,
3844
100

a putative chromodomain helicase

DNA binding protein 3 (CHD3))

217
M15800

Homo sapiens
MAL protein
308
42

218
L29554

Rattus norvegicus
alpha 2,6-
942
80

sialyltransferase

219
AL137315

Homo sapiens
hypothetical protein
983
100

220
AK026027

Homo sapiens
unnamed protein
647
100

product

221
AL137584

Homo sapiens
hypothetical protein
246
97

223
AC005498

Homo sapiens
R31665_1
1752
78

225
AC010155

Arabidopsis thaliana
F3M18.5
171
34

226
AL080276

Homo sapiens
dJ101K10.2 (regulator
1126
100

of G-protein signaling 17 (RGS17)

(RGSZ2))

227
AF042345

Homo sapiens
truncated EVI5
1815
64

228
J04214

Bos taurus
retinaldehyde-binding
504
39

protein precursor

230
AF181263

Homo sapiens
EH domain containing 2
2816
99

231
AP001660

Homo sapiens
putative gene,
1424
100

multidrug resistance associated

protein like

232
AB000910

Sus scrofa
ribosomal protein
542
100

233
AL133404

Homo sapiens
dJ238O23.9 (novel
298
100

protein similar to rat SAC (soluble

adenylyl cyclase))

234
X51397

Mus musculus
MyD88 protein (AA 1-243)
136
25

235
X01403

Homo sapiens
T-cell receptor alpha-
840
90

chain

236
X14254

Rattus rattus
invariant chain (AA
745
77

1-280)

238
U23084

Saccharomyces cerevisiae
Yn10470p
344
35

239
X03342

Homo sapiens
rpL32 (aa 1-135)
152
96

240
AF116669

Homo sapiens
PRO1828
237
100

241
U23181

Caenorhabditis elegans
final exon
135
25

in repeat region; similar to long

tandem repeat region of sialidase

(SP: TCNA_TRYCR, P23253) and

neurofilament H protein

242
AF263913

Mus musculus
fidgetin
3864
97

243
AF090892

Homo sapiens
PRO0106
290
100

244
U21310

Caenorhabditis elegans
F40H6.3 gene
153
27

product

246
AK001673

Homo sapiens
unnamed protein
3661
100

product

247
AL022603

Arabidopsis thaliana
putative
166
43

protein

248
AL023803

Homo sapiens
dJ616B8.3 (novel gene)
339
42

249
X52140

Rattus norvegicus
precursor
5429
87

polypeptide (AA −28 to 1152)

250
AB020755

Arabidopsis thaliana

139
46

gene id: MZN1.18˜unknown protein

251
AE003619

Drosophila melanogaster
CG7224 gene
186
43

product

252
AC004997

Homo sapiens
match to ESTs Z43979
388
67

(NID: g573097), R19699

(NID: g774333), and C01164

(NID: g1433394); alternatively

spliced form of H_DJ130H16.1a (C-

terminal truncation confirmed by

C01164)

254
AE003588

Drosophila melanogaster
CG13947
115
42

gene product

256
Y50934
Human fetal brain cDNA clone vc30_1
498
100

derived protein #1 [Homo sapiens]

257
AF242768

Homo sapiens
mesenchymal stem cell
1554
100

protein DSC43

259
M95779

Bos taurus
G protein gamma-5
333
98

subunit

260
AL035521

Arabidopsis thaliana
putative
145
28

protein

261
AF247501

Drosophila melanogaster
PINEAPPLE
333
36

EYE

263
AL034548

Homo sapiens
dJ1103G7.2 (novel
262
100

protein)

264
AF119851

Homo sapiens
PRO1722
143
63

265
L41834
Ensis minor nuclear protein
173
26

266
X97966

Homo sapiens
calcyphosine
963
100

267
X97966

Homo sapiens
calcyphosine
660
95

269
AF022383

Homo sapiens
complexin I
668
99

271
Y10054

Rattus norvegicus
3-hydroxy-3-
224
67

methylglutaryl CoA lyase

274
AF153201

Homo sapiens
zinc finger protein dp
179
36

275
X85738

Bos taurus
novel brain-specific
326
55

protein

277
AF250342

Arabidopsis thaliana
SMC-related
266
39

protein MSS2

278
AL080242

Homo sapiens
bA554C12.1 (RBX1 or
131
100

ROC1 (ring-box or ring finger

protein 1))

279
Z83760

Ciona intestinalis
COS41.4
1162
62

280
U41534

Caenorhabditis elegans
similar to
721
54

yeast MAK16 protein

(SP: MK16_YEAST, P10962)

281
AF272975

Gallus gallus
smoothelin-C
543
37

282
AL035414

Homo sapiens
dJ667H12.2.2 (novel
588
100

protein (isoform 2))

283
AF116661

Homo sapiens
PRO1438
145
62

285
AK001757

Homo sapiens
unnamed protein
1300
100

product

287
U20897

Homo sapiens
melanoma ubiquitous
2133
100

mutated protein

289
U09847

Homo sapiens
zinc finger protein
880
100

290
AJ000079

Trypanosoma cruzi

225
26

glycosylphosphatidylinositol-

specific phospholipase C

291
AF156549

Mus musculus
putative E1-E2 ATPase
2108
49

293
AF161345

Homo sapiens
HSPC082
439
100

294
AF116694

Homo sapiens
PRO2219
351
88

295
M74027

Homo sapiens
mucin
461
39

298
AL133640

Homo sapiens
hypothetical protein
2149
100

299
M17886

Homo sapiens
acidic ribosomal
161
76

phosphoprotein (P1)

300
Y99368
Human PRO1326 (UNQ686) amino acid
300
32

sequence SEQ ID NO: 100 [Homo

sapiens
]

303
AE003708

Drosophila melanogaster
CG6171 gene
144
27

product

304
M32639

Homo sapiens
statherin precursor
276
87

305
Z83844

Homo sapiens
dJ37E16.2 (SH3-domain
897
96

binding protein 1)

306
AE003791

Drosophila melanogaster
CG18065
120
32

gene product

307
AF135026

Homo sapiens
kallikrein-like
1392
100

protein 3 splice variant 1

310
AF198257

Felis catus
immunoglobulin kappa
678
76

light chain

311
X57725

Homo sapiens
TCR Vbeta 22a
626
100

312
AC018513

Homo sapiens
unknown
818
100

313
X03249

Bos taurus
epsilon-4 beta-globin
321
79

314
AB046099

Macaca fascicularis
unnamed protein
395
88

product

315
AC006033

Homo sapiens
T cell receptor gamma
1017
95

chain; match to S08328

(PID: g106470)

316
AB046103

Macaca fascicularis
unnamed protein
801
94

product

317
U88895

Homo sapiens
ORF2
399
81

318
U09848

Homo sapiens
zinc finger protein
242
49

319
AB003184

Homo sapiens
ISLR
880
59

320
AB036921

Chrysophrys major
maturation-
797
69

inducing protein

322
AF284422

Homo sapiens
cation-chloride
4694
100

cotransporter-interacting protein

325
AE000659

Homo sapiens
TCRAV8S2
577
100

327
R59748
T cell receptor Valpha2.3 chain
636
100

[homo sapiens]

328
AJ004871

Homo sapiens
TCR alpha chain
1328
94

329
AF043179

Homo sapiens
T cell receptor beta
1286
92

chain

330
AF090930

Homo sapiens
PRO0478
140
50

332
AF077043

Homo sapiens
60S ribosomal protein
275
87

L36

333
AL121988

Homo sapiens
dJ34M23.3 (gap
1457
100

junction protein, beta 4 (connexin

30.3))

334
D86424

Mus musculus
high-sulfur keratin
521
87

protein

335
AF090434

Fundulus heteroclitus
cytoehrome
760
40

P450 2N1

336
AF116688

Homo sapiens
PRO2133
370
98

337
X85372

Homo sapiens
Sm protein F
222
84

338
D87009

Homo sapiens
putative
1822
99

339
AE000860

Methanobacterium

631
35

thermoautotrophicum
conserved

protein

340
AL049759

Homo sapiens
dJ930L11.1 (similar to
1305
98

KIAA0397)

341
AE000004

Mycoplasma pneumonia
MG207 homolog,
141
27

from M. genitalium

342
AF151076

Homo sapiens
HSPC242
135
100

343
AB037902

Homo sapiens
truncated aldo-keto
670
100

reductase

345
M33014

Drosophila melanogaster
ubiquitin
153
62

346
AF053356

Homo sapiens
leucin rich neuronal
580
46

protein

348
AL137512

Homo sapiens
hypothetical protein
751
100

349
S68015

Homo sapiens
c6.1A
1664
100

350
AF151037

Homo sapiens
HSPC203
318
100

351
AB036432

Homo sapiens
advanced glycation
2133
100

endproducts receptor

352
AB036432

Homo sapiens
advanced glycation
2094
96

endproducts receptor

353
AC006942

Homo sapiens
R31181_2, partial
547
100

protein

354
AF125535

Homo sapiens
pp21 homolog
502
95

355
AF227130

Homo sapiens
candidate taste
1629
100

receptor T2R3

357
AB046626

Macaca fascicularis
hypothetical
291
93

protein

358
Z69597

Canis familiaris
Rod transducin
1145
100

alpha subunit

359
AE000659

Homo sapiens
TCRAV16S1
565
100

360
Y99368
Human PRO1326 UNQ686) Amino acid
2034
100

sequence SEQ ID NO: 100. [Homo

sapiens
]

362
L06499

Homo sapiens
ribosomal protein L37a
187
55

[0387]

3

TABLE 3

SEQ ID
ACCESSION

NO:
NO.
DESCRIPTION
RESULTS*

1
PR00651
5-HYDROXYTRYPTAMINE
PR00651E 10.53 4.025e−06 60-80

2B RECEPTOR

SIGNATURE

2
BL00126
3′5′-cyclic
BL00126B 15.20 7.750e−06 208-220

nucleotide

phosphodiesterases

proteins.

3
DM00892
3 RETROVIRAL
DM00892C 23.55 9.438e−07 285-319

PROTEINASE.

4
BL00895
3-hydroxyisobutyrate
BL00895B 21.14 7.061e−22 151-190

dehydrogenase
BL00895C 20.10 8.071e−22 200-236

proteins.
BL00895A 12.61 1.973e−18 42-63

5
DM00099
4 kw A55R REDUCTASE
DM00099A 5.17 5.263e−06 409-415

TERMINAL

DIHYDROPTERIDINE.

7
PF00598
Influenza Matrix
PF00598C 19.35 3.333e−07 531-563

protein (M1).

9
DM00522
499 kw TRYPSIN
DM00522A 8.30 3.250e−06 287-297

KINASE KUNITZ

PANCREATIC.

10
PR00514
5-HYDROXYTRYPTAMINE
PR00514C 11.01 9.061e−07 81-100

1D RECEPTOR

SIGNATURE

11
PR00514
5-HYDROXYTRYPTAMINE
PR00514C 11.01 9.061e−07 81-100

1D RECEPTOR

SIGNATURE

12
PR00775
90 KD HEAT SHOCK
PR00775G 10.64 3.487e−07 8-27

PROTEIN SIGNATURE

13
PR00902
VP6 BLUE-TONGUE
PR00902K 11.09 1.000e−05 176-200

VIRUS INNER CAPSID

PROTEIN SIGNATURE

14
PR00875
MOLLUSC
PR00875A 5.83 1.127e−07 159-171

METALLOTHIONEIN
PR00875D 5.00 1.000e−05 158-169

SIGNATURE

15
DM01803
1 HERPESVIRUS
DM01803C 7.00 9.200e−07 181-191

GLYCOPROTEIN H.
DM01803A 10.51 1.000e−06 178-199

DM01803C 7.00 7.337e−06 214-224

16
PF00803
3A movement protein.
PF00803D 14.15 2.622e−06 41-71

17
PR00170
SODIUM CHANNEL
PR00170G 7.74 1.000e−05 24-53

SIGNATURE

18
PR00701
60 KD INNER MEMBRANE
PR00701E 13.83 5.684e−06 117-133

PROTEIN SIGNATURE

20
DM01415
6 SALIVARY GLUE
DM01415A 6.65 6.063e−06 55-68

PROTEIN.

21
DM01418
352 FIBRILLAR
DM01418A 20.83 7.731e−06 64-112

COLLAGEN CARBOXYL-

TERMINAL.

22
BL00616
Histidine acid
BL00616D 15.83 7.268e−06 117-133

phosphatases

phosphohistidine

proteins.

23
DM00611
9 kw LECTIN HTPG
DM00611A 7.73 5.826e−06 173-181

SERINE GNTR.

24
BL00832
2′-5′-oligoadenylate
BL00832D 21.81 5.017e−06 425-449

synthetases

proteins.

25
PR00354
7FE FERREDOXIN
PR00354C 5.72 8.590e−09 543-561

SIGNATURE

26
PR00217
43 KD POSTSYNAPTIC
PR00217C 10.91 3.851e−07 89-105

PROTEIN SIGNATURE

27
PR00513
5-HYDROXYTRYPTAMINE
PR00513A 7.75 1.439e−06 168-180

1B RECEPTOR

SIGNATURE

28
DM00552
GROWTH FACTOR AND
DM00552A 11.97 1.000e−05 130-152

CYTOKINES RECEPTORS

FAMILY.

29
PR00701
60 KD INNER MEMBRANE
PR00701I 8.59 3.088e−06 102-126

PROTEIN SIGNATURE

30
DM00060
338 kw NEUREXIN
DM00060 6.92 3.284e−07 680-690

ALPHA III CYSTEINE.

34
PR00517
5-HYDROXYTRYPTAMINE
PR00517D 7.66 6.971e−07 484-496

2C RECEPTOR

SIGNATURE

35
PR00701
60 KD INNER MEMBRANE
PR00701A 14.28 4.183e−06 722-744

PROTEIN SIGNATURE

37
PR00513
5-HYDROXYTRYPTAMINE
PR00513C 10.79 8.927e−07 287-304

1B RECEPTOR

SIGNATURE

38
PR00166
AROMATIC AMINO ACID
PR00166I 11.06 1.000e−05 98-118

PERMEASE SIGNATURE

39
PR00003
4-DISULPHIDE CORE
PR00003A 14.69 3.803e−06 311-321

SIGNATURE

40
PR00003
4-DISULPHIDE CORE
PR00003A 14.69 3.803e−06 311-321

SIGNATURE

41
PR00517
5-HYDROXYTRYPTAMINE
PR00517D 7.66 8.788e−07 2-14

2C RECEPTOR

SIGNATURE

42
PR00701
60 KD INNER MEMBRANE
PR00701F 14.45 7.750e−06 25-46

PROTEIN SIGNATURE

43
DM00895
7 kw REVERSE
DM00895B 8.85 4.185e−06 157-167

TRANSCRIPTASE RNA

POLYMERASE.

44
DM00099
4 kw A55R REDUCTASE
DM00099A 5.17 5.263e−06 409-415

TERMINAL

DIHYDROPTERIDINE.

45
PR00519
5-HYDROXYTRYPTAMINE
PR00519A 8.06 8.984e−06 137-154

5B RECEPTOR

SIGNATURE

46
PR00519
5-HYDROXYTRYPTAMINE
PR00519B 9.99 1.828e−07 151-168

5B RECEPTOR

SIGNATURE

47
DM00892
3 RETROVIRAL
DM00892B 9.78 2.047e−06 21-27

PROTEINASE.

48
BL00832
2′-5′-oligoadenylate
BL00832B 15.45 6.836e−07 375-416

synthetases

proteins.

49
DM00588
8 kw CHO2 ALPHA
DM00588A 10.87 7.128e−06 20-31

ANTIGEN PARAMYOSIN.

50
DM00604
2 SHIGA/RICIN
DM00604D 13.26 8.250e−06 263-273

RIBOSOMAL

INACTIVATING TOXINS.

51
BL01193
Ribosomal protein
BL01193A 13.21 1.000e−05 19-50

S8e proteins.

52
PR00172
GLUCOSE TRANSPORTER
PR00172F 8.47 9.901e−06 69-90

SIGNATURE

53
PR00297
10 KD CHAPERONIN
PR00297A 13.91 4.740e−06 379-395

SIGNATURE

54
PR00320
G-PROTEIN BETA WD-40
PR00320A 16.74 9.710e−06 81-96

REPEAT SIGNATURE

55
PR00517
5-HYDROXYTRYPTAMINE
PR00517C 5.36 4.126e−07 352-365

2C RECEPTOR

SIGNATURE

56
PR00517
5-HYDROXYTRYPTAMINE
PR00517C 5.36 4.126e−07 352-365

2C RECEPTOR

SIGNATURE

57
DM00547
1 kw CHROMO
DM00547E 13.94 4.656e−06 229-252

BROMODOMAIN SHADOW

GLOBAL.

58
PF00506
Influenza virus
PF00506I 10.26 3.723e−06 32-68

nucleoprotein.

60
DM00522
499 kw TRYPSIN
DM00522B 9.43 7.338e−07 171-185

KINASE KUNITZ

PANCREATIC.

61
DM01123
5 kw RESISTANCE
DM01123B 20.06 3.187e−06 205-244

TETRACYCLINE

METHYLENOMYCIN

EXPORT.

62
PR00439
11-S SEED STORAGE
PR00439G 17.85 9.239e−07 82-100

PROTEIN FAMILY

SIGNATURE

63
PR00652
5-HYDROXYTRYPTAMINE
PR00652F 11.66 4.767e−06 100-122

7 RECEPTOR SIGNATURE

64
DM01785
72 PYRUVATE
DM01785A 14.90 2.196e−06 218-261

(FLAVODOXIN)

DEHYDROGENASE.

65
PR00652
5-HYDROXYTRYPTAMINE
PR00652A 8.92 5.104e−06 315-336

7 RECEPTOR SIGNATURE

67
BL00405
43 Kd postsynaptic
BL00405F 8.07 9.920e−06 13-44

protein.

68
PR00380
KINESIN HEAVY CHAIN
PR00380D 9.93 9.043e−06 44-66

SIGNATURE

69
PR00683
SPECTRIN PLECKSTRIN
PR00683D 15.87 9.571e−06 46-65

HOMOLOGY DOMAIN

SIGNATURE

70
PR00753
1-AMINOCYCLOPROPANE-
PR00753C 13.93 7.330e−06 192-213

1-CARBOXYLATE

SYNTHASE SIGNATURE

71
PF00602
Influenza RNA-
PF00602J 9.52 9.727e−06 47-102

dependant RNA

polymerase subunit

PB1.

72
DM01855
PROTEIN-GLUTAMATE O-
DM01855A 11.54 7.594e−06 27-44

METHYLTRANSFERASE.

74
BL01277
Ribonuclease PH
BL01277A 17.39 1.000e−05 50-88

proteins.

75
PR00517
5-HYDROXYTRYPTAMINE
PR00517G 16.45 6.919e−06 755-771

2C RECEPTOR

SIGNATURE

77
PF01073
3-beta
PF01073B 12.26 9.767e−07 102-147

hydroxysteriod

dehydrogenase/isomerase

family.

78
PR00351
MAS20 PROTEIN IMPORT
PR00351C 7.03 6.182e−06 99-112

RECEPTOR SIGNATURE
PR00351C 7.03 1.000e−05 5-18

79
DM00611
9 kw LECTIN HTPG
DM00611C 11.08 4.549e−06 1489-1501

SERINE GNTR.

80
DM01111
4 kw PHOSPHATASE
DM01111C 9.35 2.800e−06 44-73

TRANSFORMING 61 K

PDF1.

82
PD02407
3-
PD02407B 16.51 1.000e−06 94-111

BISPHOSPHOGLYCERATE-

INDEPENDENT

PHOSPHOGLYCER.

83
PR00116
ARGINASE SIGNATURE
PR00116D 14.91 9.850e−06 14-44

84
PR00517
5-HYDROXYTRYPTAMINE
PR00517F 11.48 7.250e−06 45-62

2C RECEPTOR

SIGNATURE

87
DM00522
499 kw TRYPSIN
DM00522B 9.43 3.535e−07 223-237

KINASE KUNITZ

PANCREATIC.

88
DM00303
6 LEA 11-MER REPEAT
DM00303A 13.20 8.034e−08 270-320

REPEAT.

91
DM01242
3 THREONINE-TRNA
DM01242B 23.57 4.672e−06 71-120

LIGASE.

92
PR00388
3′,5′-CYCLIC
PR00388E 6.66 3.797e−06 124-136

NUCLEOTIDE CLASS II

PHOSPHODIESTERASE

SIGNATURE

93
PD02407
3-
PD02407B 16.51 9.676e−06 15-32

BISPHOSPHOGLYCERATE-

INDEPENDENT

PHOSPHOGLYCER.

94
PF00506
Influenza virus
PF00506I 10.26 4.555e−06 16-52

nucleoprotein.

95
PR00551
2-S GLOBULIN FAMILY
PR00551H 11.29 8.740e−06 21-39

SIGNATURE

96
PR00756
MEMBRANE ALANYL
PR00756E 11.91 9.338e−06 68-81

DIPEPTIDASE (M1)

FAMILY SIGNATURE

97
PR00547
X OPIOID RECEPTOR
PR00547B 6.96 3.268e−06 17-36

SIGNATURE

98
PR00651
5-HYDROXYTRYPTAMINE
PR00651A 16.53 4.000e−06 653-674

2B RECEPTOR

SIGNATURE

99
PR00208
GLIADIN AND LMW
PR00208C 11.51 9.775e−06 54-71

GLUTENIN SUPERFAMILY

SIGNATURE

101
PR00451
CHITIN-BINDING
PR00451A 6.49 1.000e−05 152-161

DOMAIN SIGNATURE

102
BL00832
2′-5′-oligoadenylate
BL00832B 15.45 7.569e−06 1-42

synthetases

proteins.

103
BL00405
43 Kd postsynaptic
BL00405J 13.28 6.952e−06 142-176

protein

104
DM01242
3 THREONINE —TRNA
DM01242F 10.61 5.500e−07 187-201

LIGASE.

105
DM01834
8 HYDROGENASE (FE)
DM01834A 4.96 7.097e−06 53-60

SMALL CHAIN.

107
PR00101
ASPARTATE
PR00101E 5.52 1.000e−05 111-117

CARBAMOYLTRANSFERASE

SIGNATURE

109
PR00902
VP6 BLUE-TONGUE
PR00902K 11.09 9.922e−06 91-115

VIRUS INNER CAPSID

PROTEIN SIGNATURE

110
PR00259
TRANSMEMBRANE FOUR
PR00259A 9.27 9.716e−06 9-33

FAMILY SIGNATURE

111
BL00785
5′-nucleotidase
BL00785B 10.65 6.507e−06 53-67

proteins.

112
PR00652
5-HYDROXYTRYPTAMINE
PR00652G 10.94 5.429e−06 14-32

7 RECEPTOR SIGNATURE

113
PR00517
5-HYDROXYTRYPTAMINE
PR00517D 7.66 3.661e−06 372-384

2C RECEPTOR

SIGNATURE

114
PF00637
7-fold repeat
PF00637D 7.09 9.449e−07 32-44

proteins in

Clathrin, also in

VPS proteins.

115
DM01235
5 kw T4 55.10
DM01235 20.29 9.832e−06 77-108

METHYLCYTOSINE

TRANSCRIPTASE.

116
PR00873
ECHINOIDEA (SEA
PR00873C 6.16 9.906e−06 70-81

URCHIN)

METALLOTHIONEIN

SIGNATURE

117
PR00387
3′5′-CYCLIC
PR00387D 10.81 4.889e−06 155-172

NUCLEOTIDE

PHOSPHODIESTERASE

SIGNATURE

118
PF00598
Influenza Matrix
PF00598A 14.24 7.158e−06 211-254

protein (M1).

119
BL00895
3-hydroxyisobutyrate
BL00895B 21.14 8.036e−06 428-467

dehydrogenase

proteins.

120
PR00419
ADRENODOXIN
PR00419D 10.62 9.430e−06 18-33

REDUCTASE FAMILY

SIGNATURE

121
DM00396
5 kw INTRON COI ND4L
DM00396B 7.85 3.739e−07 381-389

ND5.

122
BL00198
4Fe-4S ferredoxins,
BL00198 10.43 5.500e−06 135-147

iron-sulfur binding

region proteins.

123
DM01554
1 THYROLIBERIN
DM01554E 10.78 4.208e−06 93-110

PRECURSOR.

124
BL00405
43 Kd postsynaptic
BL00405G 7.78 6.294e−06 130-167

protein.

125
PR00298
60 KD CHAPERONIN
PR00298D 10.23 3.847e−06 14-40

SIGNATURE

126
PR00317
EPENDYMIN SIGNATURE
PR00317A 13.39 9.897e−06 79-99

127
PR00513
5-HYDROXYTRYPTAMINE
PR00513B 17.51 3.971e−06 277-290

1B RECEPTOR

SIGNATURE

130
PR00516
5-HYDROXYTRYPTAMINE
PR00516G 15.11 8.811e−06 18-35

2A RECEPTOR

SIGNATURE

131
PR00828
FORMIN SIGNATURE
PR00828F 8.56 1.000e−05 61-81

132
DM01269
303 kw ACTIVATING
DM01269A 23.35 7.279e−06 28-56

RAN GTPASE ISOZYME.

133
PR00586
PROSTANOID EP4
PR00586B 14.97 7.322e−06 10-28

RECEPTOR SIGNATURE
PR00586H 8.65 9.791e−06 16-40

134
PF00954
S-locus glycoprotein
PF00954D 18.68 9.843e−06 9-44

family.

135
PR00018
KRINGLE DOMAIN
PR00018A 14.52 1.000e−05 120-136

SIGNATURE

136
BL00115
Eukaryotic RNA
BL00115E 14.13 9.921e−06 40-69

polymerase II

heptapeptide repeat

proteins.

137
PR00521
ANDROGEN RECEPTOR
PR00521A 17.02 9.729e−06 5-25

SIGNATURE

138
PR00701
60 KD INNER MEMBRANE
PR00701I 8.59 5.267e−07 16-40

PROTEIN SIGNATURE

139
DM01269
303 kw ACTIVATING
DM01269A 23.35 7.085e−08 93-121

RAN GTPASE ISOZYME.

140
PR00915
LUTEOVIRUS GROUP 1
PR00915D 16.14 1.000e−05 374-392

COAT PROTEIN

SIGNATURE

143
DM00522
499 kw TRYPSIN
DM00522A 8.30 4.441e−06 94-104

KINASE KUNITZ

PANCREATIC.

144
PF00598
Influenza Matrix
PF00598B 13.10 1.623e−06 89-133

protein (M1).

145
PR00217
43 KD POSTSYNAPTIC
PR00217C 10.91 6.250e−06 24-40

PROTEIN SIGNATURE

147
PR00701
60 KD INNER MEMBRANE
PR00701A 14.28 6.049e−06 266-288

PROTEIN SIGNATURE

148
PR00513
5-HYDROXYTRYPTAMINE
PR00513D 11.06 9.920e−06 103-121

1B RECEPTOR

SIGNATURE

149
PF00603
Influenza RNA-
PF00603D 8.49 9.319e−07 30-85

dependant RNA

polymerase subunit

PA.

150
DM01688
2 POLY-IG RECEPTOR.
DM01688N 11.93 9.920e−08 72-100

151
PF00637
7-fold repeat
PF00637B 10.68 6.906e−06 186-195

proteins in

Clathrin, also in

VPS proteins.

152
BL00461
6-phosphogluconate
BL00461A 15.90 1.764e−08 21-57

dehydrogenase

proteins.

153
DM01554
1 THYROLIBERIN
DM01554A 6.07 2.565e−06 589-599

PRECURSOR.

154
BL00405
43 Kd postsynaptic
BL00405E 8.84 8.125e−06 109-135

protein.

155
PF00637
7-fold repeat
PF00637A 15.49 5.179e−06 42-65

proteins in

Clathrin, also in

VPS proteins.

156
PR00517
5-HYDROXYTRYPTAMINE
PR00517D 7.66 7.339e−06 85-97

2C RECEPTOR

SIGNATURE

157
DM01688
2 POLY-IG RECEPTOR.
DM01688P 13.54 1.925e−07 44-89

DM01688L 4.36 2.367e−07 123-133

159
PR00933
B-LYTIC
PR00933D 13.92 1.000e−05 85-106

METALLOENDOPEPTIDASE

(M23) SIGNATURE

161
PR00352
3FE-4S FERREDOXIN
PR00352A 11.15 6.162e−06 94-106

SIGNATURE

163
BL00785
5′-nucleotidase
BL00785E 15.85 4.000e−06 95-111

proteins.

164
PR00916
2C ENDOPEPTIDASE
PR00916C 8.02 2.655e−06 121-133

(C24) CYSTEINE

PROTEASE FAMILY

SIGNATURE

165
BL00785
5′-nucleotidase
BL00785D 9.89 3.045e−06 154-164

proteins.

166
BL00785
5′-nucleotidase
BL00785D 9.89 3.045e−06 123-133

proteins.

167
DM01023
2 GLYCOSYL
DM01023C 13.51 6.486e−06 149-175

HYDROLASES FAMILY 5.

168
PF00803
3A movement protein.
PF00803A 15.38 8.088e−06 255-290

169
PR00282
SNAKE CYTOTOXIN
PR00282D 11.82 9.882e−06 74-85

SIGNATURE

170
PR00519
5-HYDROXYTRYPTAMINE
PR00519B 9.99 5.787e−06 83-100

5B RECEPTOR

SIGNATURE

172
DM01803
1 HERPESVIRUS
DM01803A 10.51 6.804e−06 136-157

GLYCOPROTEIN H.

173
PR00701
60 KD INNER MEMBRANE
PR00701E 13.83 9.724e−06 14-30

PROTEIN SIGNATURE

174
BL00118
Phospholipase A2
BL00118A 16.00 9.842e−06 132-145

histidine proteins.

175
DM01688
2 POLY-IG RECEPTOR.
DM01688G 16.45 1.825e−06 89-121

176
DM01930
2 kw FINGER SMCX
DM01930A 7.97 2.403e−07 146-159

SMCY YDR096W.

177
PR00510
NEBULIN SIGNATURE
PR00510F 9.88 8.552e−06 34-51

179
PF00432
Prenyltransferase
PF00432A 11.90 1.000e−05 27-39

and squalene oxidase

repeat proteins.

180
PR00537
MU OPIOID RECEPTOR
PR00537A 8.17 1.000e−05 27-41

SIGNATURE

183
PR00536
MELANOCYTE
PR00536C 8.58 8.833e−06 64-82

STIMULATING HORMONE

RECEPTOR SIGNATURE

184
DM00973
3 kw RESISTANCE
DM00973B 17.81 8.261e−06 158-184

BENOMYL YLL028W

CYCLOHEXIMIDE.

185
PR00517
5-HYDROXYTRYPTAMINE
PR00517C 5.36 4.265e−06 718-731

2C RECEPTOR

SIGNATURE

186
PR00517
5-HYDROXYTRYPTAMINE
PR00517C 5.36 4.265e−06 718-731

2C RECEPTOR

SIGNATURE

187
PR00651
5-HYDROXYTRYPTAMINE
PR00651A 16.53 6.447e−06 144-165

2B RECEPTOR

SIGNATURE

188
BL01017
Ergosterol
BL01017D 20.82 9.737e−06 21-67

biosynthesis

ERG4/ERG24 family

proteins.

191
DM00315
072 RIBONUCLEASE
DM00315B 6.84 7.459e−06 95-107

INHIBITOR.

192
PR00930
HIGH MOBILITY GROUP
PR00930E 5.98 9.740e−06 285-298

PROTEIN (HMGY)

SIGNATURE

193
BL00794
7,8-dihydro-6-
BL00794B 22.12 8.967e−06 150-191

hydroxymethylpterin-

pyrophosphokinase

proteins.

194
PR00517
5-HYDROXYTRYPTAMINE
PR00517C 5.36 5.853e−06 115-128

2C RECEPTOR

SIGNATURE

196
DM01803
1 HERPESVIRUS
DM01803A 10.51 7.031e−07 11-32

GLYCOPROTEIN H.

197
PR00171
SUGAR TRANSPORTER
PR00171B 14.73 1.000e−05 15-35

SIGNATURE

198
PD02407
3-
PD02407J 10.55 6.610e−06 69-81

BISPHOSPHOGLYCERATE-

INDEPENDENT

PHOSPHOGLYCER.

199
PF00604
Influenza RNA-
PF00604F 10.21 2.417e−06 276-331

dependant RNA

polymerase subunit

PB2.

201
PR00409
PHTHALATE
PR00409D 13.02 9.900e−06 43-58

DIOXYGENASE

REDUCTASE FAMILY

SIGNATURE

202
BL00660
Band 4.1 family
BL00660A 31.50 9.595e−06 1-54

domain proteins.

203
PR00745
GLYCOSYL HYDROLASE
PR00745D 15.85 9.700e−06 68-83

FAMILY 39 SIGNATURE

204
PD01364
MUCIN GLYCOPROTEIN
PD01364A 6.18 9.667e−06 9-16

PRECURSOR MEM.

205
BL00794
7,8-dihydro-6-
BL00794C 19.62 7.690e−07 309-347

hydroxymethylpterin-

pyrophosphokinase

proteins.

206
BL00794
7,8-dihydro-6-
BL00794C 19.62 7.690e−07 309-347

hydroxymethylpterin-

pyrophosphokinase

proteins.

207
DM00895
7 kw REVERSE
DM00895E 15.72 3.170e−06 241-266

TRANSCRIPTASE RNA

POLYMERASE.

208
PR00517
5-HYDROXYTRYPTAMINE
PR00517D 7.66 4.835e−06 59-71

2C RECEPTOR

SIGNATURE

209
PD02365
CHAIN FACTOR
PD02365C 7.89 9.719e−06 20-50

INTERLEUKIN-12 BETA

PRECURSOR IL-1.

210
PR00551
2-S GLOBULIN FAMILY
PR00551E 10.27 9.432e−06 19-34

SIGNATURE

211
DM01418
352 FIBRILLAR
DM01418B 22.51 3.289e−06 527-569

COLLAGEN CARBOXYL-

TERMINAL.

213
DM01785
72 PYRUVATE
DM01785E 12.98 6.400e−06 165-216

(FLAVODOXIN)

DEHYDROGENASE.

214
DM01834
8 HYDROGENASE (FE)
DM01834B 15.29 3.382e−06 64-90

SMALL CHAIN.

215
PR00217
43 KD POSTSYNAPTIC
PR00217C 10.91 4.583e−06 407-423

PROTEIN SIGNATURE

216
DM00547
1 kw CHROMO
DM00547F 23.43 6.538e−36 628-675

BROMODOMAIN SHADOW
DM00547E 13.94 2.400e−18 387-410

GLOBAL.
DM00547C 17.30 9.486e−16 266-288

DM00547B 11.28 9.217e−15 237-251

DM00547D 11.60 4.951e−12 357-371

DM00547A 12.38 6.455e−11 216-228

217
BL00407
Connexins proteins.
BL00407D 17.61 1.000e−05 57-87

218
BL00198
4Fe-4S ferredoxins,
BL00198 10.43 9.481e−06 74-86

iron-sulfur binding

region proteins.

219
DM01417
6 kw INDUCING XPMC2
DM01417B 15.47 3.550e−06 90-102

MUSHROOM

SPAC22G7.04.

220
PR00519
5-HYDROXYTRYPTAMINE
PR00519E 3.58 2.404e−07 184-199

5B RECEPTOR

SIGNATURE

221
PD01313
INTRON PROBABLE
PD01313B 23.27 1.000e−05 10-45

MATURASE CHLOROPLAST

MR.

222
PR00047
C4-TYPE STEROID
PR00047A 15.70 9.878e−06 99-116

RECEPTOR ZINC FINGER

SIGNATURE

223
DM01554
1 THYROLIBERIN
DM01554C 11.76 3.571e−06 255-271

PRECURSOR.

224
DM01123
5 kw RESISTANCE
DM01123B 20.06 5.206e−06 28-67

TETRACYCLINE

METHYLENOMYCIN

EXPORT.

226
BL00198
4Fe-4S ferredoxins,
BL00198 10.43 8.630e−07 28-40

iron-sulfur binding

region proteins.

227
BL00895
3-hydroxyisobutyrate
BL00895B 21.14 5.173e−06 185-224

dehydrogenase

proteins.

229
PR00907
THROMBOMODULIN
PR00907G 11.63 9.794e−06 13-40

SIGNATURE

230
PR00651
5-HYDROXYTRYPTAMINE
PR00651B 9.95 6.416e−06 62-77

2B RECEPTOR

SIGNATURE

231
DM00611
9 kw LECTIN HTPG
DM00611C 11.08 9.113e−06 214-226

SERINE GNTR.

232
PR00582
PROSTANOID EP3
PR00582B 9.74 1.000e−05 76-95

RECEPTOR SIGNATURE

233
PR00407
EUKARYOTIC
PR00407E 13.51 9.438e−06 176-192

MOLYBDOPTERIN DOMAIN

SIGNATURE

234
DM00892
3 RETROVIRAL
DM00892C 23.55 9.913e−06 6-40

PROTEINASE.

235
DM01688
2 POLY-IG RECEPTOR.
DM01688G 16.45 4.450e−06 94-126

DM01688J 14.69 6.000e−06 34-71

236
PD00930
PROTEIN GTPASE
PD00930A 25.62 1.000e−05 80-106

DOMAIN ACTIVATION.

237
PR00076
6-PHOSPHOGLUCONATE
PR00076B 11.24 6.418e−07 14-44

DEHYDROGENASE

SIGNATURE

239
PR00243
MUSCARINIC
PR00243F 16.45 9.182e−06 7-18

ACETYLCHOLINE

RECEPTOR SIGNATURE

240
BL00854
Proteasome B-type
BL00854B 10.97 1.000e−05 1-9

subunits proteins.

241
PR00513
5-HYDROXYTRYPTAMINE
PR00513B 17.51 5.263e−07 876-889

1B RECEPTOR

SIGNATURE

242
DM01111
4 kw PHOSPHATASE
DM01111I 15.32 2.473e−07 522-552

TRANSFORMING 61 K

PDF1.

243
BL00415
Synapsins proteins.
BL00415B 9.91 9.778e−06 53-89

244
BL00514
Fibrinogen beta and
BL00514E 14.28 1.000e−05 221-238

gamma chains C-

terminal domain

proteins.

245
PR00187
ARTHROPOD
PR00187B 15.70 1.000e−05 37-55

HAEMOCYANIN

SIGNATURE

246
PF00637
7-fold repeat
PF00637C 27.33 1.184e−06 368-415

proteins in

Clathrin, also in

VPS proteins.

247
BL00785
5′-nucleotidase
BL00785A 9.73 7.557e−06 57-68

proteins.

248
PR00651
5-HYDROXYTRYPTAMINE
PR00651D 12.56 2.615e−06 228-249

2B RECEPTOR

SIGNATURE

249
PR00516
5-HYDROXYTRYPTAMINE
PR00516B 10.78 1.811e−06 310-325

2A RECEPTOR

SIGNATURE

250
BL00461
6-phosphogluconate
BL00461C 18.34 9.495e−06 30-58

dehydrogenase

proteins.

251
BL00888
Cyclic nucleotide-
BL00888A 18.03 9.667e−06 20-37

binding domain

proteins.

252
DM01242
3 THREONINE--TRNA
DM01242E 23.00 6.215e−07 119-161

LIGASE.

253
DM00250
kw ANNEXIN ANTIGEN
DM00250A 10.52 6.488e−06 16-32

PROLINE TUMOR.

254
BL00291
Prion protein.
BL00291A 4.49 2.469e−07 51-86

BL00291A 4.49 6.878e−07 40-75

BL00291A 4.49 5.330e−06 22-57

BL00291A 4.49 1.000e−05 30-65

255
PF00506
Influenza virus
PF00506F 9.40 5.459e−08 17-55

nucleoprotein.

256
BL00126
3′5′-cyclic
BL00126A 27.56 6.026e−06 25-62

nucleotide

phosphodiesterases

proteins.

257
PR00003
4-DISULPHIDE CORE
PR00003D 8.10 5.131e−06 291-300

SIGNATURE

258
DM01803
1 HERPESVIRUS
DM01803C 7.00 7.061e−06 10-20

GLYCOPROTEIN H.

259
DM01803
1 HERPESVIRUS
DM01803C 7.00 8.071e−06 3-13

GLYCOPROTEIN H.

260
PR00775
90 KD HEAT SHOCK
PR00775D 8.91 3.831e−06 147-165

PROTEIN SIGNATURE

261
PR00217
43 KD POSTSYNAPTIC
PR00217C 10.91 1.167e−06 75-91

PROTEIN SIGNATURE

262
PR00388
3′,5′-CYCLIC
PR00388D 14.87 8.079e−06 69-83

NUCLEOTIDE CLASS II

PHOSPHODIESTERASE

SIGNATURE

263
PR00023
ZONA PELLUCIDA
PR00023A 17.17 9.036e−06 24-39

SPERM-BINDING

PROTEIN SIGNATURE

264
BL00024
Hemopexin domain
BL00024F 11.30 9.894e−06 3-24

proteins.

265
DM00303
6 LEA 11-MER REPEAT
DM00303A 13.20 6.294e−06 177-227

REPEAT.

266
PR00652
5-HYDROXYTRYPTAMINE
PR00652A 8.92 9.224e−07 69-90

7 RECEPTOR SIGNATURE

267
PR00652
5-HYDROXYTRYPTAMINE
PR00652A 8.92 9.224e−07 69-90

7 RECEPTOR SIGNATURE

268
DM01803
1 HERPESVIRUS
DM01803A 10.51 1.673e−06 14-35

GLYCOPROTEIN H.

270
PR00875
MOLLUSC
PR00875C 8.64 9.550e−06 65-77

METALLOTHIONEIN

SIGNATURE

271
DM01803
1 HERPESVIRUS
DM01803A 10.51 7.308e−06 21-42

GLYCOPROTEIN H.

272
PF00598
Influenza Matrix
PF00598A 14.24 4.383e−06 46-89

protein (M1).

273
PR00113
ALKALINE PHOSPHATASE
PR00113D 6.87 9.260e−06 8-19

SIGNATURE

274
PD01841
PHOSPHORYLASE KINASE
PD01841E 18.60 9.446e−06 80-118

ALPHA MUSCL.

275
PF00600
Influenza non-
PF00600A 20.40 1.563e−06 40-67

structural protein

(NS1).

276
PR00877
PLANT PEC FAMILY
PR00877B 4.74 9.878e−06 31-38

METALLOTHIONEIN

SIGNATURE

277
PR00076
6-PHOSPHOGLUCONATE
PR00076E 12.73 6.417e−06 71-99

DEHYDROGENASE

SIGNATURE

279
BL00101
Hexapeptide-repeat
BL00101A 10.95 1.000e−05 71-78

containing-

transferases

proteins.

280
DM01111
4 kw PHOSPHATASE
DM01111M 10.67 2.629e−06 163-187

TRANSFORMING 61 K

PDF1.

282
PR00049
WILM'S TUMOUR
PR00049D 0.00 9.934e−06 35-50

PROTEIN SIGNATURE

283
PR00519
5-HYDROXYTRYPTAMINE
PR00519C 9.73 1.227e−06 22-37

5B RECEPTOR

SIGNATURE

284
PR00304
TAILLESS COMPLEX
PR00304E 7.79 1.000e−05 54-67

POLYPEPTIDE 1

(CHAPERONE)

SIGNATURE

285
BL00197
2Fe-2S ferredoxins,
BL00197A 18.23 9.866e−07 49-79

iron-sulfur binding

region proteins.

286
PR00753
1-AMINOCYCLOPROPANE-
PR00753D 6.85 8.636e−06 61-83

1-CARBOXYLATE

SYNTHASE SIGNATURE

287
PR00003
4-DISULPHIDE CORE
PR00003B 7.64 1.300e−06 166-174

SIGNATURE

288
BL00940
Gamma-thionins
BL00940A 20.51 9.671e−06 16-40

family proteins.

289
PD00066
PROTEIN ZINC-FINGER
PD00066 13.92 9.609e−11 122-135

METAL-BINDI.
PD00066 13.92 1.900e−09 94-107

PD00066 13.92 2.703e−07 66-79

PD00066 13.92 1.000e−05 38-51

291
PR00516
5-HYDROXYTRYPTAMINE
PR00516F 10.18 9.609e−07 761-779

2A RECEPTOR

SIGNATURE

293
PR00651
5-HYDROXYTRYPTAMINE
PR00651E 10.53 8.487e−06 51-71

2B RECEPTOR

SIGNATURE

296
PR00635
AT1 ANGIOTENSIN II
PR00635C 7.44 8.602e−06 28-45

RECEPTOR SIGNATURE

297
PF00915
Calicivirus coat
PF00915E 5.71 1.000e−05 102-112

protein.

298
PR00519
5-HYDROXYTRYPTAMINE
PR00519B 9.99 3.968e−06 495-512

5B RECEPTOR

SIGNATURE

299
PR00784
MITOCHONDRIAL BROWN
PR00784D 15.86 9.730e−06 22-40

FAT UNCOUPLING

PROTEIN SIGNATURE

300
DM00973
3 kw RESISTANCE
DM00973A 21.17 1.273e−06 7-44

BENOMYL YLL028W

CYCLOHEXIMIDE.

301
PR00049
WILM'S TUMOR
PR00049D 0.00 8.752e−06 42-57

PROTEIN SIGNATURE

302
BL00283
Soybean trypsin
BL00283B 16.55 1.000e−05 15-30

inhibitor (Kunitz)

protease inhibitors

family.

303
DM01753
6 kw OSTEOBLAST
DM01753A 21.93 9.830e−06 59-94

MAJOR IMMUNOGENIC

MPB70.

304
PR00516
5-HYDROXYTRYPTAMINE
PR00516E 14.87 6.516e−06 20-38

2A RECEPTOR

SIGNATURE

305
DM01554
1 THYROLIBERIN
DM01554E 10.78 5.452e−07 20-37

PRECURSOR.

306
PR00331
HAEMAGGLUTININ HA2
PR00331E 18.67 1.000e−05 75-93

CHAIN SIGNATURE

307
PR00651
5-HYDROXYTRYPTAMINE
PR00651H 5.59 8.858e−06 152-175

2B RECEPTOR

SIGNATURE

308
BL00208
Plant hemoglobins
BL00208A 18.41 1.000e−05 5-47

proteins.

309
PR00240
ALPHA-1A ADRENERGIC
PR00240E 9.25 9.391e−06 36-56

RECEPTOR SIGNATURE

310
PR00701
60 KD INNER MEMBRANE
PR00701C 10.53 8.255e−06 55-76

PROTEIN SIGNATURE

311
PR00423
CELL DIVISION
PR00423B 7.15 1.000e−05 5-26

PROTEIN FTSZ

SIGNATURE

312
PF00602
Influenza RNA-
PF00602C 12.16 2.068e−07 26-66

dependant RNA

polymerase subunit

PB1.

313
PR00246
SOMATOSTATIN
PR00246D 7.36 1.000e−05 4-14

RECEPTOR SIGNATURE

314
BL00216
Sugar transport
BL00216A 13.29 9.526e−06 26-38

proteins.

316
PF00721
Tobacco mosaic virus
PF00721A 14.59 9.845e−06 131-167

coat.

317
PD00489
PROTEIN
PD00489A 15.57 1.000e−05 55-71

TRANSMEMBRANE

TRANSPORT C.

318
PR00163
RUBREDOXIN SIGNATURE
PR00163A 10.47 9.888e−06 59-76

319
PR00513
5-HYDROXYTRYPTAMINE
PR00513A 7.75 9.149e−07 205-217

1B RECEPTOR

SIGNATURE

320
DM01418
352 FIBRILLAR
DM01418C 20.48 5.142e−06 377-419

COLLAGEN CARBOXYL-

TERMINAL.

321
BL00067
3-hydroxyacyl-CoA
BL00067D 21.49 7.441e−06 9-42

dehydrogenase

proteins.

322
DM01857
5 kw NUCLEOSIDE
DM01857B 14.94 7.821e−08 52-80

TRANSPORT DEPENDENT

NA.

323
DM01803
1 HERPESVIRUS
DM01803C 7.00 5.133e−06 43-53

GLYCOPROTEIN H.

324
PD01672
+ TRANSPORT
PD01672B 15.16 1.000e−05 6-55

EXCHANGER NA H

TRANS.

325
DM01688
2 POLY-IG RECEPTOR.
DM01688J 14.69 5.538e−06 31-68

327
DM00372
CARCINOEMBRYONIC
DM00372A 19.18 1.000e−05 9-54

ANTIGEN PRECURSOR

AMINO-TERMINAL

DOMAIN.

328
DM01688
2 POLY-IG RECEPTOR.
DM01688J 14.69 4.308e−06 31-68

329
DM01930
2 kw FINGER SMCX
DM01930A 7.97 2.403e−07 144-157

SMCY YDR096W.

330
PF00685
Sulfotransferase
PF00685A 19.12 9.370e−06 49-82

proteins.

331
PR00347
PATHOGENESIS-RELATED
PR00347A 13.98 9.649e−06 55-68

PROTEIN SIGNATURE

332
PR00538
MUSCARINIC M1
PR00538F 10.59 8.667e−06 30-48

RECEPTOR SIGNATURE

334
PR00159
2FE-2S FERREDOXIN
PR00159A 9.58 1.153e−06 23-32

SIGNATURE

336
PR00554
ADENOSINE A2B
PR00554B 12.52 9.778e−06 41-50

RECEPTOR SIGNATURE

337
DM01111
4 kw PHOSPHATASE
DM01111G 10.39 7.250e−06 3-44

TRANSFORMING 61 K

PDF1.

338
PR00516
5-HYDROXYTRYPTAMINE
PR00516B 10.78 7.649e−06 214-229

2A RECEPTOR

SIGNATURE

340
PR00388
3′,5′-CYCLIC
PR00388A 10.45 5.050e−06 141-160

NUCLEOTIDE CLASS II

PHOSPHODIESTERASE

SIGNATURE

342
DM01664
kw.
DM01664D 16.63 1.000e−05 22-47

343
PD01841
PHOSPHORYLASE KINASE
PD01841K 14.81 1.000e−05 65-95

ALPHA MUSCL.

344
PR00416
EUKARYOTIC DNA
PR00416D 12.12 9.772e−06 23-40

TOPOISOMERASE I

SIGNATURE

345
BL00726
AP endonucleases
BL00726C 19.90 1.000e−05 7-33

family 1 proteins.

346
BL00305
11-S plant seed
BL00305D 21.08 4.566e−06 465-507

storage proteins.

347
PR00332
HISTIDINE TRIAD
PR00332A 10.15 9.890e−06 16-33

FAMILY SIGNATURE

348
PR00518
5-HYDROXYTRYPTAMINE
PR00518A 8.62 7.807e−06 19-36

5A RECEPTOR

SIGNATURE

349
BL00305
11-S plant seed
BL00305D 21.08 4.736e−06 276-318

storage proteins.

350
PR00503
BROMODOMAIN
PR00503C 19.84 9.731e−06 28-47

SIGNATURE

351
DM01688
2 POLY-IG RECEPTOR.
DM01688K 17.19 9.066e−07 81-120

352
DM01688
2 POLY-IG RECEPTOR.
DM01688K 17.19 9.066e−07 81-120

353
DM01415
6 SALIVARY GLUE
DM01415B 13.78 5.273e−06 99-147

PROTEIN.

354
PR00216
OSTEOPONTIN
PR00216F 11.79 9.913e−06 50-69

SIGNATURE

356
DM00895
7 kw REVERSE
DM00895G 3.62 9.913e−06 62-72

TRANSCRIPTASE RNA

POLYMERASE.

357
BL00126
3′5′-cyclic
BL00126B 15.20 6.329e−06 35-47

nucleotide

phosphodiesterases

proteins.

358
PR00512
5-HYDROXYTRYPTAMINE
PR00512G 6.54 3.139e−06 3-19

1A RECEPTOR

SIGNATURE

359
PD02455
ELEMENT TRANSPOSABLE
PD02455D 18.65 1.000e−05 58-77

INSERTION PROTEIN

TRANSPOSITION DNA.

360
DM01415
6 SALIVARY GLUE
DM01415A 6.65 3.250e−07 16-29

PROTEIN.

361
BL00794
7,8-dihydro-6-
BL00794C 19.62 9.702e−06 27-65

hydroxymethylpterin-

pyrophosphokinase

proteins.

362
PR00866
RNA-DEPENDENT DNA-
PR00866B 9.86 9.786e−06 60-73

POLYMERASE (MSDNA)

SIGNATURE

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

[0388]

4

TABLE 4

SEQ ID
pFAM

pFAM

NO:
NAME
DESCRIPTION
p-value
SCORE

2
PGAM
Phosphoglycerate
2.5e−05
23.4

mutase family

6
Ubie—
ubiE/COQ5 methyl-
0.035
−133.9

methyltran
transferase

family

8
Plexin—
Plexin repeat
0.03
18.4

repeat

13
K_tetra
K+ channel
2.3e−31
117.6

tetramerisation

domain

14
EGF
EGF-like domain
7.8e−14
59.4

16
Armadillo—
Armadillo/beta-
1.3e−05
32.1

seg
catenin-like

repeats

19
Ribosomal—
Ribosomal pro-
1.7e−46
167.9

S5
tein S5

21
gpdh
glyceraldehyde
1.3e−230
773.2

3-phosphate

dehydrogenases

24
GCV_T
Glycine cleavage
9.3e−156
530.9

T-protein

(aminomethyl tran

25
zf-C3HC4
Zinc finger, C3HC4
0.015
12.5

type (RING finger)

26
zf-C3HC4
Zinc finger, C3HC4
1.6e−10
38.4

type (RING finger)

33
urease
Urease
0.014
11.0

35
tRNA-
tRNA synthetases
0.0091
12.1

synt_le
class I (C)

37
LRRNT
Leucine rich repeat
0.00049
26.8

N-terminal domain

39
SH3
SH3 domain
3.4e−60
213.4

40
SH3
SH3 domain
3.4e−60
213.4

41
PBD
P21-Rho-binding
1e−08
42.4

domain

42
GDA1—
GDA1/CD39 (nucle-
9e−94
324.9

CD39
oside phosphatase)

family

43
Band_7
SPFH domain / Band
1.7e−21
84.9

7 family

46
Rhodanese
Rhodanese-like
2.9e−24
94.0

domain

47
zf-C2H2
Zinc finger, C2H2
6.2e−32
119.5

type

50
ZAP
ZAP domain
1.6e−50
181.3

52
sushi
Sushi domain (SCR
9.5e−27
102.3

repeat)

55
zf-C2H2
Zinc finger, C2H2
0.047
20.3

type

56
zf-C2H2
Zinc finger, C2H2
0.00021
28.1

type

59
PH
PH domain
2.6e−06
27.6

60
PHD
PHD-finger
2e−09
44.8

64
IQ
IQ calmodulin-binding
6.4e−42
152.7

motif

66
ank
Ank repeat
2.7e−23
90.8

69
eIF-1a
Eukaryotic initiation
0.0047
−2.4

factor 1A

74
Ribosomal—
Ribosomal protein S17
6e−43
148.6

S17

75
LIM
LIM domain containing
0.00067
19.0

proteins

80
Phospho-
Type I phosphodies-
2.7e−49
177.2

diest
terase/nucleotide py

81
transmem-
Transmembrane 4 family
6.6e−61
197.7

brane4

84
zf-C2H2
Zinc finger, C2H2 type
1.6e−64
227.8

85
zf-C2H2
Zinc finger, C2H2 type
1.4e−07
38.6

89
ank
Ank repeat
4e−31
116.8

93
L15
Ribosomal protein L15
3.5e−21
61.9

98
Band_41
FERM domain (Band 4.1
0.00015
16.7

family)

101
Nol1—
NOL1/NOP2/sun family
4.5e−19
68.6

Nop2_Sun

103
LIM
LIM domain containing
1.3e−30
113.2

proteins

113
WD40
WD domain, G-beta
0.00018
28.3

repeat

115
pro—
Cyclophilin type
5.3e−34
120.4

isomerase
peptidylprolyl cis-tr

116
DUF25
Domain of unknown
1.1e−11
46.9

function DUF25

118
Band_41
FERM domain (Band 4.1
3.2e−77
242.4

family)

119
rrm
RNA recognition motif.
1.1e−33
125.4

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

120
SH3
SH3 domain
3e−05
30.9

125
Ribosomal
Ribosomal L29 protein
1.6e−15
65.0

L29

126
NTF2
Nuclear transport
7.6e−06
32.2

factor 2 (NTF2) domain

129
rrm
RNA recognition motif.
0.0016
25.2

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

130
Fork_head
Fork head domain
1e−28
108.8

132
PC4
Transcriptional Co-
2.1e−38
141.0

activator p15 (PC4)

133
RGS
Regulator of G protein
2.6e−45
164.0

signaling domain

137
COX7a
Cytochrome c oxidase
2.3e−40
147.5

subunit VIIa

139
rrm
RNA recognition motif.
3.2e−15
64.0

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

141
lectin_c
Lectin C-type domain
5.1e−05
30.0

142
lectin_c
Lectin C-type domain
5.1e−05
30.0

147
ig
Immunoglobulin domain
9.1e−07
26.9

150
ank
Ank repeat
8.6e−09
42.6

161
Ribosomal—
Ribosomal protein L7Ae
0.03
0.8

L7Ae

162
HMG_box
HMG (high mobility
8e−53
188.9

group) box

163
PH
PH domain
3e−13
52.4

168
Peptidase—
Helper component
0.0056
7.9

C6
proteinase

175
ig
Immunoglobulin domain
2.3e−09
35.2

176
ig
Immunoglobulin domain
9.2e−09
33.3

178
WW
WW domain
0.054
17.2

180
Ribosomal—
Ribosomal protein S12e
1.9e−38
141.1

S12e

185
myb_DNA-
Myb-like DNA-binding
0.00011
29.1

binding
domain

186
myb_DNA-
Myb-like DNA-binding
0.00011
29.1

binding
domain

187
pkinase
Eukaryotic protein
3.4e−26
98.4

kinase domain

189
ER_lumen—
ER lumen protein
3.9e−144
492.2

recept
retaining receptor

190
ER_lumen—
ER lumen protein
2.1e−88
307.1

recept
retaining receptor

195
EMP24—
emp24/gp25L/p24 family
6.9e−06
28.1

GP25L

199
zf-B_box
B-box zinc finger.
5.2e−07
36.7

211
HECT
HECT-domain (ubiquitin-
1.1e−115
397.8

transferase).

213
Rhomboid
Rhomboid family
4.2e−42
153.3

214
LIM
LIM domain containing
8.8e−35
127.8

proteins

215
Ricin_B—
Similarity to lectin
0.0015
19.2

lectin
domain of ricin

216
chromo
‘chromo’ (CHRromatin
2.1e−09
37.1

Organization MOdifier

218
Sialyl-
Sialyltransferase
7.3e−20
79.4

transf
family

219
PG—
Putative peptido-
5e−06
33.5

binding_2
glycan binding domain

223
zf-C2H2
Zinc finger, C2H2 type
1.5e−104
360.7

226
RGS
Regulator of G protein
5.1e−52
186.2

signaling domain

227
TBC
TBC domain
7.2e−35
129.3

228
CRAL—
CRAL/TRIO domain.
4.5e−47
158.6

TRIO

232
Ribosomal—
Ribosomal protein L44
1e−48
175.3

L44

235
ig
Immunoglobulin domain
3.5e−08
31.4

236
thyro-
Thyroglobulin type-1
3.9e−24
93.6

globulin_1
repeat

238
TBC
TBC domain
1.2e−54
195.0

241
zf-C2H2
Zinc finger, C2H2
3.8e−08
40.5

type

242
AAA
ATPases associated
2.1e−43
157.6

with various cellular

act

249
integrin_A
Integrin alpha cyto-
0.091
18.0

plasmic region

256
PAP2
PAP2 superfamily
0.00084
22.8

257
zf-C2H2
Zinc finger, C2H2 type
1.2e−60
214.9

259
G-gamma
GGL domain
5.5e−30
108.3

266
efhand
EF hand
3.4e−07
37.4

267
efhand
EF hand
3.4e−07
37.4

274
zf-C2H2
Zinc finger, C2H2 type
0.00014
28.6

277
RecF
RecF protein
0.036
11.1

281
CH
Calponin homology (CH)
7.9e−22
86.0

domain

285
cyclin
Cyclin
3.9e−07
28.5

289
zf-C2H2
Zinc finger, C2H2 type
1.9e−21
84.7

290
PI-PLC-X
Phosphatidylinositol-
0.073
10.8

specific phospholipase

299
60s—
60s Acidic ribosomal
4.1e−07
25.8

ribosomal
protein

307
trypsin
Trypsin
6.9e−81
257.3

310
ig
Immunoglobulin domain
1.3e−10
39.3

311
ig
Immunoglobulin domain
6.1e−07
27.4

313
globin
Globin
3.8e−21
78.2

315
ig
Immunoglobulin domain
1.6e−05
22.8

318
zf-C2H2
Zinc finger, C2H2 type
9e−19
75.8

319
ig
Immunoglobulin domain
0.01
13.8

320
BTB
BTB/POZ domain
5e−17
70.0

322
aa_per-
Amino acid permease
0.0058
−262.2

meases

325
ig
Immunoglobulin domain
1.6e−10
38. 9

327
ig
Immunoglobulin domain
1.9e−09
35.5

328
ig
Immunoglobulin domain
2.9e−09
34.9

329
ig
Immunoglobulin domain
7.4e−14
49.7

332
Ribosomal—
Ribosomal protein L36e
6.3e−17
69.7

L36e

333
connexin
Connexin
7.6e−148
504.6

335
p450
Cytochrome P450
2.1e−100
347.0

337
Sm
Sm protein
0.00012
28.8

338
zf-C2H2
Zinc finger, C2H2 type
0.0025
24.5

343
aldo—
Aldo/keto reductase
2.4e−53
190.7

ket_red
family

345
ubiquitin
Ubiquitin family
3.1e−13
45.5

346
CH
Calponin homology (CH)
0.0017
23.8

domain

351
ig
Immunoglobulin domain
4.8e−18
63.2

352
ig
Immunoglobulin domain
4.8e−18
63.2

358
G-alpha
G-protein alpha subunit
4.5e−148
505.3

359
ig
Immunoglobulin domain
8.9e−09
33.3

362
Ribosomal—
Ribosomal L37ae protein
0.00083
−3.0

L37ae
family

[0389]

5

TABLE 5

POSITION OF

SIGNAL IN
maxS

SEQ ID
AMINO ACID
(MAXIMUM
meanS

NO:
SEQUENCE
SCORE)
(MEAN SCORE)

2
1-29
0.942
0.664

12
1-15
0.909
0.589

14
1-17
0.974
0.943

20
1-22
0.932
0.802

25
1-16
0.988
0.881

28
1-13
0.896
0.771

37
1-21
0.992
0.929

42
1-46
0.978
0.754

52
1-34
0.954
0.756

63
1-31
0.960
0.773

71
1-45
0.981
0.652

80
1-22
0.982
0.882

81
1-42
0.993
0.715

83
1-30
0.966
0.767

95
1-18
0.997
0.971

102
1-13
0.981
0.764

107
1-45
0.890
0.631

110
1-27
0.992
0.969

138
1-33
0.961
0.864

144
1-45
0.987
0.658

145
1-20
0.992
0.967

175
1-20
0.957
0.874

176
1-21
0.989
0.945

179
1-42
0.980
0.577

184
1-20
0.972
0.771

189
1-28
0.941
0.755

190
1-28
0.941
0.755

191
1-12
0.907
0.779

195
1-21
0.958
0.779

200
1-15
0.970
0.875

211
1-20
0.895
0.595

215
1-31
0.987
0.895

218
1-30
0.971
0.889

225
1-17
0.884
0.588

235
1-23
0.965
0.817

237
1-29
0.933
0.725

249
1-28
0.972
0.870

251
1-17
0.966
0.905

260
1-26
0.921
0.587

270
1-20
0.938
0.631

283
1-18
0.901
0.763

288
1-20
0.940
0.693

293
1-26
0.937
0.784

295
1-22
0.972
0.745

296
1-15
0.930
0.748

297
1-35
0.906
0.600

300
1-29
0.981
0.864

307
1-19
0.976
0.916

308
1-27
0.973
0.931

309
1-29
0,950
0.629

310
1-19
0,969
0.913

311
1-21
0.956
0.823

315
1-17
0.976
0.938

317
1-19
0.943
0.837

319
1-18
0.991
0.978

324
1-26
0.968
0.806

325
1-20
0.972
0.828

326
1-27
0.893
0.567

327
1-21
0.994
0.959

328
1-20
0.945
0.891

329
1-21
0.984
0.858

330
1-27
0.891
0.593

333
1-40
0.955
0.703

347
1-22
0.968
0.806

351
1-23
0.982
0.945

352
1-23
0.982
0.945

355
1-32
0.955
0.617

356
1-23
0.936
0.677

359
1-20
0.937
0.859

360
1-29
0.956
0.765

361
1-23
0.968
0.819

	Number	Date	Country
Parent	09714936	Nov 2000	US
Child	10115635	Apr 2002	US

Novel nucleic acids and polypeptides

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Continuations (1)