CA2460611A1 - Novel nucleic acids and secreted polypeptides - Google Patents

Abstract

The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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NOVEL NUCLEIC ACIDS AND SECRETED
POLYPEPTIDES
1. CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the priority benefit of U.S. Provisional Application Serial No.
60/323,349 filed September 18, 2001 entitled "Novel Nucleic Acids and Secreted Polypeptides", Attorney Docket No. 808, which is a continuation-in-part application of PCT
Application Serial No. PCT/LTS00/35017 filed December 22, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 784CIP3A/PCT, which in turn is a continuation-in-part application of U.S. Application Serial No. 09/552,317 filed April 25, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No.
784C1P, which in turn is a continuation-in-part application of U.S.
Application Serial No.
09!488,725 filed January 21, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 784; PCT Application Serial No.
PCTlCTSOI/02623 filed January 25, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 785CIP3/PCT, which in turn is a continuation-in-part application of U.S.
Application Serial No. 09/491,404 filed January 25, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 785; PCT Application Serial No.
PCT/TJSO1/03800 filed February 5, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Doclcet No. 787CIP3lPCT, which in turn is a continuation-in-part application of U.S. Application Serial No. 09/560,875 filed April 27, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 787CIP, which in turn is a continuation-in-part application of U.S. Application Serial No. 09/496,914 filed February 03, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 787;
PCT Application Serial No. PCT/USO1/04927 filed February 26, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 788CIP3/PCT, which in turn is a continuation-in-part application of U.S. Application Serial No.
09/577,409 filed May 18, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 788CIP, which in turn is a continuation-in-part application of U.S.
Application Serial No. 09/515,126 filed February 28, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 788; PCT Application Serial No. PCT/USO1/04941 filed March 5, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 789CIP3/PCT, which in turn is a continuation-in-part application of U.S.
Application Serial No. 09/574,454 filed May 19, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 789CIP, which in turn is a continuation-in-part application of U.S. Application Serial No. 09/519,705 filed March 07, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 789; PCT Application Serial No.
PCT/LTSO1/08631 filed March 30, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 790CIP3/PCT, which in turn is a continuation-in-part application of U.S. Application Serial No. 09/649,167 filed August 23, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 790CIP, which in turn is a continuation-in-part application of U.S. Application Serial No. 09/540,217 filed March 31, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 790;
PCT Application Serial No. PCT/USOl/08656 filed April 18, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 791CIf3/PCT, which in turn is a continuation-in-part application of U.S. Application Serial No. 09/770,160 filed January 26, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No.
791CIP, which is in turn a continuation-in-part application of U.S.
Application Serial No.
09/552,929 filed April 18, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 791; and PCT Application Serial No. PCT/IJSOl/14827 filed Mayl6, 2001 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No.
792CIP3/PCT, which in turn is a continuation-in-part application of U.S.
Application Serial No. 09/577,408 filed May 18, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No. 792; all of which are incorporated herein by reference in their entirety.
2. BACKGROUND OF THE INVENTION
2.1 TECHNICAL FIELD
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.
2.2 BACKGROUND
Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, circulating soluble factors, chemolcines, and interleulcins) 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 lcnown biological activity.
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.

3. SUMMARY OF THE INVENTION
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.
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.
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 m NO: 1-151, or 303-399 and are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A
is adenine; C is cytosine; G is guanine; T is thyrnine; and N is any of the four bases or unknown. In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.
The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ m NO: 1-151, or 303-399 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
m NO: 1-151, or 303-399. A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ m NO: 1-151, or 303-399 or a degenerate variant or fragment thereof. The identifying sequence can be 100 base pairs in length.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ m NO: 1-151, or 303-399.
The sequence information can be a segment of any one of SEQ m NO: 1-151, or 303-399 that uniquely identifies or represents the sequence information of SEQ m NO: 1-151, or 303-399.
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 anay. In one embodiment, segments of sequence information are 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.
This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors contaiung 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.
In a preferred embodiment, the nucleic acid sequences of SEQ m NO: 1-151, or 399 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well lcnov~m in the art. W a particularly preferred embodiment, the nucleic acid sequences of SEQ m NO: 1-151, or 303-399 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well lrnown 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.
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
~ NO: 1-151, or 303-399; a polynucleotide comprising any of the full length protein coding sequences of SEQ m NO: 1-151, or 303-399; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of SEQ m NO: 1-151, or 303-399. 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 m NO: 1-151, or 303-399; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in SEQ m NO: 1-151, or 303-399; (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homologue (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 SEQ m NO: 152-302, or 400-496, or Tables 3, 4A, 4B, 5, or 6.
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 m NO: 1-151, or 303-399; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically 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.

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.
The invention also provides host cells transformed or transfected with a polynucleotide of the invention.
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 processes is a mature form of the protein.
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., ira situ hybridization.
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.
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.
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.

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.
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 fornz 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.
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.
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 fox 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 that binds to a polypeptide of the invention is identified.

The methods of the invention also provide 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 affect such modulation either on the level of target gene/protein expression or target protein activity.
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 Tables 2A and 2B);
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 Tables 4A and 4B). If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.
4. DETAILED DESCRIPTION OF THE INVENTION
4.1 DEFINITIONS
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.
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.
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.
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 certain portions) 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.
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 Bells (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.
The term "expression modulating fragment," EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.
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.
The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" or "oligonucleotide" 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, G is cytosine, T is thymine, G
is guanine and N is A, C, G, or T (L~ or unknown. 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.
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 NO: 1-151, or 303-399.
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 lcnown in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambroolc, 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 NY, both of which are incorporated herein by reference in their entirety.
The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ >D NO: 1-151, or 303-399.
The sequence information can be a segment of any one of SEQ m NO: I-151, or 303-399 that uniquely identifies or represents the sequence information of that sequence of SEQ 1D NO:
1-151, or 303-399, or those segments identified in Tables 3, 4A, 4B, 5, 6, or 8. 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 42° 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.
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 x 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.
The teen "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.
The teens "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 iii 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.
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.
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.
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, Iipidation and acylation.
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.
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.
The term "derivative" refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.
The term "variant"(or "analog") refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, a 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.
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 prokaryotib 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 degradationturnover rate.
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 malting insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
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.
The terns "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).

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 nozmally present in a solution of the same. The terms "isolated" and "purified" do not encompass nucleic acids or polypeptides present in their natural source.
The teen "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" def nes 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.
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 stz~uctural or coding sequence which is transcribed into mRNA
and translated into protein, and (3) appropriate transcription initiation and tezmination sequences.
Stz-uctural 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 rnay or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
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.
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. Interleulcin-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. Iminunol. 16:27-SS) 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.
The teen "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 O.S M NaHPOQ, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 6S°C, and washing in O.1X SSC/0.1% SDS at 68°C), and moderately stringent conditions (i.e., washing in 0.2X SSC/0.1% SDS at 42°C). Other exemplary hybridization conditions are described herein in the examples.
In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6X SSC/O.OS% sodium pyrophosphate at 37°C (for I4-base oligonucleotides), 48°C (for 17-base oligonucleotides), SS°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).
As used herein, "substantially equivalent" or "substantially similar" can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to have 65% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 2S%
(7S% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity). Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90%
sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity.
Substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.
Preferably, the nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a new stop codon) should be disregarded. Sequence identity may be determined, e.g., using'the Jotun Hein method (Hero, 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.
The term "totipotent" refers to the capability of a cell to differentiate into all of the cell types of an adult organism.
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.
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.
Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.
4.2 NUCLEIC ACIDS OF THE INVENTION
Nucleotide sequences of the invention are set forth in the Sequence Listing.
The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 1-151, or 303-399; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 1-151, or 303-399; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polynucleotides of any one of SEQ ID NO: 1-151, or 303-399. 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 m NO: 1-1S l, or 303-399; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing, or Table 8; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homologue 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 )D NO:
1S2-302, or 400-496 (for example, as set forth in Tables 3, 4A, 4B, S, 6, or 8). 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.
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 entire coding region of the cDNA or may represent a portion of the coding region of the cDNA.
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:
1-151, or 303-399 can be obtained by screening appropriate cDNA or genomic DNA
libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-151, or 303-399 or a portion thereof as a probe. Alternatively, the polynucleotides of SEQ ID NO:
1-151, or 303-399 may be used as the basis for suitable primers) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.
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.
The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.
Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99% sequence identity to a polynucleotide recited above.
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 m NO: 1-151, or 303-399, 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 pol5mucleotides 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.
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 m NO: 1-151, or 303-399, a representative fragment thereof, or a nucleotide sequence at least 90%
identical, preferably 95% identical, to SEQ m NO: 1-151, or 303-399 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.
The nearest neighbor or homology results for the nucleic acids of the present invention, including SEQ m NO: 1-151, or 303-399 can be obtained by searching a database using an algoritlun or a program. Preferably, a BLAST (Basic Local Aligmnent Search Tool) program 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 may be performed.
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.
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.
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.
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.,17NA 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 (I982). 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, primers) 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.
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 tinder stringent conditions.
Polynucleotides encoding preferred polypeptide truncations of the invention could be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.
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.
In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-151, or 303-399, 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.
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 lcnown 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.
The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ >D NO: 1-15I, or 303-399 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 NO: 1-151, or 303-399 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 fox 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, pNHBa, pNHl6a, pNHIBa, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia); Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
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.
Kaufinan, Methods in Enzyynology 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.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV
immediate early, HSV thymidine lcinase, 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 TRP 1 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 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 typhiynu~-ium and various species within the genera Pseudon~onas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
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, WI, 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.

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 intra-muscular injection of the DNA. The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.
4.3 ANTISENSE
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-151, or 303-399, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules axe provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 1-151, or 303-399 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ m NO: 1-151, or are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence of the invention. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence of the invention. The term "noncoding region" refers to 5' and 3' sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ m NO: 1-151, or 303-399, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of an mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of an mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanne, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil; 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetie acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II
or pol III
promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual a.-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15:
6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (moue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (moue et al. (1987) FEBS Lett 215: 327-330).
4.4 RIBOZYMES AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Natuf°e 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of an mRNA. A ribozyrne having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-151, or 303-399). For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,I I6,742.
Alternatively, mRNA of the invention can be used to select a catalytic RNA having a specific nibonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter andlor enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See generally, Helene.
(1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Aran. N. Y. Acad.
Sci.
660:27-36; and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chenz. 4: 5-23). As used herein, the terms "peptide nucleic acids"
or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al.
(1996) PNAS 93:
14670-675.
PNAs of the invention can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.
(1996) above);
or as probes or primers for DNA sequence and hybridization (Hyrup et al.
(1996), above;
Perry-O'Keefe (1996), above).
In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, e.g., RNase H and DNA polymerases, to interact With the DNA

portion while the PNA portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linlcers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Firm et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
(1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al.
(1996) above). Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment. See, Petersen et al. (1975) Biooyg Med Cher~z Lett 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. U.S.A.
86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.
W089/10134).
In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., I~rol et al., 1988, BioTechraiques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Phar~m. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
4.5 HOSTS
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 fuxther 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.

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 occurnng 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. W094/12650, PCT International Publication No. W092/20808, and PCT International Publication No. W09I/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.
The host cell can be a higher eulcaryotic 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, DEAF, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods i32 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.
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 Sf~ 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, New York (1989), the disclosure of which is hereby incorporated by reference.
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 kicliley 293 cells, human epidermal A431 cells, human Co1o205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitj°o 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 genorne, 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.
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 Sacclaa~°omyces cerevisiae, Schizosaccharomyces pombe, Kluyvef°onayces strains, Caradida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Esclaerichia coli, Bacillus subtilis, Salmofaella typlaimuriurn,. 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.

hi 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, and 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.
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 occurnng 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.

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.
Patent No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (W093/09222) by Selden et al.; and International Application No.
PCT/LTS90/06436 (W091/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.6 POLYPEPTIDES OF THE INVENTION
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 m NO: 152-302, or 400-496 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ )D NO: 1-151, or 303-399 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 m NO: 1-151, or 303-399 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ >D NO: 152-302, or 400-496 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 )D NO: 152-302, or 400-496 or the corresponding full length or mature protein; and "substantial equivalents" thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ )D NO: 152-302, or 400-496.
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. Fragments are also identified in Tables 3, 4A, 4B, 5, 6, or 8.
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 predicted signal sequence is set forth in Table 6.
The mature form of such protein may be obtained and confirmed by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell and sequencing of the cleaved product. One of skill in the art will recognize that the actual cleavage site may be different than that predicted in Table 6. 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, pact 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 (See, e.g., Sakal et al., Prep. Biochem. Biotechnol. (2000), 30(2), pp. 107-23, incorporated herein by reference).
Protein compositions of the present invention may further comprise an acceptable Garner, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
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.
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.
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.
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.
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 Purificatiora: Principles arad 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.

The purified polypeptides can be used in irz 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 111 l32 VdVO 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.
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: 152-302, or 400-496.
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.
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 slcilled 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 fox 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 acids) in biological activity. Regions of the protein that are important for protein function may be determined by the eMATRTX program.

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.
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 MaxBatTM kit), and such methods are well known in the art, as described in Smnmers 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."
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-toyopearlTM or Cibacrom blue 3GA
SepharoseTM;
one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or irmnunoaffinity chromatography.
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.).
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."
The polypeptides of the invention include analogs (variants). This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted. Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability.
Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells. Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.
4.6.1 DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE
IDENTITY AND SIMILARITY
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(I):387 (I984); Genetics Computer Group, University of Wisconsin, Madison, WI), 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 (Sonmhammer 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, I57, pp. I05-31 (1982), the GeneAtlas software (Molecular Simulations Inc. (MSI), San Diego, CA) (Sanchez and Sali (1998) Proc. Natl.
Acad. Sci., 95, 13597-13602; Kitson DH et al, (2000) "Remote homology detection using structural modeling- an evaluation" Submitted; Fischer and Eisenberg (1996) Protein Sci.
5, 947-955), Neural Network SignalP V1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark) incorporated herein by reference).
Polypeptide sequences were examined by a proprietary algorithm, SeqLoc that separates the proteins into three sets of locales: intracellular, membrane, or secreted.
This prediction is based upon three characteristics of each polypeptide, including percentage of cysteine residues, Kyte-Doolittle scores for the first 20 amino acids of each protein, and Kyte-Doolittle scores to calculate the longest hydrophobic stretch of the said protein. Values of predicted proteins are compared against the values from a set of 592 proteins of known cellular localization from the Swissprot database (http:/lwww.expas .~prot).
Predictions are based upon the maximum likelihood estimation.
Presence of transmembrane regions) was detected using the TMpred program (http:/lwww.ch.embnet.or~/software/TMPRED form.html).
The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.
NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-(1990).
4.7 CHIMERIC AND FUSION PROTEINS
The invention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises a polypeptide of the invention operatively linked to another polypeptide. Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention. In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention. W another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention. Within the fusion protein, the term "operatively linked" is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other. The polypeptide can be fused to the N-terminus or C-terminus, or to the middle.
For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.

Tn another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST
(i.e., glutathione S-transferase) sequences.
In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprise one or more domains fused to sequences derived from a member of the immunoglobulin protein family.
The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction ih vivo. The innnunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand. Tnhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e.g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.
A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for Iigation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.

4.8 GENE THERAPY
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, ifa situ, or ire 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:

(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 112 VdVO 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.
Other methods inlubiting 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 l~nown 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.
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.
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.
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-attaclunent 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.
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 occurnng 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.
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. Patent No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International Application No.
PCT/LTS92/09627 (WO93/09222) by Selden et al.; and International Application No.
PCT/LTS90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.
4.9 TRANSGENIC ANIMALS
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. Patent 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.
Patent No 5,489,743 and PCT Publication No. W094/28122, incorporated herein by reference.

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.
The polynucleotides of the present invention also malce 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 axe useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.
In preferred methods to determine biological functions of the polypeptides of the invention ifa 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 LJ.S. Patent 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.
Patent No 5,489,743 and PCT Publication No. W094128122, incorporated herein by reference.
Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

4.10 USES AND BIOLOGICAL ACTIVITY
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.
The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.
4.10.1 RESEARCH USES AND UTILITIES
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.
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.
Any or all of these research utilities are capable of being developed into reagent grade or lcit format for commercialization as research products.
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, Bergen S. L. and A. R. Kimmel eds., 1987.
4.10.2 NUTRITIONAL USES
Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
4.10.3 CYTOKINE AND CELL PROLIFERATION/DIFFERENTIATION
ACTIVITY
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, B9/1 l, BaF3, MC9/G, M+(preB M+), 2E8, RBS, DAl, 123, T1 I6S, HT2, CTLL2, TF-1, Mo7e, CMK, HWEC, and Caco. Therapeutic compositions of the invention can be used in the following:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in linmunology, 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 Yit~o assays for Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunologic studies in Humans); Talcai et al., J. linmunol.
137:3494-3500, 1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular finmunology 133:327-34I, 1991; Bertagnolli, et al., I. Immunol. I49:3778-3783, 1992;
Bowman et al., I.
Immunol. 152:1756-1761, 1994.
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-y, 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.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleulcin 2 and Interleulcin 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. W Current Protocols in Lrrmmunology. 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 Inununology. 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.
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 Vits~o assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.
Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988.
4.10.4 STEM CELL GROWTH FACTOR ACTIVITY
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.
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 Iigand (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 f broblast growth factor (bFGF).
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. Patent No. 5,690,926).
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.
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.
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:
Prirr.ciples of Tissue Erzgifaeering 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.
In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity. Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad.
Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines. The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g. as described by Bernstein et al., Blood, 77: 2316-2321 (1991).
4.10.5 HEMATOPOIESIS REGULATING ACTIVITY
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.
Therapeutic compositions of the invention can be used in the following:
Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
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.

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 Yorlc, 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. Freslmey, 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. VoI pp. I63-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.
4.10.6 TISSUE GROWTH ACTIVITY
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.
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.
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.
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 tendoWligament-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.
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.
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.
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.
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.
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.
Therapeutic compositions of the invention can be used in the following:
Assays for tissue generation activity include, without limitation, those described in:
International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No.
W091/07491 (skin, endothelium).
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. Tnvest.
Dermatol 71:382-84 (1978).
4.10.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY
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 immunodef ciency (SCJD)), 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.
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, 1990, skin prick test (Hoffinann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and marine local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).
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.
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.
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.

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 erytlunatosis 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 hmmunology, Raven Press, New York, 1989, pp.
840-856).
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.
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.

A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.
In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and (32 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.
The activity of a protein of the invention may, among other means, be measured by the following methods:
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); Hemnann 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. Irmnunol. 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.
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 Thl/Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Imrnunol. 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.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl 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.
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.
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; Ttoh 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.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et aL, Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995;
Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
4.10.8 ACTIVIN/INHIBIN ACTIVITY

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.
The activity of a polypeptide of the invention may, among other means, be measured by the following methods.
Assays for activinlinhibin activity include, without limitation, those described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
4.10.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY
A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neuti~ophils, 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.
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.
Therapeutic compositions of the invention can be used in the following:
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. I~ruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.
Greene Publishing Associates and Wiley-lnterscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.I2.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995;
Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Imimunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.
4.10.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY
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).
Therapeutic compositions of the invention can be used in the following:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-I40, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
4.10.11 CANCER DIAGNOSIS AND THERAPY
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.
Dancer 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.
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.
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 HCI, Doxorubicin HCI, 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 HCI, Octreotide, Plicamycin, Procarbazine HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.
W 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.
Iya vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment. These ira vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (lgg'7) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY
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., W t1. J. Dev.
Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999), respectively.
Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.
4.10.12 RECEPTOR/LIGAND ACTIVITY
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, cytolcine 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.
The activity of a polypeptide of the invention may, among other means, be measured by the following methods:
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.

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

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).
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, CZIYY. Opiya. Biotechsxol. 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., CuY~ Opin CheyyZ Biol, 1(1):114-19 (1997); Dorner et al., Bioo~gMed Chem, 4(5):709-15 (1996) (alkylated dipeptides).
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 i~ 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.
The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention. Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.
4.10.14 ASSAY FOR RECEPTOR ACTIVITY
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 responses of the two cell populations to the addition of Iigands(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 Iigand(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.
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 extxacellular portion of the chimeric protein, thereby activating the chimeric receptor. Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.
phosphorylation. Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.
4.10.15 ANTI-INFLAMMATORY ACTIVITY
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 inlubiting 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 (S1RS)), 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 l, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoirmnune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.
4.10.16 LEUKEMIAS
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 rnyelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).
4.10.17 NERVOUS SYSTEM DISORDERS
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:
(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;
(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;
(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;
(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;
(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 B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
(vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;
(vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (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.
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:
(i) increased survival time of neurons in culture;
(ii) increased sprouting of neurons in culture or in vivo;
(iii) increased production of a neuron-associated molecule in culture or ifa viva, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.
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 Aralcawa 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.
In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
4.10.18 OTHER ACTIVITIES
A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites;
effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms;
effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s);
effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects;
promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
4.10.19 IDENTIFICATION OF POLYMORPHISMS
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 irmnune 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.
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
dependinglon 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.
Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.
4.10.20 ARTHRITIS AND INFLAMMATII~N
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 inj ection, 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 mglkg. The control consists of administering PB S only.
The procedure for testing the effects of the test compoiuld would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24. At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis of the data would reveal that~the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.
4.11 THERAPEUTIC METHODS

The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods. Examples of therapeutic applications include, but are not limited to, those exemplified herein.
4.11.1 EXAMPLE
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 ~,g/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 ~g/kg to 10 mg/kg of patient body weight. For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.
The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.
4.12 PHARMACEUTICAL FORMULATIONS AND ROUTES OF
ADMINISTRATION
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, lympholcines, 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-S, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, 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-a and TGF-(3), insulin-like growth factor (IGF), as well as cytokines described herein.
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-lRa, IL-1 Hyl, 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.
Asian 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.
W practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated. Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
4.12.1 ROiJTES OF ADMINISTRATION
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.
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.
The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action. The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art. Preferably for wound treatment, one administers the therapeutic compound directly to 'the site. Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.
Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.
4.12.2 COMPOSITIONSIFORMULATIONS
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 o'f 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.
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 inj ection 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 pharmace~itical 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.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such earners 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.
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.
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 tout may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain fonnulatory agents such as suspending, stabilizing and/or dispersing agents.
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.
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.
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:SW) consists of VPD diluted 1:1 with a S% 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.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such Garners 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.
The pharmaceutical composition of the invention may be in the form of a complex of the proteiri(s) or other active ingredients) 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 antigens) 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.
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' Garners, 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. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.
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 ~,g to about 100 mg (preferably about 0.1 pg to about 10 mg, more preferably about 0.1 pg 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 inj ected iri 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.

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.
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, polyethylene glycol), polyoxyethylene oxide, carboxyvinyl polyner and polyvinyl 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-a and TGF-/3), and insulin-like growth factor (IGF).
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 andlor repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
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 fox expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
4,12.3 EFFECTIVE DOSAGE
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 subj ect 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 ICSO 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.
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 LDso (the dose lethal to 50%
of the population) and the EDso (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 LDSO and EDSO. 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 EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of achninistration 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. l . 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 ih 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.
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 SO-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 ~g/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 ~.g/kg to 25 mg/lcg 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.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subj ect's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
4.12.4 PACKAGING
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 Garner may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
4.13 ANTIBODIES
Also included in the invention are antibodies to proteins, or fragments of proteins of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab° and F~ab~~z fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
Certain classes have subclasses as well, such as IgGI, IgGz, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference fo all such classes, subclasses and types of human antibody species.
An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO: 152-302, or 400-496, or Tables 3, 4A, 4B, 5, 6, or ~, or 9, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least I S
amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a surface region of the protein, e.g., a hydrophilic region. A
hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 324-3828; Kyte and Doolittle 1982, J. Mol.
Biol. I57: 105-142, each of wluch is incorporated herein by reference in its entirety.
Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
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, NY
(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 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. ~ Fits 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.
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.
The labeled antibodies of the present invention can be used for in. vitro, in vivo, and ira 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 vity~o, in vivo, and irl situ assays as well as for immuno-affinity purification of the proteins of the present invention.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies:
A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
4.13.1 POLYCLONAL ANTIBODIES
For the production ofpolyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be irrununized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.
An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the imxnunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., alumimun hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants that can be employed include MPL-TDM
adjuvant (monophosphoryl Lipid A, s3mthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
4.13.2 MONOCLONAL ANTIBODIES
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256, 495 (I975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia.
Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (I~ozbor, J. Imlnunol., 133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. S1-63).
The culture medium in wluch the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107, 220 (1980).
Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA
also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous marine sequences (U.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
4.13.3 HUMANIZED ANTIBODIES
The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-327 (1988); Verhoeyen et al., Science, 239, 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
BioL, 2, 593-596 (1992)).
4.13.4 HUMAN ANTIBODIES
Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies"
herein. Human monoclonal antibodies can be prepared by the trioma technique;
the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80, 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227, 381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368, 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol.
13, 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals that are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT
publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT
publications WO
96/33735 and WO 96/34096. This animal produces B cells that secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, fox example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S.
Patent No.~ 5,939,598. It can be obtained by a method including deleting the J
segment genes from at least one endogenous heavy chain locus in an embfyonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT
publication WO 99/53049.
4.13.5 FAB FRAGMENTS AND SINGLE CHAIN ANTIBODIES
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246, 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an antibody molecule;
(ii) an Fab fragment generated by reducing the disulfide bridges of an Flab'>2 fragment; (iii) an Fav fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F,, fragments.
4.13.6 BISPECIFIC ANTIBODIES
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chainllight-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305, 537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10, 3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the fixst heavy-chain constant region (CHl) containing the site necessary fox light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and axe co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121, 210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229, 81 (1985) describe a procedure wherein intact antibodies axe proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med, 175, 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T.
cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Tinmunol.
148(5), 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
USA 90, 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners has also been reported.
See, Gruber et al., J. Immunol. 152, 5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. finmunol. 147, 60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc~yR), such as Fc~yRI (CD64), FcyRII (CD32) and Fc~yRIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

4.13.7 HETEROCONJUGATE ANTIBODIES
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, imtnunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No. 4,676,980.
4.13.8 EFFECTOR FUNCTION ENGINEERING
It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residues) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176, 1191-1195 (1992) and Shopes, J. Immunol., I48, 2918-2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53, 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3, 219-230 (1989).
4,13.9 IMMUNOCONJUGATES
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A
chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include zl2Bi, i3ih 131In, 9oY, and lg~Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
4.14 COMPUTER READABLE SEQUENCES
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 fox recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.
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.
By providing any of the nucleotide sequences SEQ ID NO: 1-151, or 303-399 or a representative fragment thereof; or a nucleotide sequence at least 95%
identical to any of the nucleotide sequences of SEQ ID NO: 1-151, or 303-399 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 (I993)) 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.
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 fox 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.
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 seaxches 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.
As used herein, "a target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the sequences) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.
There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
4.15 TRIPLE HELIX FORMATION
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, FL (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA
hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.
4.16 DIAGNOSTIC ASSAYS AND KITS
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 Iabel.
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 polynucleatide 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.

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.
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.
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 slcilled 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 hnmunocytochemistry, Academic Press, Orlando, FL 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 extxacts 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.
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.

Tn 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 recogluze that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
4.17 MEDICAL IMAGING
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 ifa vivo at the target site.
4.18 SCREENING ASSAYS
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 NO: 1-151, or 303-399, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of:
(a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.
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.
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.
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.
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.
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.
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," W 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.
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.
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 Tnhibitors of Gene Expression, CRC Press, Boca Raton, FL (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.
Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.

4.19 USE OF NUCLEIC ACIDS AS PROBES
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 NO: 1-151, or 303-399. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from any of the nucleotide sequences SEQ ID NO: 1-151, or 303-399 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.
Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described in US Patents 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.
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 ifs 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 aI (I988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY.
Fluorescent ifZ 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 (265:1981 f). 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 subj ect invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.
4.20 PREPARATION OF SUPPORT BOUND OLIGONUCLEOTIDES
Oligonucleotides, i.e., small nucleic acid segments, may be readilyprepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
Support bound oligonucleotides may be prepared by airy 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; Mornssey &
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.
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, CA).
Nmic Laboratories {Naperville, Ih) 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 bridgeheads for fzuther 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).
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 CovaLiuc and then streptavidin used to bind the probes.
More specifically, the linlcage method includes dissolving DNA in water (7.5 ng/p,l) 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-MeIm~), is then added to a final concentration of 10 mM 1-MeIm~.
A ss DNA solution is then dispensed into CovaLink NH strips (75 p,l/well) standing on ice.
Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved ill 10 mM 1-MeTm~, is made fresh and 25 p.1 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).
It is contemplated that a fiwther 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.
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(I2) 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.
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.

One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described byPease et al., (1994) Proc. Nat'1. Acad.
Sci., 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 lugh-density, miniaturized arrays, utilize photolabile 5'-protected N acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies. A
mafirix of 256 spatially defined oligonucleotide probes may be generated in this manner.
4.21 PREPARATION OF NUCLEIC ACID FRAGMENTS
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).
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.
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.
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.
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.

The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA
fragments form the small molecule pUCl9 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUCl9 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z
minus Ml3 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.
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 p,g instead of 2-5 pg); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed).
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 quiclcly to 2°C to prevent renaturation of the DNA
fragments before they are contacted with the chip. Phosphate groups must also be removed from genomic DNA by methods known in the art.
4.22 PREPARATION OF DNA ARRAYS
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 n1 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 mmz, 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. Fox 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 x 12 cm membrane. Subarrays may contain 64 samples, one from each patient.

Where the 96 subarrays are identical, the dot span may be 1 mmz and there may be a 1 mm space between subarrays.
Another approach is to use membranes or plates (available from NUNC, Naperville, Illinois) 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.
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.
All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.
5.0 EXAMPLES
5.1 EXAMPLE 1 Novel Nucleic Acid Seguences Obtained From Various Libraries 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.

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 (AB17 sequencer to obtain the novel nucleic acid sequences.
5.2 EXAMPLE 2 Assemblage of Novel Nucleic Acids The contigs or nucleic acids of the present invention, designated as SE(~ m NO: 303-399 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, gb pri, and UniGene, and exons from public domain genomic sequences predicated by GenScan) that belong to this assemblage. The algorithm terminated when there were no additional sequences from the above databases that would extend the assemblage. Further, 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%.
The novel predicted polypeptides (including proteins) encoded by the novel polynucleotides (SEQ m NO: 303-399) of the present invention, and their corresponding translation start and stop nucleotide locations to each of SEQ 1D NO: 303-399 were obtained using one of two methods. Polypeptides were obtained by using a software program called FASTY (available from http://fasta.bioch.vir~inia,edu) which selects a polypeptide based on a comparison of the translated novel polynucleotide to known polynucleotides (W.R. Pearson, Methods in Enzymology, 183:63-98 (1990), herein incorporated by reference).
Alternatively, polypeptides were obtained by using a software program called GenScan for human/vertebrate sequences (available from Stanford University, Office of Technology Licensing) that predicts the polypeptide based on a probabilistic model of gene structure/compositional properties (C.
Burge and S. Marlin, J. Mol. Biol., 268:78-94 (1997), incorporated herein by reference).
Method C refexs to a polypeptide obtained by using a Hyseq proprietary software program that translates the novel polynucleotide and its complementary strand into six possible amino acid sequences (forward and reverse frames) and chooses the polypeptide with the longest open reading frame.

5.3 EXAMPLE 3 Novel Nucleic Acids The novel nucleic acids of the present 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 (Hyseq's database containing EST sequences, dbEST, gb pri, and UniGene) 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%.
Using PHRAP (LTniv. 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 colons were corrected by hand editing. During editing, the sequences were checked using FASTY and/or BLAST against Genebank (i.e., dbEST, gb pri, UniGene, and Genpept) and the Geneseq (Derwent). Other computer programs which may have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). The full-length nucleotide and amino acid sequences, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NO: 1-302.
SEQ ID NO: 1-16 were classified as secreted according to their predicted cellular localization using the Pfam software program (Sonnhammer et al., Nucleic Acids Res., Vol.
26(1) pp. 320-322 (1998), and http://pfam.wustl.eduJ, herein incorporated by reference).
SEQ ID NO: 17-107 were determined to contain signal peptide sequences and their cleavage sites using Neural Network SignalP V1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark). The process for identifying prolcaryotic 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. l, pp. 1-6 (1997), incorporated herein by reference.
A maximum S score and a mean S score, as described in the Nielson et al reference, was obtained for the polypeptide sequences.

SEQ ID NO: 108-151 were determined to be secreted polypeptides using a proprietary algorithm, SeqLocTM (Hyseq Inc.). SeqLocTM classifies the proteins into three sets of locales: intracellular, membrane, or secreted. This prediction is calculated using maximum likelihood estimation of three characteristics of each polypeptide, 1) percentage of cysteine residues, 2) Kyte-Doolittle scores for the first 20 amino acids of each protein (J.
Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference), and 3) Kyte-Doolittle scores to calculate the longest hydrophobic stretch (LHS) of the said protein (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference). The LHS is calculated by finding the stretch~of 20 amino acid residues in tl2e protein that have the highest sum of Kyte-Doolittle hydrophobicity values.
Table 1 shows the various tissue sources of SEQ ID NO: 1-151.
The homologs for polypeptides SEQ 1D NO: 152-302, that correspond to nucleotide sequences SEQ ID NO: 1-151 were obtained by a BLASTP search against Genpept release 124 arid Geneseq (Derwent) release 200117 and against Genpept release 129 and Geneseq (Derwent) release (July 18, 2002). The results showing homologues for SEQ m NO: 152-302 from Genpept 124 axe shown in Table 2A. The results showing homologues for SEQ ID
NO: 152-302 from Genpept 129 are shown in Table 2B.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al., J.
Comp. Biol., Vol. 6, 219-235 (1999), http://motif.stanford.edulematrix-search/
herein incorporated by reference), all the polypeptide sequences were examined to determine whether they had identifiable signature regions. Scoring matrices of the eMatrix software package are derived from the BLOCKS, PRINTS, PFAM, PRODOM, and DOMO
databases. Table 3 shows the accession number of the homologous eMatrix signature found in the indicated polypeptide sequence, its description, and the results obtained which include accession number subtype; raw score; p-value; and the position of signature in amino acid sequence.
Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res., Vol.
26(1) pp. 320-322 (1998) herein incorporated by reference) alI the polypeptide sequences were examined for domains with homology to certain peptide domains. Table 4A
shows the name of the Pfam model found, the description, the e-value and the Pfam score for the identified model within the sequence as described in United States priority application serial number 60/323,349, filed September 18, 2001, herein incorporated by reference in its entirety. Table 4B shows the name of the Pfam model found, the description, the e-value and the Pfam score for the identified model within the sequence using Pfam version 7.2.
Further description of the Pfam models can be found at http://pfam.wustLedul.
The GeneAtlasr"' software package (Molecular Simulations Inc. (MSI), San Diego, CA) was used to predict the three-dimensional structure models for the polypeptides encoded by SEQ D.? NO: 1-151 (i.e. SEQ ID NO: 152-302). Models were generated by (1) PSI-BLAST which is a multiple alignment sequence profile-based searching developed by Altschul et al, (Nucl. Acids. Res. 25, 3389-3408 (1997)), (2) High Throughput Modeling (HTM) (Molecular Simulations Inc. (MSI) San Diego, CA,) which is an automated sequence and structure searching procedure (http://www.msi.com/), and (3) SeqFoldT"' which is a fold recognition method described by Fischer and Eisenberg (J. Mol. Biol. 209, 779-791 (1998)).
This analysis was carned out, in part, by comparing the polypeptides of the invention with the lcnown NMR (nuclear magnetic resonance) and x-ray crystal three-dimensional structures as templates. Table 5 shows: "PDB ID", the Protein DataBase (PDB) identifier given to template structure; "Chain ID", identifier of the subcomponent of the PDB
template structure; "Compound Information", information of the PDB template structure and/or its subcomponents; "PDB Function Annotation" gives function of the PDB template as annotated by the PDB files (http:/www.rcsb.or~/PDB/); start and end amino acid position of the protein sequence aligned; PSI-BLAST score, the verify score, the SeqFold score, and the Potentials) of Mean Force (PMF). The verify score is produced by GeneAtlasT"' software (M51), is based on Dr. Eisenberg's Profile-3D threading program developed in Dr. David Eisenberg's laboratory (US patent no. 5,436,850 and Luthy, Bowie, and Eisenberg, Nature, 356:83-85 (1992)) and a publication by R. Sanchez and A. Sali, Proc. Natl.
Acad. Sci. USA, 95:13597-12502. The verify score produced by GeneAtlas normalizes the verify score for proteins with different lengths so that a unified cutoff can be used to select good models as follows:
Verify score (normalized) _ (raw score -1/2 high score)/(1/2 high score) The PFM score, produced by GeneAtlasT'" software (MSI), is a composite scoring function that depends in part on the compactness of the model, sequence identity in the aligmnent used to build the model, pairwise and surface mean force potentials (MFP). As given in table 5, a verify score between 0 to I.O, with 1 being the best, represents a good model. Similarly, a PMF score between 0 to 1.0, with 1 being the best, represents a good model. A SeqFoldTM score of more than 50 is considered significant. A good model may also be determined by one of skill in the art based all the information in Table 5 taken in totality.
Table 6 shows the position of the signal peptide in each of the polypeptides and the maximum score and mean score associated with that signal peptide using Neural Network SignaIP V 1.1 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 Brunalc, and Gunnar von Heijne in the publication " Identification of proltaryotic and eulcaryotic signal peptides and prediction of their cleavage sites" Protein Engineering, Vol.
10, no. l, pp. 1-6 (1997), incorporated herein by reference. A maximum S score and a mean S score, as described in the Nielson et al reference, was obtained for the polypeptide sequences.
Table 7 correlates each of SEQ ID NO: 1-151 to a specific chromosomal location.
Table 8 is a correlation table of the novel polynucleotide sequences SEQ ID
NO: 1-151, their corresponding polypeptide sequences SEQ ID N~: 152-302, their corresponding priority contig nucleotide sequences SEQ II7 NO: 303-399, their corresponding priority contig polypeptide sequences SEQ ID NO: 400-496, and the US serial number of the priority application (all of which are herein incorporated in their entirety), in which the contig sequence was filed.
Table 9 is a correlation table of the novel polynucleotide sequences SEQ ID
NO: 1-151, the novel polypeptide sequences SEQ ID NO: 152-302, and the corresponding SEQ m NO in which the sequence was filed in priority US application bearing serial number 60/323,349, filed September 18, 2001.

Table 1 Tissue Origin Library/RNA HYSEQ Library SEQ ID NOS:

Source Name adrenal gland Clontech ADR002 2 9 20-21 26 30 40 adult bladder Invitrogen BLD001 27 54 96 114 116 adult brain BioChain ABR012 96 adult brain BioChain ABR013 7I-72 96 adult brain Clontech ABR001 46-47 S4 78 96 142 adult brain Clontech ABR006 7 9 1S 31 54 61 63 adult brain Clontech ABR008 6-7 9 17 21 26-27 adult brain Clontech ABRO11 8 96 adult brain GIBCO AB3001 7 20 22 26-27 49 adult brain GIBCO ABD003 3 7 9 18 21-22 26 adult brain Invitrogen ABR014 18 54 96 adult brain Invitrogen ABRO15 7 26 30 71-72 96 adult brain Invitrogen ABR016 27 32 109 125 adult brain Invitrogen ABT004 9 24-25 27-28 40 adult cervix BioChain CVX001 2-3 6 10 18 20-21 adult colon Invitrogen CLN001 3 20 24 26-28 32 adult heart GIBCO AHR001 18 24 26-28 31-32 adult kidney GIBCO AKD001 4-5 18 20-22 24 26-27 adult kidney Invitrogen AKT002 4-5 15 18 21 26-27 adult liver Clontech ALV003 24 31 40 46-47 49 adult liver Tnvitrogen ALV002 10 17 20 24 27-28 adult lung GIBCO ALG001 4-5 18 27 49 96 111 adult ovary Invitrogen AOV001 2-3 6 17 I9 21-22 adult placentaClontech APL001 27 44 59 96 148 adult s leen Clontech SPLc01 7 18 9S-97 139-140 adult spleen GIBCO ASP001 18 20 24 26-27 29 adult testis GIBCO ATS001 9 24 27 32 35 53 Table 1 Tissue Origin Library/RNA HYSEQ Library SEQ ID NOS:

' Source Name bone marrow Clontech BMD001 1 3 18-19 21 23 26-27 bone marrow Clontech BMD007 42 bone marrow GF BMD002 1 12-13 18 21 24 Burkitt's LymphomaCA-46 DGD001 72 cultured Stratagene ADP001 20 24 27 49 119 131 preadipocytes endothelial Stratagene EDT001 I 6 17 20-21 24-27 cells 29-31 38-39 fetal brain Clontech FBR001 2 21 S4 67 106 fetal brain Clontech FBR004 2 19 54 115 fetal brain Clontech FBR006 2 7 17 21 24-27 29 fetal brain GIBCO HFB001 17 20-21 25 27 29-30 fetal brain Invitro en FBT002 6 9 26 109 133 136 fetal heart Invitrogen FHR001 6 17-18 20 24 26-27 fetal kidney Clontech FKD001 2 65 110 fetal kidney Clontech FKD002 6-7 17-18 24 29 42 fetal kidney Invitro en FKD007 6 fetal liver Clontech FLV002 24 42 110 141 fetal liver Clontech FLV004 18 23 29 31 40 42 fetal liver Invitro en FLV001 23-24 27-28 38 42 fetal liver-spleenColumbia UniversityFLSOOI 2-3 6 17-19 21 23-32 fetal liver-spleenColumbia UniversityFLS002 2-6 17-18 20-30 34 SO

fetal liver-spleenColumbia UniversityFLS003 23 25-28 39 42 46-47 fetal lung Clontech FLG001 38 57 84 109 150 fetal lung Invitrogen FLG003 27-28 34 84 96 109 fetal muscle Invitro en FMS001 105 131 fetal muscle Invitrogen FIvIS002 7 21 24 42 50 64 fetal skin Invihogen FSK001 3 6 20 26-27 32 40 fetal skin Invitrogen FSK002 2 6 8 17 27 29-30 _ 50 52 60 92 95 100-101 fetal spleen BioChain FSP001 18 ~

Table 1 Tissue Origin Library/RNA HYSEQ Library SEQ ID NOS:

Source Name fibroblast Strata ene LFB001 21-22 27 38 46-47 induced neuron-cellsStrata ene NTD001 21 25-26 45 54 142 infant brain Columbia UniversityIB2002 2 6-7 19-21 25-30 infant brain Columbia UniversityIB2003 21 25 27 30-31 40 infant brain Columbia UniversityIBM002 20 infant brain Columbia UniversityIBS001 34 48 115 150 leukocyte Clontech LUC003 1 18 27 51 64 71-72 leukocyte GIBCO LUC001 1 12-13 18 20-21 lung 21-22 27 38 46-47 lung tumor Invitrogen LGT002 4-6 11 17-18 21-22 lymphocytes ATCC LPC001 18 20 25 28 34 38 macrophage Invitro en HMP001 18 21 27 38 67 97 mammary gland Invitrogen MMG001 3 10 17 20 24 26-28 82 87 95-97 109-l melanoma from-Clontech MEL004 1 21 24 26 31-32 cell-line-ATCC-#CRL-1424 mixture of various vendorsSUP002 18 20 27 31 40 43 tissues/mRNA 122 141 146 mixture of various vendorsSUP005 18 46-47 54 112 tissueslmRNA

mixture of various vendorsSUP008 7 29 99 tissues/mRNA

mixture of various vendorsSUP009 96 tissueslmRNA

mixed EST clones CGd010 3 42 132 mixed BioChain/Invitrogen/CGd011 3162 Clontech mixed BioChain/Invitrogen/CGd012 ~ 3 7 10 18 20-21 26 Clontech 62 64 68 71-72 75-76 neuronal cellsStrata ene NTU001 21 25-26 40 42-43 pituitary glandClontech PIT004 9 14 21 24 32 43 placenta Clontech PLA003 18 32 147 150 placenta Invitro en APL002 25-26 96 rostate Clontech PRT001 15 17 22 27 31 38 rectum Invitrogen REC001 3 20 38 40 109 retinoic acid-Stratagene NTR001 8 30 40 induced-neuronal-cells salivary glandClontech SAL001 3 21 27 43 71-72 Table 1 Tissue OriginLibrary/RNA HYSEQ Library SEQ ID NOS:

Source Name skeletal muscleClontech SKM001 15 21 24 27 34 10 _ small intestineClontech S1N001 _ _ _ spinal cord Clontech SPC001 _ _ stomach Clontech STO001 19 22 68 106 111 thalamus Clontech THA002 24 28 40 42 49 54 thymus Clontech THM001 6 21 thymus Clontech THMc02 _ thyroid glandClontech THR001 _ trachea Clontech TRCOOI 20 26-27 38 81 143 umbilical BioChain FUC001 3 6 21 24 27-28 40 cord 42 45 48 50 uterus Clontech UTR001 20 43 young liver GIBCO ALV001 3-5 20 27 42 46-47 *The 16 tissue/mRNAs and their vendor sources are as follows: 1 ) Normal adult brain mRNA
(Invitrogen), 2) Normal adult kidney mRNA (Invitrogen), 3) Normal fetal brain mRNA (Invitrogen), 4) Normal adult liver mRNA (Invitrogen), 5) Normal fetal kidney mRNA (Invitrogen), 6) Normal fetal liver mRNA
(Invitrogen), 7) normal fetal skin mRNA (Invitrogen), 8) human adrenal gland mRNA (Clontech), 9) Human bone marrow mRNA (Clontech), 10) Human leukemia lymphoblastic mRNA (Clontech), 11) Human thymus mRNA (Clontech), I2) human lymph node mRNA (Clontech), 13) human so\spinal cord mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA (BioChain), 16) human conceptional umbilical cord mRNA (BioChain).

Table 2A
SE(~ AccessionSpecies Description Score ID No. Identity NO:

153 1159657 Ascaris collagen 240 51 suum 153 g11280073CaenorhabditisSimilar to cuticle collagen238 51 ele ans 153 g12746786CaenorhabditisSimilar to cuticular collagen;253 53 coded for by elegans C. elegans cDNA yk68e1 I.S;
coded for by C. ele ans cDNA yk68e11.3 I54 AAY72715 Homo SapiensHFICU08 clone human attractin-like1855 77 rotein.

154 AAY72716 Homo sapiensHMEIR04 clone human attractin-like1779 94 protein.

154 g13449294Rattus MEGF6 1717 67 norve icus 155 g134659 Homo sapiensHuman mRNA for macrophage 219 97 inflammatory protein-2a1 ha (MIP2al ha).

155 g1183627 Homo SapiensHuman gro-beta mRNA, com 219 97 lete cds.

155 g1183629 Homo SapiensHuman cytokine (GRO-beta) 219 97 mRNA, complete cds.

156 g134659 Homo SapiensHuman mRNA for macrophage 341 97 inflammatory rotein-2alpha (MIP2alpha).

156 g1183627 Homo sapiensHuman gro-beta mRNA, com 341 97 fete cds.

156 g1183629 Homo sapiensHuman cytokine (GRO-beta) 341 97 mRNA, com lete cds.

157 g135087 Homo SapiensHuman mRNA for neurite outgrowth-757 97 romoting protein.

157 g1182651 Homo sa Human midkine mRNA, complete757 97 tens cds.

157 g1219929 Homo sapiensHuman midkine ene, complete757 97 cds.

I58 g112330704Mus musculuscell recognition molecule 6099 86 158 g16694278Homo Sapienscell recognition molecule 3241 48 Caspr2 (CASPR2) mRNA, complete cds.

158 g113624214Homo Sapienscontactin-associated protein3241 48 2 (CNTNAP2) mRNA, com fete cds.

159 g13449306Homo sa mRNA for MEGF7, artial cds.4182 99 tens 159 g16681362Rattus MEGF7 2589 97 norve icus 159 1438007 Gallus al ha-2-macro lobulin rece 2141 40 gallus for 160 g112654463Homo Sapienschromogranin A (parathyroid2334 100 secretory protein 1), clone MGC:2080, mRNA, complete cds.

160 g113623667Homo Sapienschromogranin A (parathyroid2334 100 secretory protein 1), clone MGC:1781, mRNA, com fete cds.

160 g1180527 Homa SapiensHuman chromogranin A mRNA, 2334 100 complete cds.

161 g131419 Homo sapiensH.sa tens mRNA for fibulin-1441 33 C.

161 AAR11150 Homo sapiensFibulin C. 441 33 161 AAW27600 Homo SapiensHuman fibulin a 1 isoform 441 33 (variant C).

162 g11732121Homo SapiensHuman cartilage matrix protein1786 99 (CMP) ene, exon 8.

162 AAR57349 Homo sa Human cartilage matrix rotein.1786 99 tens 162 g1180654 Homo SapiensHuman cartilage matrix protein1725 99 (CMP) mRNA, exons 3-8.

163 g11754611Homo SapiensDNA for SCM-lalpha precursor,483 95 complete _ cds.

163 gi895847 ~ omo SapiensH.sapiens mRNA for ATAC 483 95 - protein.

Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

163 gi902002 Homo SapiensHuman lymphotactin precursor483 95 mRNA, com fete cds.

164 gi1754611Homo SapiensDNA for SCM-lalpha precursor,545 100 complete cds.

164 1895847 Homo sa H.sapiens mRNA for ATAC 545 100 iens protein.

164 g1902002 Homo SapiensHuman lymphotactin precursor545 100 mRNA, com lete cds.

165 g1575501 Homo Sapiensthyrotropin beta-subunit 767 100 (TSHB) gene, exon 3.

165 g1339998 Homo SapiensHuman thyrotropin beta 767 100 (TSH-beta) submit gene, exons 2 and 3.

165 g1340002 Homo SapiensHuman thyrotropin beta 767 100 subunit gene, exons 2 and 3.

I66 AAY97697 Homo sa Human EGFH2 roteiii se 615 99 iens uence.

166 g14530130Mus musculusneuregulin-4 short isoform479 74 166 AAY05451 Homo SapiensHuman here ulin-like factor111 39 se uence.

167 AAB19923 Homo SapiensHuman interleukin-1 Hy2 582 71 (extended form, partial se uence).

167 AAB19924 Homo sa Human interleukin-1 Hy2 582 71 iens (long version .

167 AAB19922 Homo sa Human interleukin-1 Hy2 580 74 iens (short version).

168 AAY28919 Homo SapiensHuman re ulatory protein 1641 81 HRGP-5.

168 g114211570Homo Sapienscega mRNA for conserved 1633 80 ERA-like GTPase, com lete cds.

168 g13415109Homo SapiensEra GTPase A protein (HERA-A)1633 80 mRNA, artial cds.

169 g132433 Homo SapiensHuman mRNA for hematopoetic459 100 proteogl can core protein.

169 g1190420 Homo sapiensHuman secretory granule 459 100 proteoglycan eptide core mRNA, complete cds.

169 g1338062 6,7,8 Human (clone lambda-PG) 459 100 secretory granule 170 AAY73496 Homo sapiensHuman secreted protein 467 92 clone ys 10-1 protein se uence SEQ ID
N0:214.

170 g112751092Homo SapiensPNAS-123 mRNA, complete 51 42 cds.

171 AAB54178 Homo SapiensHuman pancreatic cancer 1025 99 antigen protein se uence SEQ ID N0:630.

171 g17321824Drosophila out at first 510 38 melanogaster 171 g12443448Drosophila out at first 508 39 virilis 172 g112001958Homo Sapiensclone OOSa05 My009 protein1335 98 mRNA, com fete cds.

172 AAB88335 Homo SapiensHuman membrane or secretory1331 98 protein clone PSEC0045.

172 AAB57113 Homo SapiensHuman prostate cancer antigen1308 96 protein se uence SEQ ID N0:1691.

173 g112803251Homo SapiensTat-interacting protein 1245 100 (30kD), clone MGC:1318, mRNA, complete cds.

173 12618733 Homo sa CC3 (CC3) mRNA, complete 1239 99 iens cds.

173 AAW 12697Homo SapiensCC3 polypeptide derived 1239 99 from small cell carcinoma.

174 AAB39216 Homo sapiensHuman secreted protein 1109 99 sequence encoded b ene 38 SEQ ID N0:96.

174 AAB24063 Homo SapiensHuman PR0809 protein sequence652 99 SEQ ID

Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

N0:23.

174 AAY66691 Homo sa Membrane-bound rotein PR0809.652 99 iens 175 gi4105190Homo Sapiensperoxisomal short-chain 719 80 alcohol dehydrogenase (SCAD-SRL) mRNA, com lete cds.

175 gi12804321Homo Sapiensperoxisomal short-chain 718 80 alcohol dehydrogenase, clone MGC:4052, mRNA, complete cds.

175 AAY96729 Homo sa PR01800, a Hep27 homolo 718 80 iens ue.

176 gi13111903Homo Sapiensclone IMAGE:3357127, mRNA, 166499 partial cds.

176 gi11993865Mus musculuscdk3-binding protein ik3-1 871 64 176 gi11320875Mus musculuscdk-binding protein 866 64 177 gi7020622Homo sa cDNA FLJ20487 fis, clone 755 92 iens KAT08245.

177 AAB36622 Homo SapiensHuman FLEXHT-44 protein 702 92 sequence SEQ

ID N0:44.

177 AAG01349 Homo sa Human secreted protein, 567 100 iens SEQ ID NO: 5430.

178 gi289402 Bos tauruscathepsin B 114379 178 gi1524328Rattus cathepsin b 111676 norvegicus 178 1309152 Mus musculuscathepsin B 110776 179 AAW75196 Homo SapiensHuman secreted protein encoded330 98 by gene 1 clone HCEAB46.

179 1553886 Mus musculusal ha-1 a IV collagen SO 37 179 AAY14488 Homo SapiensFragment of human secreted 63 60 protein encoded by gene 33.

180 g113623271Homo SapiensSimilar to RIKEN cDNA 2600005P051332100 gene, clone MGC:11321, mRNA, complete cds.

180 AAG00724 Homo SapiensHuman secreted rotein, SEQ 478 100 ID NO: 4805.

180 11762316 Mus musculusuracil-DNA glycosylase 66 35 181 114336711Homo sa 16 13.3 se uence section 103996 iens 3 of 8.

181 AAY88278 Homo sa Human TANGO 188 protein. 103996 iens 181 AAA39947_Homo SapiensHuman TANGO 188 cDNA. 103596 aal 182 g11922287Homo sapiensH.sapiens gene encoding 117499 enoyl-CoA

hydratase, exon 1(and 'oined CDS).

182 g114286220Homo Sapiensenoyl Coenzyme A hydratase,117499 short chain, 1, mitochondrial, clone MGC:1513, mRNA, complete cds.

182 g1433413 Homo SapiensmRNA for mitochondrial short-chain115797 enoyl-CoA hydratase, com fete cds.

183 g112052902Homo SapiensmRNA; cDNA DKFZp564I2178 244298 (from clone DKFZ 564I2178); com lete cds.

183 17020859 Homo sa cDNA FLJ20628 fis, clone 243697 iens KAT03903.

183 AAG01972 Homo sa Human secreted rotein, SEQ 586 92 iens ID NO: 6053.

184 AAY86443 Homo SapiensHuman gene 42-encoded protein739 100 fragment, SEQ ID N0:358.

184 AAB33773 Homo SapiensHuman secreted protein BLAST62 52 search rotein SEQ ID NO: 117.

184 g1841378 SaccharomycesGpi2p 80 34 cerevisiae 185 AAA64426_Homo SapiensDNA encoding a human TANGO 968 87 aal variant pol a tide.

Table 2A
SE(~ AccessionSpecies Description Score ID No. Identity NO:

185 AAB08641 Homo sa A human TANGO 223 01 epode.968 87 iens 185 AAB08658 Homo sa A human TANGO 223 variant 968 87 iens of a tide.

186 AAY12948 Homo SapiensAmino acid sequence of a 194 90 human secreted a tide.

186 gi329669 Hepatitis S/S protein 37 61 B

virus 186 AAB56409 Homo SapiensHuman prostate cancer antigen37 72 protein se uence SEQ ID N0:987.

187 gi10440992Homo sa Bv8 rotein (BV8 mRNA, aroal621 97 iens cds.

187 gi6524947Mus musculusBv8 variant 1 recursor 556 86 187 gi9957625Mus musculussecretory rotein BV8 556 86 188 AAY91443 Homo SapiensHuman secreted protein sequence1029 100 encoded by ene 39 SEQ ID N0:164.

188 110439951Homo sa cDNA: FLJ23311 fis, clone 128 27 iens HEP11681.

188 g15917666Zea ma extensin-like rotein 118 27 s 189 g14929557Homo sa CGI-44 rotein mRNA, complete1347 96 iens cds.

189 AAB75509 Homo SapiensHuman secreted protein sequence1345 100 encoded by ene 4 SEQ ID N0:63.

189 g15738222Mus musculusflavo-bindin rotein 1163 85 190 AAB34624 Homo sapiensHuman secreted protein sequence260 100 encoded by ene 48 SEQ ID N0:108.

190 g114009530unculturedparticulate methane monooxygenase-like62 30 bacterium 190 g1472902 Caenorhabditiscarrier protein (c1) 66 33 elegans 191 g113477197Homo SapiensSimilar to quinolinate 955 87 phosphoribosyltransferase (nicotinate-nucleotide pyrophosphorylase (carboxylating)), clone MGC:12951, mRNA, complete cds.

191 g11060907Homo SapiensmRNA fox quinolinate phosphoribosyl941 80 transferase, com Iete cds.

191 g1836802 SaccharomycesYFR047C 393 46 cerevisiae 192 AAY38394 Homo SapiensHuman secreted protein encoded213 100 by gene No. 9.

192 g119358 LycopersiconTDRS 41 50 esculentum 192 g13879131Caenorhabditiscontains similarity to Pfam49 54 domain:

elegans PF01461 (7TM chemoreceptor), Score=-54.8, E-value=4.7e-12, N=1 193 g1555594 Homo SapiensHuman dihydroorotate dehydrogenase1602 100 mRNA, 3' end.

193 14379422 Ratlus dihydroorotate dehydro enase1443 89 rattus 193 18784074 Mus musculusdihydroorotate deh dro enase1453 89 194 AAW 17971Homo SapiensRAC protein kinase C-terminal132 83 binding rotein C-terminal re ion.

194 11580714 EscherichiaYbaW 83 32 coli 194 g1496680 SaccharomycesA-270 protein 89 20 cerevisiae 195 g113436389Homo Sapienssecreted protein, acidic, 1664 100 cysteine-rich (osteonectin), clone MGC:3651, mRNA, com fete cds.

195 g114124970Homo Sapienssecreted protein, acidic, 1664 100 cysteine-rich TahlP 7.A
SEQ AccessionSpecies Description Score ID No. Identity NO:

(osteonectin), clone MGC:15881, mRNA, com lete cds.

195 gi29462 Homo SapiensHuman mRNA for extracellular1664 100 matrix rotein BM-40.

196 AAB27587 Homo sa Human secreted protein 689 100 iens SEQ ID NO: 88.

196 gi14250466Homo SapiensSimilar to RIKEN cDNA 5530601I19610 100 gene, clone MGC:9743, mRNA, complete cds.

196 gi7106878Homo sa HSPC244 154 40 iens 197 gi181356 Homo sapiensHuman cytochrome P450IIE1 2307 100 (ethanol-inducible) ene, com lete cds.

197 gi181360 Homo SapiensHuman cytochrome P-450j 2307 100 mRNA, com fete cds.

197 AAR72362 Homo SapiensHuman cytochrome P450 molecular2307 100 species 2E1 rotein.

198 gi181356 Homo SapiensHuman cytochrome P450IIE1 1477 100 (ethanol-inducible) ene, complete cds.

198 gi181360 Homo SapiensHuman cytochrome P-450j 1477 100 mRNA, com lete cds.

198 AAR72362 Homo SapiensHuman cytochrome P450 molecular1477 100 species 2E1 rotein.

199 13387914 Homo sa clone 24688 cotel mRNA, 1634 100 iens artial cds.

199 g12564916Homo Sapiensclk2 kinase (CLK2), propinl,1112 85 cotel, glucocerebrosidase (GBA), and metaxin genes, complete cds; metaxin pseudogene and glucocerebrosidase pseudogene; and thrombospondin3 (THBS3) gene, partial cds.

199 113938585Homo Sapiensclone MGC:4509, mRNA, complete1112 85 cds.

200 AAW80748 Homo SapiensHuman mitochondrial chaperone98 100 protein (Hmt-GrpE).

200 g111139093Homo SapiensGrpE-like protein cochaperone93 100 mRNA, nuclear gene for mitochondrial product, partial cds.

200 AAY07463 Homo SapiensFragment of human tumour 71 37 suppressor protein TS10 23.3.

201 g114456615Homo SapiensPIG-T mRNA for phosphatidyl1747 96 inositol glycan class T, complete cds.

201 AAB88407 Homo SapiensHuman membrane or secretory1747 96 protein clone PSEC0163.

201 AAY66731 Homo sa Membrane-bound rotein PR01379.1730 96 iens 202 AAV81204-Homo sapiensHuman CD7 cDNA. 1129 96 aal 202 g114424540Homo sapiensCD7 antigen (p41), clone 1128 96 MGC:16622, mRNA, complete cds.

202 g1732757 Homo sa Human mRNA for CD7 anti 1128 96 iens en ( 40).

203 g112483900Rattus zinc forger protein HIT-4 1000 59 norve icus 203 g11769491Homo SapiensHuman kruppel-related zinc552 38 forger protein (ZNF184) mRNA, artial cds.

203 15679576 Homo sa mRNA for zinc fin er 41 554 39 iens (ZNF41 ene).

204 AAY86455 Homo SapiensHuman gene 45-encoded protein417 100 fragment, SEQ ID N0:370.

204 AAY86259 Homo sa Human secreted rotein HAGDI35,237 100 iens SEQ ID

Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

N0:174.

204 AAY76156 Homo sapiensHuman secreted protein 62 33 encoded by gene 33.

205 gi56752 Rattus Neurofilament protein middle3394 82 (NF-M) norve icus 205 1297529 Mus musculusNF-M 3393 82 205 g1205688 Ratlus NF-M protein 3381 82 norve icus 206 17576448 Homo SapiensmRNA for ALEX3, com fete 733 41 cds.

206 g113528786Homo SapiensALEX3 protein, clone MGC:12199,733 41 mRNA, com fete cds.

206 AAY17220 Homo sa Human secreted rotein (clone732 41 iens fj283-11).

207 AAY17227 Homo SapiensHuman secreted rotein (clone500 31 yal-1).

207 AAY94938 Homo SapiensHuman secreted protein 227 25 clone ye78-1 rotein se uence SEQ ID
N0:82.

207 g112831176Agelaius gamma filamin protein 217 23 phoeniceus 208 AAB38568 Homo SapiensHuman secreted protein 330 100 sequence encoded by ene 47 SEQ ID N0:105.

208 g110802913ThraustochytriuNADH dehydrogenase subunit54 33 m aureum 208 g113785657Mus musculuscandidate taste rece for 77 36 209 AAY87298 Homo SapiensHuman signal peptide containing57 38 protein HSPP-75 SEQ ID N0:75.

209 g1833941 Felis NADH dehydrogenase 1 {cytoplasxnic54 44 catus=domesticclone pCmt.l2}

cats, lymphocytes, fibroblasts, Peptide Mitochondria) Partial, 115 as 209 g14261871Felis catusNADH dehydrogenase 1 54 44 210 g113477235Homo SapiensSinular to RII~EN cDNA 917 100 gene, clone MGC:13061, mRNA, complete cds.

210 g1458726 Homo SapiensHuman mRNA for estrogen 230 37 responsive fin er rotein, complete cds.

210 11088467 Mus musculusestro en-res onsive finger230 32 rotein 211 g113785516Rattus RGPR-p117 720 64 norvegicus 211 g16634121Drosophila BAB-I protein 95 37 melanogaster 211 g15420389Leishmania proteophosphoglycan 131 22 ma'or 212 g11778061Saccharomycestranscription/repair factor83 31 TFIIH sub~uut cerevisiae Ttb3 212 g1927727 SaccharomycesTfb3p: TFIIH subunit Ttb3;83 31 cerevisiae 212 AAG01831 Homo sa Human secreted protein, 75 26 iens SEQ ID NO: 5912.

213 AAR60619 Homo sa Carcinoembr onic antigen 456 34 iens glyco rotein.

213 AAB43687 Homo SapiensHuman cancer associated 456 34 protein sequence SEQ ID N0:1132.

~ AAR98519 Ghimeric Immunogenic carcinoembryonic456 34 213 ~ Homo ~ antigen. ~
~

Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

Sapiens 214 g18272464Homo Sapienshuman endogenous retrovirus226 42 W gagC3.37 G a (ga ) ene, com lete cds.

214 AAB07704 Homo SapiensProtein encoded by the endogenetic226 42 fragment of HERV-W.

214 g15726238multiple gag polyprotein 218 42 sclerosis associated retrovirus element 215 g1338766 Homo SapiensHuman T-cell receptor rearranged967 72 alpha-chain V-region (V-D-J) mRNA, complete cds.

215 g13089419Homo SapiensSSCl l rearranged T cell 936 70 receptor alpha chain TCRAV 17) gene, com lete cds.

215 AAY69995 Homo sapiensHuman receptor-associated 919 72 protein from W cyte clone 1361202.

216 g1456384 Blastocrithidiaapocytochrome B 41 50 culicis 2I6 AAB20695 Homo SapiensPolymeric immunoglobulin 60 55 receptor bindin domain eptide SEQ
ID NO:I 1.

216 g114030701ArabidopsisAt2g28370/T1B3.11 74 26 thaliana 217 g1950422 Homo SapiensHuman cell surface glycoprotein1217 89 (CD44) gene, exon I8, 3' end of short tailed isoform.

217 g1950420 Homo SapiensHuman cell surface glycoprotein1217 89 (CD44) ene, 3' end of lon tailed isoform.

217 g11101786Homo SapiensHuman cell surface glycoprotein1214 89 mRNA, com lete cds.

218 AAY86222 Homo sapiensHuman secreted protein HCUDD24,233 83 SEQ

ID NO:137.

2I8 AAY86355 Homo SapiensHuman gene 8-encoded protein142 69 fragment, SEQ ID N0:270.

218 g1457140 Lactococcusabi416 68 28 lactis 219 1387012 Homo sa Human pe sino en ene, exon 1145 99 iens 9.

219 g1387013 Homo SapiensHuman pepsinogen A (I5.0) 1148 99 gene, exon 9, clone c HGP2.

219 g138069 Macaca re rope sino en A-2/3 1091 94 fuscata 220 AAG00471 Homo sa Human secreted protein, 285 93 iens SEQ ID NO: 4552.

220 g1298489 Papio SP-10=intraacrosomal protein136 34 f alternatively hamadryas spliced}

220 g1298488 Papio SP-10=intraacrosomal protein136 34 hamadr as 221 12315138 Mus musculusIGFBP-like protein 1170 77 221 AAR79102 Homo sa Prosta landin I2 (PGI2) 571 45 . iens rodn. romoter.

221 g1861521 human, prostacyclin-stimulating 570 45 factor cultured diploid fibroblast cells, mRNA, 1124 nt]. [Homo sa iens 222 g1339010 Homo SapiensHuman T-cell receptor active1063 80 beta-chain Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

mRNA, com fete cds.

222 gi3002931Homo SapiensT cell receptor beta chain 1051 79 (TCRBV2S1-TCRBJ2S5) mRNA, com fete cds.

222 gi1100182Homo SapiensT-cell receptor beta (TCRB)927 70 mRNA (HLA-A1, 24; B7, 8; DR 1, 3 , com Iete cds.

223 gi339010 Homo SapiensHuman T-cell receptor active1305 83 beta-chain mRNA, complete cds.

223 gi3002931Homo SapiensT cell receptor beta chain 1269 81 (TCRBV2S1-TCRBJ2S5) mRNA, complete cds.

223 gi1100182Homo SapiensT-cell receptor beta (TCRB)1169 75 mRNA (HLA-A1, 24; B7, 8; DR 1, 3), complete cds.

224 AAY25457 Homo SapiensHuman secreted protein 5 706 98 derived from extended cDNA.

224 AAY36051 Homo SapiensExtended human secreted 706 98 protein sequence, SEQ ID NO. 436.

224 AAY35888 Homo SapiensExtended human secreted 706 98 protein sequence, SEQ ID NO. 25.

225 AAB38361 Homo SapiensHuman secreted protein encoded204 100 by gene 41 clone HTOHG09.

225 1525302 Bacillus orfY 60 55 subtilis 225 12635283 Bacillus ysxD 60 55 subtilis 226 1623157 Macaca T-cell rece for al ha 708 85 mulatta 226 1555719 Macaca T-cell receptor al ha chain651 88 mulatta 226 g1339419 Homo SapiensHuman TCR variable region 630 90 Va30 subfamily gene (VA30, JA, CA segments), 5' end.

227 AAY69995 Homo SapiensHuman receptor-associated 1010 74 protein from . Incyte clone 1361202.

227 g11223888synthetic T cell receptor alpha chain998 72 construct 227 g1338766 Homo SapiensHuman T-cell receptor rearranged943 68 alpha-chain V-region (V-D-J) xnRNA, complete cds.

229 g15305335Mycobacteriumproline-rich mucin homolog 221 33 tuberculosis 229 g15734705ArabidopsisF24J5.4 193 32 thaliana 229 g1535586 Medicago proline rich protein I9I 36 sativa 230 AAB33444 Homo SapiensHuman PR01346 protein IJNQ7012298 100 SEQ ID

NO:152.

230 AAY66727 Homo SapiensMembrane-bound protein PRO1346.2298 100 230 AAB65250 Homo sapiensHuman PR01346 protein sequence2298 100 SEQ ID

N0:314.

231 g11850831Homo SapiensH.sapiens TRGV10 gene, allele526 98 V10*A2.

231 g11850832Homo SapiensH.sapiens TRGV10 gene, allele520 98 V10*A1.

231 g11657829Bos taurusT cell receptor gamma chain346 61 variable region BVG3.2 232 AAR24442 Homo sa Sequence of antibody molecule492 63 iens I Gl.

232 g133451 Homo sapiensHuman mRNA for IgM heavy 481 60 chain com lete se uence.

232 g1185362 Homo sapiensHuman (hybridoma H210) anti-hepatitis476 60 A

IgG variable region, constant region, com lementarity-determinin re ions Table 2A
SEQ AccessionSpecies Description Score TD No. Identity NO:

mRNA, com fete cds.

233 16288984 Homo sa acid-labile subunit gene, 244 39 iens com lete cds.

233 g1184808 Homo SapiensHuman IGF binding protein 244 39 complex acid-labile subunit a mRNA, com lete cds.

233 g17105624Caenorhabditiscontains similarity to Pfam238 38 families elegans PF00560 (Leucine Rich Repeat-2 copies, score=157.2, E=2.8e-43, N=13), PF01463 Leucine rich repeat C-terminal domain, score=17.5, E=0.023, N=1) and PF01462 (Leucine rich repeat N-terminal domain, score=13.7 E=1, 234 AAY30835 Homo sapiensHuman secreted protein encoded329 100 from gene 25.

234 g15107309nitrogen-fixingvanadium dinitrogenase delta64 25 subunit bacterium DUl 234 g1608495 Homo SapiensHuman AMP deaminase isoform52 34 L, alternatively spliced (AMPD2) mRNA, exons 1B, 2 and 3, partial cds.

235 gi6453S12Homo SapiensmRNA; cDNA DKFZp434I1117 2693 83 (from clone DKFZp434I1117).

235 g16807969Homo sapiensmRNA; cDNA DKFZp4340192 248 33 (from clone DKFZp434O192); partial cds.

235 g112018147Chlamydomonavegetative cell wall protein127 34 gpl s reinhardtii 236 112652661Homo sa clone MGC:3279, mRNA, complete1313 100 iens cds.

236 AAB2407S Homo SapiensHuman PR01182 protein sequence1313 100 SEQ ID

N0:51.

236 AAY79510 Homo SapiensHuman carbohydrate-associated1313 100 protein CRBAP-6.

237 g113605426Rattus wingless-type MMTV integration57 35 site norvegicusfamily member 2 237 g1415362 Homo SapiensH.sapiens Ig germline kappa-chain66 42 gene variable region (LlOa).

237 g14467841Homo SapiensHuman gexmline fragment 66 42 for immunoglobulin kappa light chain (sub roup III) variable re ion ene V(h).

238 113446922Homo sa ClqR/ 32 ene, complete cds.714 100 iens 238 g112653333Homo Sapienscomplement component 1, 714 100 q subcomponent binding protein, clone MGC:8368, mRNA, com fete cds.

238 1472956 Homo sa H.sapiens mRNA for gCIq-R. 714 100 iens 239 AAW88580 Homo SapiensSecreted protein encoded 182 100 by gene 47 clone HCMSX86.

239 AAY25724 Homo SapiensHuman secreted protein encoded59 44 from gene 14.

239 g13929189Certhia NADH dehydrogenase subunit 40 32 III

familiaris 240 g113625176Homo Sapiensclone 1 thrombospondin mRNA,574 46 complete cds.

240 AAW85607 Homo sa Secxeted rotein clone da228S74 46 iens 6.

240 g114041961Homo SapienscDNA FLJI4440 fis, clone 572 46 HEMBB 1000915, weakly similar to SUBTILISIN-LIKE PROTEASE

PRECURSOR (EC 3.4.21.-).

Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

241 gi67400I3Homo Sapiensclone cDSCl Down syndrome 6370 60 cell adhesion molecule (DSCAM) mRNA, com lete cds.

241 AAW42086 Homo SapiensHuman Down syndrome-cell 6344 62 adhesion molecule DS-CAM1.

241 g111066998Mus musculusDown syndrome cell adhesion6341 60 molecule 242 112007410Mus musculusP2 olfactory rece for 441 44 242 17638409 Mus musculusolfactory rece for P2 441 44 242 g13153225Rattus olfactory receptor-like 444 44 protein norvegicus 243 14126467 Mus musculusmyosin 15 533 43 243 g16224683Homo Sapiensunconventional myosin-15 537 43 mRNA, com fete cds.

243 16224685 Mus musculusunconventional myosin-15 533 43 244 g110880453Homo SapiensGP3ST mRNA for glycoprotein623 41 beta-Gal 3'-sulfotransferase, com fete cds.

244 g17363258Mus musculuscerabroside sulfotransferase593 39 244 g16714628Homo SapiensCST gene for cerebroside 582 39 sulfotransferase, complete cds.

245 AAW74756 Homo SapiensHuman secreted protein 368 98 encoded by gene 26 clone HE6EH18.

245 gi49I002 synthetic EBV related protein p90. 86 32 construct 245 g16180093Cafeteria NADH dehydrogenase subunit79 28 roenbergensis 246 g15822850StreptococcusM protein 67 70 yogenes 246 g11373133Borrelia ErpB 44 42 burgdorferi 246 g12982984Aquifex small protein B 65 39 aeolicus 247 g112963879Homo Sapiensprostaglandin D synthase 579 98 mRNA, complete cds.

247 g113543568Homo Sapiensprostaglandin D2 synthase 579 98 (2lkD, brain), clone MGC:14559, mRNA, com lete cds.

247 g1189772 Homo SapiensHuman prostaglandin D2 579 98 synthase gene, exons 2 through 6 and com Iete cds.

248 AAB58937 Homo SapiensBreast and ovarian cancer 528 98 associated antigen protein sequence SEQ ID 645.

248 1293675 Mus musculusIFN-response element binding401 56 factor 2 248 g1606799 Homo SapiensHuman albumin D-box binding122 36 protein (DBP) mRNA, com lete cds.

249 111878220Xeno us transcri tional re ressor 479 61 laevis 249 g16910966Homo Sapienstranscriptional repressor 481 61 CTCF (CTCF) ene, exon 10 and com lete cds.

249 g1924760 Homo SapiensHuman transcriptional repressor481 6I
(CTCF) mRNA, com Iete cds.

250 g15457357Homo SapiensmRNA for ara 1e in-like 736 69 rotein.

250 AAW64469 Homo SapiensHuman secreted protein 732 69 from clone CW795 2.

250 AAB90744 Homo SapiensHuman CW795 2 protein sequence732 69 SEQ ID

188.

251 AAY96963 Homo SapiensWound healing tissue peptidoglycan1848 95 reco 'tion rotein-like rotein.

251 AAY96962 Homo SapiensKeratinocyte peptidoglycan777 62 ~ ~ recognition Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

rotein-like rotein.

251 AAY76124 Homo sa Human secreted rotein encoded777 62 iens by ene 1.

252 AAY96963 Homo SapiensWound healing tissue peptidoglycan1848 95 recognition rotein-like rotein.

252 AAY96962 Homo SapiensKeratinocyte peptidoglycan777 62 recognition rotein-like rotein.

252 AAY76124 Homo sa Human secreted rotein encoded777 62 iens by gene 1.

253 gi13236176PhotorhabdusNgrB 93 41 luminescens 253 gi1045676Mycoplasmathiophene and furan oxidation97 29 protein enitalium (tdhF) 253 17020951 Homo sa cDNA FLJ20693 fis, clone 79 31 iens KAIA2667.

254 13747097 Homo SapiensC1 -related factor mRNA, 620 64 com fete cds.

254 g114250666Homo SapiensClq-related factor, clone 620 64 MGC:3776, mRNA, com lete cds.

254 110566471Mus musculusGliacolin 508 67 255 g13747097Homo sa C1 -related factor mRNA, 908 71 iens complete cds.

255 g114250666Homo SapiensClq-related factor, clone 908 7I
MGC:3776, mRNA, complete cds.

255 g13747099Mus musculusC1 -related factor 792 72 256 g113528939Homo sa clone MGC:12301, mRNA, 200 100 iens com fete cds.

256 g12340091Homo SapiensHuman BAC clone CTA-20803,70 36 complete sequence.

256 g114250377Homo Sapienssmall inducible cytokine 70 36 subfamily A (Cys-Cys), member 13, clone MGC:17134, mRNA, complete cds.

257 g11769491Homo SapiensHuman kruppel-related zinc1551 50 finger protein (ZNF184) mRNA, artial cds.

257 113752754Homo sa zinc forger 1111 mRNA, 1551 49 iens com lete cds.

257 g110436789Homo SapienscDNA FLJ14345 fis, clone 1548 51 THYR01001189, weakly similar to ZINC

' FINGER PROTEIN 91.

258 AAY53038 Homo SapiensHuman secreted protein 487 97 clone fm3_1 protein se uence SEQ ID
N0:82.

258 g110862976Veratrum maturase 69 42 stamineum 258 g11321830Hepatitis HBsAg 70 31 B

virus 259 12633819 Bacillus transcriptional re lator 40 26 subtilis 259 g13493605VitreoscillaT repressor binding rotein57 42 Sp.

259 g114089811MycoplasmaTYPE III RESTRICTION- 52 31 pulmonis MODIFICATION SYSTEM:

METHYLASE

260 g19187610Homo SapiensmRNA full length insert 1050 99 cDNA clone EUROIMAGE 1669387.

260 g12921342Plasmodiummerozoite surface protein 61 43 falci arum 260 g11236783Teladorsagiacuticular collagen 96 29 circumcincta 261 AAY03227 Homo SapiensAmino acid sequence of 347 100 human clone ar54 1 1.

261 AAB 10279Homo SapiensHuman adult retina protein347 100 fragment AR54 1 1.

261 g1487147 Mus musculuscellular disintegrin-related63 54 protein z3z Table 2A
SEQ AccessionSpecies Description Score ID No. , Identity NO:

262 AAY48346 Homo SapiensHuman prostate cancer-associated289 94 protein 43.

262 16671200 Sus scrofat a I collagen al hal 63 47 262 g1306845 Homo sapiensHuman Hanukah factor serine54 57 protease (HuHF) mRNA, com Iete cds.

263 g112804141Homo Sapiensclone IMAGE:3956179, mRNA,1226 98 partial cds.

263 g14688938Leopoldamyspancreatic ribonuclease 76 28 edwarsi 264 g17582286Homo SapiensBM-007 591 99 264 AAG02907 Homo sa Human secreted rotein, 469 97 iens SEQ ID NO: 6988.

264 g13878572CaenorhabditisMO1F1.6 127 27 elegans 265 g113325190Homo Sapiensclone IMAGE:3640200, mRNA,215 97 partial cds.

265 g114024258Mesorhizobiumpyridoxamine kinase 65 46 loti 265 g1186468 Homo SapiensHuman insulin receptor 39 53 gene allele A, exon 18.

266 AAB32376 Homo SapiensHuman secreted protein 449 100 sequence encoded by gene 6 SEQ ID N0:62.

266 AAB32430 Homo SapiensHuman secreted protein 229 100 sequence encoded by ene 6 SEQ ID N0:116.

266 AAB32428 Homo SapiensHuman secreted protein 208 69 sequence encoded by ene 6 SEQ ID N0:114.

267 g110645308LeishmaniaL8453.1 252 26 ' major 267 g112018147Chlamydomonavegetative cell wall protein243 26 gpl s reinhardtii 267 g11572721Homo SapiensHuman megakaryocyte stimulating224 25 factor mRNA, complete cds.

268 g113623607Homo Sapienszinc finger protein 136 1874 63 (clone pHZ-20), clone MGC:12711, mRNA, com fete cds.

268 g1487785 Homo SapiensHuman zinc forger protein 1874 63 ZNF136.

268 g16467206Homo sapiensGIOT-4 mRNA for gonadotropin1798 58 inducible transcription repressor-4, com lete cds.

269 12252496 Homo sa mRNA for SKAP55 rotein. 84 48 iens 269 AAW37959 Homo SapiensAmino acid sequence of 84 48 the human SKAP55.

269 1495659 Pisum sativumas artate carbamoyltransferase78 31 270 16118396 Pan troglodytesbeta-defensin 1 55 35 270 g11293651Homo Sapiensbeta-defensin-1 gene, exon55 35 2 and complete cds.

270 g11617088Homo sa H.sapiens mRNA for hBD-1 55 35 iens protein.

271 g13786430Caenorhabditiscontains similarity to 269 36 protein kinases elegans (Pfam: kinase.hmm, score:
149.36) 271 g17268518ArabidopsisNAK like protein kinase 230 35 thaliana 271 g12351097ArabidopsisATMRK1 226 32 thaliana 272 1554377 Mus musculusT-cell rece for al ha 152 58 272 1201145 Mus musculusT-cell rece for al ha chainISO 61 272 1433691 Rattus T cell rece for V-alpha 150 47 rattus J-al ha 274 15441615 Canis familiariszinc fm er rotein 1295 50 Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

274 113752754Homo sa zinc finger 1111 mRNA, com 1294 57 iens lets cds.

274 g11769491Homo SapiensHuman kruppel-related zinc 1290 57 finger protein (ZNF184) mRNA, artial cds.

275 AAY36342 Homo SapiensFragment of human secreted 61 35 protein encoded by gene 1.

275 152591 Mus musculusWM65 immuno Iobulin aroma 61 54 one 275 AAY597I7 Homo SapiensSecreted rotein 58-49-3-G10-FLI.56 42 276 g13800742Rattus RGC-32 623 89 norve icus 276 g16137108Homo SapiensRGC32 (RGC32) mRNA, com 569 100 lets cds.

276 g1624685 Canis familiarisheat-shock rotein 88 27 277 g11769491Homo sapiensHuman kruppel-related zinc 1388 S1 finger protein (ZNF184) mRNA, artial cds.

277 g12970038Homo sa HKL1 mRNA, com lets cds. 1373 53 iens 277 g14164083Homo Sapienszinc linger protein EZNF 1372 53 (EZNF) mRNA, complete cds.

278 g113990957Homo SapiensmRNA for CREBIATF family 1326 94 transcription factor, com lets cds.

278 AAY59682 Homo sa Secreted protein 108-009-5-0-A2-FL.155 32 lens 278 AAY01635 Homo SapiensHuman PS214 derived of a 155 32 tide.

279 g110436801Homo SapienscDNA FLJ14351 fis, clone 749 100 THYR01001828.

279 AAB28066 Homo SapiensHuman secreted protein BLAST58 30 search rotein SEQ ID NO: 114.

279 g110584326HalobacteriumVng6287h 57 33 s . NRC-1 280 AAY48424 Homo SapiensHuman prostate cancer-associated244 97 protein 121.

280 g14512353Bacillus similar to B. subtilis ycgJ63 31 haloduransgene(35%-identity) 280 g17417236Trypanoplasmaribosomal protein L3 65 60 borreli 281 g11017722Homo SapiensHuman repressor transcriptional2212 71 factor (ZNF85) mRNA, complete cds.

281 g1186774 Homo SapiensHuman Kruppel related zinc 2138 70 finger protein (HTF10) mRNA, complete cds.

281 14454678 Homo sa zinc forger protein 4 2071 72 lens 282 AAB43919 Homo SapiensHuman cancer associated 1129 49 protein sequence SEQ ID N0:1364.

282 AAY87343 Homo SapiensHuman signal peptide containing1128 49 protein HSPP-120 SEQ ID N0:120.

282 AAG01572 Homo sa Human secreted protein, 312 42 lens SEQIDNO:

3.

283 AAY76219 Homo Sapiens_ 336 94 _ Human secreted protein encoded by gene 96.

283 AAY30164 Homo sa Human doxsal root receptor I26 34 lens 6 hDRR6.

283 AAZ10071_Homo SapiensHuman dorsal root receptor 122 33 aa1 5 hDRRS
nucleotide se uence.

284 g11669370Homo SapiensHuman BAC clone RG083M05 348 100 from 7q21-7q22, corn lets sequence.

284 g110438898Homo SapienscDNA: FLJ22489 fis, clone 340 98 HRC10951.

284 12980673 Mus musculusbeta-d strobrevin 67 35 285 g15262560Homo SapiensmRNA; cDNA DKFZpS72P0920 IS33 6I
(from clone DKFZ 572P0920); artial cds.

~ 28S g110434856Homo SapienscDNA FLJ13032 fis, clone 1519 61 ~ ~ ~ ~

Table 2A
SEQ AccessionSpecies Description Score ID No. Identity NO:

NT2RP3001120, moderately similar to ZINC FINGER PROTEIN 136.

285 gi13623354Homo SapiensSimilar to zinc forger 1519 63 protein 136 (clone pHZ-20), clone MGC:10647, mRNA, com Iete cds.

286 AAB27956 Homo sa Human secreted rotein SEQ 527 98 iens ID NO: 110.

286 AAY59778 Homo SapiensHuman normal ovarian tissue65 33 derived rotein 55.

286 gi10880708Hepatitis core protein 61 72 C

virus 287 gi5817048Homo SapiensmRNA; cDNA DKFZp586C1324 657 100 (from clone DKFZ 586C1324 ; artial cds.

287 AAY9I519 Homo SapiensHuman secreted protein 63 39 sequence encoded by gene 69 SEQ ID NO:192.

287 gi552372 Oryctolaguscytochrome P-450 58 31 cuniculus 288 gi14042863Homo SapienscDNA FLJ14966 fis, clone 544 33 THYR01000034, weakly similar to TRICHOHYAL1N.

288 gi12654519Homo Sapiensclone IMAGE:3504989, mRNA,270 39 partial cds.

288 gi10440520Homo sa mRNA for FLJ00109 protein,291 37 iens artial cds.

289 gi13592175Leishmaniappg3 112 24 maj or 289 gi12053243Homo SapiensmRNA; cDNA DKFZp434J0450 92 37 (from clone DI~FZp434J0450);
com lete cds.

289 il?32444 Homo sa mRNA for DRPLA protein, 86 24 iens complete cds.

290 gi13544007Homo Sapiensclone 1MAGE:3678120, mRNA,1049 91 partial cds.

290 11488047 Xeno us RING fm er protein 246 24 laevis 290 g1214915 Xeno us xnf7 335 25 laevis 291 g113544007Homo sapiensclone IMAGE:3678I20, mRNA,1335 100 partial cds.

291 g1458726 Homo SapiensHuman mRNA for estrogen 207 27 responsive fin er rotein, com lete cds.

291 g1 1088467Mus musculusestro en-responsive fm 195 31 er protein 292 AAY36113 Homo SapiensExtended human secreted 346 84 protein sequence, SEQ ID NO. 498.

292 AAY36114 Homo SapiensExtended human secreted 344 84 protein sequence, SEQ ID NO. 499.

292 g1487805 Danio rerioLIM domain homeobox rotein79 31 293 g15881782CercopithecusHSBP1-like protein 139 87 aethiops 293 g13283409Homo sapiensheat shock factor binding 81 50 protein 1 HSBP 1 mRNA, complete cds.

293 g113960151Homo Sapiensheat shock factor binding 81 50 protein 1, clone MGC:4536, mRNA, com Iete cds.

294 g11421664Human envelope glycoprotein 57 30 immunodeficie ncy virus a 1 294 g14154751HelicobacterABC transporter, ATP-binding67 27 protein pylori 294 g17544641SaccharomyeesRF2 protein 56 29 bayanus Table 2A
SEQ AccessionSpecies Description Score ~

ID No. Identity NO:

295 gi1390023Homo SapiensHuman testis specific leucine161 35 rich repeat rotein (TSLRP), com fete cds.

295 AAW 19394Homo sa Human cal astatin (clone 161 35 iens C-2 roduct).

295 gi3661587Mus musculustestis s ecific rotein 155 32 296 gi14042415Homo SapienscDNA FLJ14710 fis, clone 1984 65 NT2RP3000632, weakly similar to ZINC

FINGER PROTEIN 84.

296 gi13543419Homo sapiensSimilar to zinc finger 1816 53 protein 304, clone MGC:4079, mRNA, com fete cds.

296 gi9968290Homo SapiensmRNA fox zinc finger protein1723 48 (ZNF304 gene).

297 gi8777428Arabidopsiscontains similarity to 201 51 pyridoxamine 5-thaliana hos hate oxidase~gene id:K9P8.11 297 AAY79507 Homo SapiensHuman carbohydrate-associated185 43 protein CRBAP-3.

297 gi3979940CaenorhabditisY18D10A.3 119 35 ele ans 298 gi14211816Homo SapiensHBV pX associated protein 2739 98 8 large isoform XAP8aI ha) mRNA, complete cds.

298 112001942Homo SapiensMy001 rotein mRNA, com 2281 99 lete cds.

298 g110803733Homo SapiensHBV pX associated protein-81443 96 mRNA, complete cds.

299 112804833Homo Sapiensclone MGC:4827, mRNA, complete378 60 cds.

299 g19650982Homo SapiensmRNA for testis-specific 344 57 RING Finger rotein, com lete cds.

299 113938457Homo sa clone MGC:16175, mRNA, 126 48 iens complete cds.

300 g13643791Gloydius salmosin3 76 31 halys brevicaudus 300 AAB61146 Homo sa Human NOV 15 protein. 66 36 iens 300 g14584257Vitis viniferaSINA2 73 34 301 g112804771Homo Sapienstransmembrane trafficking 717 90 protein, clone MGC:3798, mRNA, complete cds.

301 g13288463Homo SapiensmRNA for integral membrane717 90 protein Tm 21-I (p23).

301 g11359886Homo SapiensH.sapiens mRNA for transmembrane717 90 protein Tmp21-I.

302 AAG03040 Homo SapiensHuman secreted protein, 79 40 SEQ ID NO: 7121.

302 AAG03133 Homo sa Human secreted protein, 78 40 iens SEQ ID NO: 7214.

302 AAB37411 Homo SapiensHuman secreted protein 76 38 BLAST search rotein SEQ ID NO: I21.

Table 2B
SEQ Hit ID Species Description S Percent ID scoreidentit 152 AAXI4999 Homo SapiensUNMI DNA encoding an 509 100 aal interluekin-8 IL-8 protein.

152 AAI67660 Homo sapiensGENA- Human interleukin509 100 aal (IL)-8 of eptide codin se uence.

152 AAT67659 Homo SapiensGENA- Nucleotide sequence509 100 aal of h uman interleukin (IL
-8 ene.

153 AAU12212 Homo SapiensGETH Human PR04395 2271 100 of a tide se uence.

153 ~gi13649767Mus musculuscollagen-like Alzheimer262 S3 amyloid la ue component precursor a I

153 gi15383905 Gallus collagen XVIII 260 S3 gallus 154 AAY7271 Homo SapiensHUMA- HFICU08 clone 2152 73 S human attractin-like rotein.

154 gi3449294 Rattus MEGF6 1780 61 norvegicus 154 AAY72716 Homo SapiensHUMA- HMEIR04 clone 1779 94 ' human aitractin-like protein.

155 AAX15002 Homo SapiensUNMI DNA encoding a 296 63 aa1 growth related oncogene-beta (GRO-beta).

155 AAA74871 Homo SapiensNEOR- Human chemokine 296 63 aal coding S sequence EQ ID NO: 31.

155 AAM25812 Homo SapiensHYSE- Human protein 296 63 sequence SEQ ID N0:1327.

156 AAX15002_aalHomo SapiensUNMI DNA encoding a 41S 70 growth related oncogene-beta (GRO-beta).

156 AAA74871 Homo SapiensNEOR- Human chemokine 415 70 aal coding S sequence EQ ID NO: 31.

156 AAM258I2 Homo SapiensHYSE- Human protein 415 70 sequence SEQ ID N0:1327.

157 AAG64863 Homo SapiensKYOW Heart muscle cell7S4 97 differentiation related protein SEQ

ID NO: 67.

157 AAB99934 Homo SapiensKYOW Human Midkine 754 97 protein sequence SEQ ID N0:67.

157 AAU28068 Homo SapiensHYSE- Novel human secretory7S4 97 protein, Seq ID No 237.

158 118390059 Homo sa cell recognition rotein6797 100 iens CASPR4 158 g112330704 Mus musculuscell recognition molecule6099 86 158 g117986216 Homo sa cell recognition molecule4821 70 iens CASPR3 159 g117224416 Mus musculusLDLR dan 5918 96 159 AAU28I66 Homo SapiensHYSE- Novel human secretory4545 99 protein, Se ID No 335.

159 AAU28354 Homo SapiensHYSE- Novel human secretory4216 99 protein, Se ID No 711.

160 g1180527 Homo Sapienschromogranin A recursor2334 100 160 g113623667 Homo Sapienschromogranin A (parathyroid2334 100 secreto rotein 1 I60 12072129 Homo Sapienschromogranin A 2326 99 161 AAE01436 Homo SapiensHUMA- Human gene 1 2110 100 encoded secreted protein HWLFJ10, SEQ

ID N0:91.

161 AAE01464 Homo SapiensHUMA- Human gene 1 2106 99 encoded secreted protein HWLFJ10, SEQ

ID N0:119.

161 'AAE01515 Homo SapiensHUMA- Human gene 1 1823 99 encoded Table 2B
SEQ Hit ID Species Description S Percent ID score identit secreted protein fragment, SEQ ID

NO:172.

162 AAR57349 Homo SapiensGEHO Human cartilage 1786 99 matrix rotein.

162 11732121 Homo sa cartila a matrix rotein1786 99 iens 162 g11163179 Mus musculuscartilage matrix rotein1686 90 precursor 163 AAR79051 Homo sa SCHE Human AI O-4 483 95 iens thymokine.

163 11754611 Homo sa SCM-lalpha recursor 483 95 iens 163 g1927651 Homo sa cytokine 483 95 iens 164 AAR79051 Homo SapiensSCHE Human A10-4 thymokine.545 100 164 g11754611 Homo sa SCM-lalpha precursor 545 100 iens 164 g1927651 Homo sa cytokine 545 100 iens 165 g1340002 Homo sapiensthyrotro in beta subunit767 I00 165 g17690113 Homo Sapiensthyroid-stimulating 764 99 hormone beta subunit 165 AAR99419 Homo SapiensGENZ TSH beta subunit.751 99 I 66 AAY97697 Homo SapiensCHIR Human EGFH2 protein615 99 se uence.

166 117068379 Homo sa Similar to neure ulin615 99 iens 4 166 14530130 Mus musculusneure 11n-4 short _ 74 isoform _ 167 g119068192 Mus musculusIL-IFIO _ 92 167 AAB84999 Homo sapiensCURA- Human interleukin-1_ 78 receptor antagonist (NOVINTRA

A) pol eptide.

167 ~AAM50219 Homo SapiensAMGE- Interleukin-1 584 68 receptor antagonist related protein splice variant.

168 AAB94547 Homo SapiensHELI- Human protein 2134 95 sequence SEQ ID N0:15303.

168 AAY28919 Homo SapiensINCY- Human regulatory2134 95 protein HRGP-5.

168 AAM42084 Homo SapiensHYSE- Human polypeptide2130 95 SEQ

ID NO 7015.

169 AAB56784 Homo SapiensROSE/ Human prostate 690 87 cancer antigen protein sequence SEQ ID

NO:1362.

169 AAR39393 Homo SapiensBGHM Serglycin - proteoglycan690 87 eptide core.

169 AAR05249 Homo SapiensBRIG- Proteoglycan 690 87 peptide core encoded by consensus nucleotide sequenchuman leukennia promyelocytic cell line HL-60 derived cDNAs.

170 AAY73496 Homo SapiensGEMY Human secreted 467 92 protein clone ysl0_1 protein sequence SEQ

ID N0:214.

170 gi~18579608~ref~Homo SapiensTJ6 protein 553 100 'XP 096586.1 171 AAU83188 Homo SapiensZYMO Novel secreted 1443 100 protein Z887042G3P.

171 AAB85336 Homo sa CHIR Human oaf protein144 100 iens se uence. 3 171 AAE03851 Homo SapiensHUMA- Human gene 8 _ 99 encoded 1437 secreted protein HBIOH81, SEQ ID

N0:97.

172 g112001958 Homo sa My009 rotein 1715 88 iens Table 2B
SEQ Hit ID Species Description S Percent ID score identit 172 AAB88335 Homo SapiensHELI- Human membrane 1711 88 or secretory rotein clone PSEC0045.

172 AAY94893 Homo SapiensPROT- Human protein 1308 96 clone HP 10392.

173 gi15929874 Homo SapiensHIV-1 Tat interactive1245 100 protein 2, 30 kDa 173 gi1280325I Homo sa Tat-interactin rotein1245 100 iens (30kD) 173 AAW 12697 Homo SapiensSYST- CC3 polypeptide1239 99 derived from small cell carcinoma.

174 AAB39216 Homo SapiensHUMA- Human secreted 1109 99 protein sequence encoded by gene 38 SEQ

ID N0:96.

174 AAU83666 Homo sapiensGETH Human PRO protein,886 68 Seq ID

No 150.

174 AAB65214 Homo SapiensGETH Human PR0809 886 68 (UNQ464) rotein se uence SEQ
ID N0:223.

175 AAY96729 Homo sapiensGETH PR01800, a Hep271178 87 homolo ue.

175 gi12804321 Homo Sapiensperoxisomal short-chain1178 87 alcohol dehydro enase 175 AAB93414 Homo SapiensHELI- Human protein 1174 87 sequence SEQ ID N0:12620.

176 gi12964242 Homo sa dJ908M14.2 (novel 1469 93 iens protein) 176 119070529 Homo SapiensCABLES 1111 62 176 g111993865 Mus musculuscdk3-bindin protein 993 59 1k3-1 177 AAG81289 Homo sapiensZYMO Human AFP protein762 92 se uence SEQ ID N0:96.

177 AAM79384 Homo SapiensHYSE- Human protein 762 92 SEQ ID NO

3030.

177 AAM78400 Homo SapiensHYSE- Human protein 762 92 SEQ ID NO

1062.

178 AAR90616 Homo SapiensIDEK Anti-procathepsin1795 94 B

monoclonal antibody.

178 ' 1I81192 Homo sa preprocathe sin B 1795 94 iens 178 g1291888 Homo Sapienscathe sin B 1792 94 179 AAW75196 Homo SapiensHUMA- Human secreted 330 98 protein encoded by gene 1 clone HCEAB46.

179 AAM86806 Homo sapiensHUMA- Human 76 34 immuneJhaematopoietic antigen SEQ ID N0:14399.

179 ABB 11593 Homo sapiensNYSE- Human secreted 71 32 protein homolo ue, SEQ ID
N0:1963.

180 AAM48455 Homo SapiensBODE- Human ATP-dependent1635 100 serine protein hydrolase 34.

180 AA016947 Homo SapiensKYOW Human NF-kappaB 1628 99 activity enhancin protein SEQ
ID NO: 3.

180 g118043288 Mus musculusmitochondria) ribosomal1346 79 protein 181 AAY88278 Homo SapiensMILL- Human TANGO 1123 82 protein.

_ 181 g114336711 Homo Sapienssimilar to C. Elegans1123 82 protein F17C8.5 181 AAA39947 Homo SapiensMILL- Human TANGO 1119 82 aal 188 cDNA.

Table 2B
SEQ Hit ID Species Description S Percent ID scoreidentit 182 11922287 Homo sa enoyl-CoA hydratase 1354 93 iens 182 g114286220 Homo Sapiensenoyl Coenzyme A hydratase,1350 93 short chain, 1, mitochondria) 182 g1433413 Homo Sapiensmitochondria) short-chain1337 92 enoyl-CoA h dratase 183 ABB05699 Homo SapiensGEHU- Human metabolism2447 98 protein clone fbr2 78121.

183 AAM40365 Homo SapiensHYSE- Human polypeptide2447 98 SEQ

ID NO 3510.

183 AAM42151 Homo SapiensHYSE- Human polypeptide1952 99 SEQ

ID NO 7082.

184 AAY86443 Homo SapiensHUMA- Human gene 42-encoded739 I00 rotein fra ment, SEQ
ID N0:358.

184. g114530178 PlasmodiumKrueppel-like protein 73 38 falciparum 184 g12621995 Methanothermconserved protein 73 23 obacter thermautotrop hicus str.
Delta H

185 AAA64426_aalHomo sapiensMILL- DNA encoding 1226 92 a human TANGO 223 variant olype tide.

185 AAB08658 Homo SapiensMILL- A human TANGO 1226 92 variant pol a tide.

185 AAB08641 Homo sapiensMILL- A human TANGO 1226 92 pol epode.

186 AAY12948 Homo SapiensHLTMA- Amino acid sequence191 90 of a human secreted eptide.

186 g118558601 Mus musculusvomeronasal rece for 66 40 186 gi~20821790~re~Mus musculussimilar to vomeronasal66 40 1 receptor, XP 144714.1 C3 187 g115022155 Homo sa Bv8 protein 627 97 iens 187 g19957625 Mus musculussecretory rotein BV8 544 86 187 g16524947 Mus musculusBv8 variant 1 recursor544 86 188 AAY91443 Homo SapiensHUMA- Human secreted 1029 100 protein sequence encoded by gene 39 SEQ

ID N0:164.

188 g116741724 Homo sapiensSimilar to growth accentuating616 100 rotein 43 188 121321072 Danio reriotbx6 related protein 110 23 189 .ABB57454 Homo SapiensHUMA- Human secreted 2231 95 protein encoding polypeptide SEQ TD NO

100.

189 AAB75509 Homo SapiensROSE/ Human secreted 2231 95 protein sequence encoded by gene 4 SEQ

ID N0:63.

I89 g116877130 Homo SapiensCGI-44 protein; sulfide2231 95 dehydrogenase like ( east) 190 AAB34624 Homo SapiensHLTMA- Human secreted 260 100 protein sequence encoded by gene 48 SEQ

ID NO:I08.

191 g113477197 Homo SapiensSimilar to quinolinate1484 94 phosphoribosyltransferase (nicotinate-nucleotide Table 2B
SEQ Hit ID Species Description S Percent ID score identit o hos horylase (carboxylating) 19I g11060907 Homo Sapiensquinolinate phosphoxibosyl1460 92 transferase 191 gi1694S374 Neurosporaprobable nicotinate-nucleotide604 44 crassa yro hos horylase (carboxylating) ~

192 AAY38394 Homo SapiensHUMA- Human secreted 213 100 protein encoded b ene No.

9.

193 iSSSS94 Homo sa dihydroorotate dehydrogenase1790 83 iens 193 14379422 Rattus dihydroorotate dehydro1593 7S
rattus enase 193 g18784074 Mus musculusdihydroorotate dehydro1586 74 enase 194 11S866714 Homo sa C-terminal modulator 1273 100 iens protein 194 AAG81369 Homo SapiensZYMO Human AFP protein777 100 sequence SEQ ID N0:2S6.

194 ABB89S30 Homo SapiensHUMA- Human polypeptide734 100 SEQ

ID NO 1906.

19S AAU75314 Homo SapiensGLAX Human SPARC rotein.1664 100 195 AAG66546 Homo SapiensPHAR- Human interferon-alpha1664 100 induced of eptide, SPARC.

19S g129462 Homo Sapiensextracellular matrix 1664 100 protein BM-40 (AA 1 - 303) 196 AAB27S 87 Homo SapiensHUMA- Human secreted 689 100 protein SEQ ID NO: 88.

196 AAG89301 Homo SapiensGEST Human secreted 610 100 protein, SEQ ID NO: 421.

196 gi142S0466 Homo sapiensSimilar to RIKEN cDNA610 100 SS30601I19 ene 197 AAB73899 Homo SapiensCALY Human derived 2512 96 cytochrome P4S0 2E1 monooxy enase.

197 AABS336S Homo SapiensHUMA- Human colon 2512 96 cancer antigen protein sequence SEQ ID

N0:90S.

197 AAR81467 Homo SapiensSUMO Human derived 2S 96 cytochrome 12 P4S02E 1.

198 AAB73899 Homo sapiensCALY Human derived 2510 96 cytochrome P450 2E1 monooxy enase.

198 AAB53365 Homo SapiensHUMA- Human colon 2S 96 cancer 10 antigen protein sequence SEQ ID

NO:90S.

198 AAR81467 Homo SapiensSUMO Human derived 2510 96 cytochrome P4S02E1.

199 gi2S64916 Homo sa cotel 1792 92 iens 199 g110800049 Gallid ICP4 protein 114 32 he esvirus 199 163898 Gallus Zyxin 104 26 allus 200 AAH7SS71 Homo sapiensSHAN- Human GrpE protein98 100 aal encoding cDNA.

200 AAG64S77 Homo sa SHAN- Human E rotein.98 100 iens 200 AAW80748 Homo SapiensINCY- Human mitochondrial98 100 cha erone rotein (Hmt-G
E).

201 AAB88407 Homo SapiensHELI- Human membrane 1744 96 or secretory rotein clone PSEC0163.

201 gi144S661S Homo sa phosphatidyl inositol1744 96 iens g1 can class T

201 AAB652S4 Homo SapiensGETH Human PR01379 1727 96 (UNQ716) protein sequence SEQ

ID N0:340.

Table 2B
SEQ Hit ID Species Description S Percent ID score identit 202 AAV81204 Homo sa GEHO Human CD7 cDNA. 1130 92 aal iens 202 AAB36657 Homo SapiensIMMV Human CD7 protein1130 92 se uence SEQ ID N0:2.

202 AAU02438 Homo SapiensGEHO Human lymphocyte1130 92 cell surface anti en CD7 0l a tide.

203 gi12483900 Rattus zinc forger protein 1001 57 norvegicus 203 gi3294544 Homo sa C2H2- a zinc forger 571 32 iens rotein 203 gi5757625 Homo sa C2H2 zinc fin er rotein571 32 iens 204 AAY86455 Homo SapiensHUMA- Human gene 45-encoded417 100 rotein fra ment, SEQ
ID N0:370.

204 AAY86259 Homo sapiensHUMA- Human secreted 237 100 protein HAGDI35, SEQ ID NO:174.

204 ~gi13429972Homo sa mosaic serine protease73 35 iens 205 135046 Homo sa NF-M 4094 91 iens 205 g1297529 Mus musculusNF-M 2922 71 205 g13641350 Bos taurusneurofilament-M subunit2917 77 206 g120987928 Mus musculusSimilar to RIKEN cDNA1159 73 1200004E24 ene 206 ABB55699 Homo SapiensFECH/ Human polypeptide724 40 SEQ ID

NO 4.

206 AAU38990 Homo sapiensGEMY Human secreted 724 40 protein f283 11.

207 AAU81998 Homo SapiensINCY- Human secreted 2393 91 protein SECP24.

207 ABB55706 Homo SapiensFECH/ Human polypeptide495 31 SEQ ID

NO 18.

207 AAU38997 Homo SapiensGEMY Human secreted 495 31 protein yall 1.

208 AAB38568 Homo SapiensHUMA- Human secreted 330 100 protein sequence encoded by gene 47 SEQ

ID N0:105.

208 AA009777 Homo SapiensHYSE- Human polypeptide72 35 SEQ

ID NO 23669.

208 115147679 Mus musculustaste receptor T1R1 68 36 210 g113477235 Homo sapiensSimilar to RIKEN cDNA917 100 0610037N03 gene 210 g119484119 Mus musculusSimilar to RIKEN cDNA560 68 2410006N06 gene 210 AAM47991 Homo sapiensBODE- Human dih droorotase239 97 12.

211 114517637 Homo sapiensRGPR- 117 1064 92 211 g113785516 Rattus RGPR-p117 714 64 norve icus 211 g114517635 Mus musculusRGPR-p117 712 66 212 g118676660 Homo sa FLJ00229 rotein 1691 100 iens 212 AAM40342 Homo sapiensHYSE- Human polypeptide1136 63 SEQ

ID NO 3487.

212 AAM42128 Homo SapiensHYSE- Human polypeptide447 69 SEQ

ID NO 7059.

213 AAB70880 Homo SapiensPECH/ Human carcino-embryonal453 34 antigen rotein.

213 AAB43687 Homo sapiensHUMA- Human cancer 453 34 associated protein se uence SEQ
ID N0:1132.

213 AAW22844 Homo SapiensSUNK Human carcinoembryonic453 34 anti en.

Table 2B
SEQ Hit ID Species Description S Percent ID score identit 214 AAE05302 Homo SapiensMILL- Human TANGO 1929 100 rotein.

214 AAE05303 Homo SapiensMILL- Human mature 1805 100 rotein.

214 AAE05305 Homo SapiensMILL- Human TANGO 1260 100 rotein cyto lasmic domain.

215 1338766 Homo sa T-cell rece for precursor965 72 iens 215 13089419 Homo sa T cell rece for alpha935 70 iens chain 215 g119343617 Homo SapiensSimilar to T cell 931 70 receptor alpha locus 216 AAE01313 Homo SapiensHUMA- Human gene 2 77 24 encoded secreted protein fragment, SEQ ID

N0:178.

216 AAE01259 Homo SapiensHUMA- Human gene 2 77 24 encoded secreted protein HMVAV54, SEQ

ID N0:121.

216 AAE01233 Homo SapiensHUMA- Human gene 2 77 24 encoded secreted protein HMVAV54, SEQ

ID N0:95.

217 g1950420 Homo sa cell surface lycoprotein1211 89 iens CD44 217 g121429241 Homo sa CD44 antigen 1211 89 iens 217 11101786 Homo sa cell surface Tyco 1208 89 iens rotein CD44 218 'AAY86222 Homo SapiensHUMA- Human secreted 233 83 protein HCUDD24, SEQ ID N0:137.

218 AAY86355 Homo sapiensHUMA- Human gene 8-encoded142 69 protein fra ment, SEQ ID N0:270.

218 ABB06092 Homo SapiensCOMP- Human NS protein76 24 sequence SEQ ID N0:184.

219 g1387013 Homo sa pepsino en A 1148 99 iens 219 g1387012 Homo sa epsino en 1145 99 iens 219 g120810074 Homo SapiensSimilar to pepsinogen1145 99 5, group I

( a siiiogen A

220 AAG00471 Homo SapiensGEST Human secreted 285 93 protein, SE ID NO: 4552.

220 g1298489 Papio SP-10 133 34 hamadryas 220 g1452582 Vulpes fox sperm acrosomal 132 34 vulpes protein FSA-Acr. l 221 g117384405 Homo SapiensbA113024.1 (similar 1516 100 to insulin-like owth factor binding rotein) 221 AAE15654 Homo SapiensCUBA- Human growth 1468 97 factor bindin protein-like protein, NOVS.

221 12315138 Mus musculusIGFBP-like rotein 1169 76 222 ABB 11922 Homo SapiensHYSE- Human T cell 1329 98 receptor beta chain homologue, SEQ
ID

N0:2292.

222 1339010 Homo SapiensT-cell rece for beta 1054 80 chain 222 g13002931 Homo sa T cell rece for beta 1045 79 iens chain 223 ABB 11922 Homo SapiensHYSE- Human T cell 1571 98 receptor beta chain homologue, SEQ
ID

N0:2292.

223 1339010 Homo sa T-cell receptor beta 1296 83 iens chain 223 13002931 Homo sa T cell receptor beta 1263 81 iens chain 224 AAY35888 Homo SapiensGEST Extended human 706 98 secreted rotein se uence, SEQ
ID NO. 25.

Table 2B
SEQ Hit ID Species Description S Percent ID scoreidentit 224 AAY36051 Homo SapiensGEST Extended human 706 98 secreted rotein se uence, SEQ
ID NO. 436.

224 AAY25457 Homo sapiensGEST Human secreted 706 98 protein 5 derived from extended cDNA.

225 AAB38361 Homo SapiensHUMA- Human secreted 204 100 protein encoded by gene 4I
clone HTOHG09.

226 g1623157 Macaca T-cell receptor alpha 707 85 mulatto 226 g1555719 Macaca T-cell receptor alpha 648 88 chain mulatto 226 g1339419 Homo SapiensTCR 630 90 227 g119343617 Homo SapiensSimilar to T cell receptor1395 98 alpha locus 227 AAY69995 Homo SapiensINCY- Human receptor-associated1006 74 rotein from Incyte clone 1361202.

227 g11223888 synthetic T cell receptor alpha 993 72 chain construct 228 g19294050 Arabidopsisprotein kinase-like 84 32 protein thaliana 228 giI5983765 ArabidopsisAT3g24550/MOB24 8 84 32 thaliana 228 g113877617 Arabidopsisprotein kinase-like 84 32 protein thaliana 229 g115291913 DrosophilaLD31582p 214 24 melanogaster 229 g16523547 Volvox hydxoxyproline-rich 208 28 carteri glycoprotein f. nagariensisDZ-HRGP

229 g15305335 Mycobacteriuproline-rich mucin 203 33 homolog m tuberculosis 230 AAB65250 Homo sapiensGETH Human PR01346 2298 100 protein sequence SEQ ID N0:314.

230 AAY66727 Homo SapiensGETH Membrane-bound 2298 100 protein PR01346.

230 AAB33444 Homo SapiensGETH Human PR01346 2298 100 protein UNQ701 SEQ ID N0:152.

231 g11850831 Homo so TRGV 10 526 98 iens 231 g1537394 OryctolagusT-cell receptor gamma 343 62 chain V2-cuniculus P2-C precursor 231 g11657829 Bos taurusT cell receptor gaxrnna341 61 chain variable region BVG3.2 232 AAR24442 Homo SapiensGEHO Sequence of antibody490 63 molecule IgGl.

232 133451 Homo Sapiensprecursor (AA -15 to 480 60 612) 232 g119684012 Homo SapiensSimilar to ixnmunoglobulin480 60 heavy constant anima 3 (G3m marker) 233 gi193440I0 Homo Sapiensinsulin-like growth 242 38 factor binding rotein, acid labile subunit 233 g1184808 Homo Sapiensinsulin-like growth 241 38 factor binding rotein com lex 233 16288984 Homo so acid-labile subunit 241 38 iens 234 AAY30835 Homo SapiensHUMA- Human secreted 329 100 protein encoded from ene 25.

234 g114575530 Homo Sapiensleishmanolysin-like 280 92 peptidase, variant 1 Table 2B
SEQ Hit ID Species Description S Percent ID scoreidentit 234 giI57490 Drosophilagerm cell-less protein68 28 melano aster 235 AAM95470 Homo SapiensHUMA- Human reproductive561 63 system related antigen SEQ ID NO:

4128.

235 g12754696 Gallus high molecular mass 142 20 gallus nuclear anti en 235 g16433936 Homo sa aczonin . 142 23 iens 236 ABB56397 Homo SapiensFUSO Human collectin 1313 100 collagen-like domain SEQ ID
NO 11.

236 ABB84900 Homo SapiensGETH Human PR01182 1313 100 protein se uence SEQ ID N0:168.

236 AAB65261 Homo SapiensGETH Human PR01182 1313 100 (UNQ596) protein sequence SEQ

ID N0:357.

237 g117984796 Brucella XANTHINElURACIL 70 28 melitensisPERMEASE

237 'gi~20856691~ref~Mus musculusRIKEN cDNA 9430034F23 68 31 XP 125979.1 238 AAB58262 Homo SapiensROSE/ Lung cancer associated1355 93 pol eptide sequence SEQ ID 600.

238 AAW05534 Homo SapiensTANO- Clq receptor. 1355 93 238 g1472956 Homo SapiensCI -R 1355 93 239 ABB50347 Homo SapiensHUMA- Human secreted 182 100 protein encoded by gene 47 SEQ ID

N0:295.

239 AAW88580 Homo SapiensHUMA- Secreted protein182 100 encoded by ene 47 clone HCMSX86.

240 AAB99220 Homo sa BIOR- Human thrombos 568 46 iens ondin-30.

240 AAM78328 Homo SapiensHYSE- Human protein 568 46 SEQ ID NO

990.

240 AAE13163 Homo SapiensKIRI Human secreted 568 46 protein from clone DA228 6.

24I g120067221 Homo SapiensDown syndrome cell 1074499 adhesion molecule 2 241 g118033452 Homo SapiensDown syndrome cell 1073799 adhesion molecule DSCAMLl 241 AAM39040 Homo sapiensHYSE- Human polypeptide9116 100 SEQ

ID NO 2185.

242 AAG72603 Homo SapiensVEDA Human OR-like 1441 89 polypeptide uery se uence, SEQ
ID NO: 2284.

242 AAG71515 Homo SapiensVEDA Human olfactory 1441 89 receptor polype tide, SEQ ID
NO: 1196.

242 118480466 Mus musculusolfactory receptor 610 44 243 16224683 Homo sapiensunconventional myosin-15539 43 243 16224685 Mus musculusunconventional m osin-15535 43 243 g14126467 Mus musculusmyosin 15 535 43 244 g114625435 Homo sa galactose-3-O-sulfotransferase2271 99 iens 244 g114571498 Homo Sapiensbeta-galactose-3-O-sulfotransferase2271 99 244 g110880453 Homo Sapiensglycoproteinbeta-Gal3'-602 40 sulfotransferase 245 AAW74756 Homo sapiensHUMA- Human secreted 368 98 protein encoded by gene 26 clone HE6EH18.

Table 2B
SEQ Hit ID Species Description S Percent ID score identit 245 gi491002 synthetic EB V related protein 83 32 p90.

construct 245 gi6180093 Cafeteria NADH dehydrogenase 74 28 subunit 5 roenber ensis 246 AAM65980 Homo SapiensMOLE- Human bone marrow266 100 expressed probe encoded protein SEQ ID NO: 26286.

246 AAM53601 Homo SapiensMOLE- Human brain 266 100 expressed single exon probe encoded protein SEQ ID NO: 25706.

246 gi47371 StreptococcusML2.2 67 70 yogenes 247 1189772 Homo sa rosta landin D2 s 945 84 iens thase 247 112963879 Homo sa rostaglandin D s thase945 84 iens 247 g113543568 Homo Sapiensprostaglandin D2 synthase945 84 (211tD, brain) 248 AAB58937 Homo SapiensHUMA- Breast and ovarian528 98 cancer associated antigen protein sequence SEQ ID 645.

248 g1293675 Mus musculusIFN-response element 401 56 binding factor 2 248 1606799 Homo sa albumin D-box bindin 112 36 iens rotein 249 120805280 Homo sa nuclear DNA binding 922 100 iens factor 249 g113121344 Homo SapiensdJ579F20.2.2 (novel 837 100 protein similar to transcriptional repressor CTCF, isoform 2) 249 g18217649 Homo SapiensdJ579F20.2. l (novel 572 100 protein similar to transcriptional repressor CTCF, isoform 1) 250 g114549664 Mus musculusATP-dependent zinc 1448 68 metallo rotease 250 AAM79377 Homo sapiensHYSE- Human protein 1321 62 SEQ ID NO

3023.

250 AAM78393 Homo SapiensHYSE- Human protein 1321 62 SEQ ID NO

1055.

251 g115590686 Homo Sapienspeptidoglycan recognition1953 98 protein-I-beta recursor 251 AAY96963 Homo SapiensHUMA- Wound healing 1845 95 tissue peptidoglycan recognition protein-like rotein.

25I g115590684 Homo Sapienspeptidoglycan recognition1226 62 protein-I-alpha precursor 252 g115590686 Homo Sapienspeptidoglycan recognition1953 98 protein-I-beta recursor 252 AAY96963 Homo SapiensHiJMA- Wound healing 1845 95 tissue peptidoglycan recognition protein-like rotein.

252 g115590684 Homo Sapienspeptidoglycan recognition1232 61 protein-I-alpha recursor 253 ~gi1045676 Mycoplasmathiophene and furan 94 33 oxidation genitaliumprotein (tdhF) 253 g113236176 PhotorhabdusNgrB 87 40 luminescens 253 gi[20542599[refsHomo Sapienssimilar to Macrophage1617 99 scavenger XP 095545.2[ receptor types T and II (Macrophage z4s Table 2B
SEQ Hit ID Species Description S Percent ID score identit _ acetylated LDL rece for I and II) 2s4 13747099 Mus musculusCI -related factor 616 61 2s4 114278927 Mus musculusliacolin 61s 64 2s4 110S66471 Mus musculusGliacolin 61s 64 2ss 13747099 Mus musculusCI -related factor 904 68 2ss 13747097 Homo sa C1 -related factor 901 69 iens 2ss 114278927 Mus musculusgliacolin 892 70 2s7 is0807s8 Homo sa BC331191 1 1660 49 iens 2s7 AAM80207 Homo SapiensHYSE- Human protein 1645 s0 SEQ ID NO

38s3.

2s7 AAM79223 Homo SapiensHYSE- Human protein 164s s0 SEQ ID NO

188s.

2s8 AAYS3038 Homo SapiensGEMY Human secreted 487 97 protein clone fin3 1 protein sequence SEQ

_ ID N0:82 2s8 ABB 1 s27s Homo SapiensHUMA- Human nervous 42s 96 system related polypeptide SEQ ID NO

3932.

2s8 gi~14s17838~gb~Hepatitis SHBs 67 29 B

AAK64389.1~AFvirus 369s41 1 260 g11236751 Teladorsagiacuticularcollagen 99 29 circumcincta 260 AAM93276 Homo SapiensHELI- Human polypeptide,96 28 SEQ ID

NO: 2748.

260 gi298117s Homo Sapiensdeltex 91 28 261 AAB 10279 Homo sapiensGEMY Human adult retina347 100 protein fra went ARS4 1 1.

261 AAY03227 Homo SapiensGEMY Amino acid sequence347 100 of human clone ars4 1i.

261 g120976810 DrosophilaGHOlsl7p 7s 32 melano aster 262 AAY48346 Homo sapiensMETA- Human prostate 289 94 cancer-associated rotein 43.

263 .gi~2086234s~ref~Mus musculusRIKEN cDNA 1110038D171251 9s XP_12s766.1 ~

263 gi~21293101~gb~EAnopheles agCP6889 317 36 AA05246.1) gambiae str.

PEST

263 gi~1841s044~ret1Arabidopsisexpressed protein 27s 33 NP s681 thaliana s9. l ~

264 AAG81367 Homo SapiensZYMO Human AFP protein877 99 se uence SEQ ID N0:2s2.

264 gi1808834s Homo sapiensSimilar to RIKEN cDNA860 99 1110066001 ene 264 g120381141 Mus musculusRIKEN cDNA 111006600I670 7s gene 266 AAB32376 Homo SapiensHLTMA- Human secreted449 100 protein sequence encoded by gene 6 SEQ

ID N0:62.

266 AAB32430 Homo sapiensHLTMA- Human secreted229 100 protein sequence encoded by gene 6 SEQ

ID NO:116.

266 AAB32428 Homo SapiensHLTMA- Human secreted206 69 protein sequence encoded by gene 6 SEQ

ID N0:114.

TahlP 7.R
SEQ Hit ID Species Description S Percent ID score identit 267 g112018147 Chlamydomonvegetative cell wall 255 25 protein gpl as reinhardtii 267 g117945382 DrosophilaRE17165p 211 27 melanogaster 267 g15917666 Zea mays extensin-like rotein 208 25 268 g120306657 Homo Sapienssimilar to zinc finger3114 100 protein 136 (clone HZ-20) 268 g1487785 Homo sa zinc fm er rotein 1871 63 iens ZNF136 268 g113623607 Homo Sapienszinc forger protein 1871 63 136 (clone pHZ-20) 269 AAB94716 Homo SapiensHELI- Human protein 922 100 sequence SEQ ID N0:15724.

269 116118245 Homo SapiensARAP2 841 100 269 g116974764 Homo sa PARX protein 841 100 iens 270 g12257485 Schizosaccharp1005 70 23 omyces ombe 270 g1490625 synthetic antigen 70 35 construct 270 g1158902 Eimeria transhydrogenase 70 35 acervulina 271 .AAE16259 Homo SapiensINCY- Human kinase 3062 99 protein.

271 AAU 11287 Homo sapiensBODA- Human transducin1925 99 polype tide 41.

271 AAM25477 Homo SapiensHYSE- Human protein 833 99 sequence SEQ ID N0:992.

272 g1554377 Mus musculusT-cell receptor alpha152 58 272 1201145 Mus musculusT-cell rece for al 150 61 ha chain 272 1433691 Rattus T cell receptor V-al 147 54 rattus ha J-alpha 273 g120306351 Homo Sapienssimilar to gonadotropin2456 98 inducible transcri tion re ressor-1 273 g16467200 Homo Sapiensgonadotropin inducible1812 98 transcri tion repressor-1 273 ABB50184 Homo Sapiens1NCY- Human transcription1493 55 factor TRFX-35.

274 AAM70526 Homo SapiensMOLE- Human bone marrow2634 100 expressed probe encoded protein SEQ ID NO: 30832.

274 AAM58080 Homo sapiensMOLE- Human brain 2634 100 expressed single exon probe encoded protein SEQ ID NO: 30185.

274 AAM69575 Homo SapiensMOLE- Human bone marrow2559 84 expressed probe encoded protein SEQ ID NO: 29881.
, 276 g118182820 Homo SapiensbA157L14.2 (RGC32, 684 100 a novel gene induced by complement activation in oli odendrocytes) 276 g13800742 Rattus RGC-32 623 89 norve icus 276 114582295 Mus musculusresponse gene to complement610 86 277 12970038 Homo SapiensHKL1 1498 53 277 AAY58624 Homo SapiensINCY- Protein regulating1497 53 gene ex ression PRGE-17.

277 g14164083 Homo sa zinc fm er protein 1497 53 iens EZNF

278 g113990957 Homo sa CREB/ATF family transcri1324 94 iens tion Table 2B
SEQ Hit ID Species Description S Percent ID scoreidentit factor 278 AAM25719 Homo sapiensHYSE- Human protein 939 74 sequence SEQ ID N0:1234.

278 13548791 Homo sa 833590 1 939 74 iens 279 AAB95866 Homo sapiensHELI- Human protein 749 100 sequence SEQ ID N0:18937.

279 .gi~20891333~ref~Mus musculussimilar to DEAD-box 72 21 protein XP 146975.1 ~

280 AAY48424 Homo sapiensMETA- Human prostate 244 97 cancer-associated rotein 121.

281 g11017722 Homo sa re ressor transcri 2261 70 iens tional factor 281 12739353 Homo SapiensZNF91L 2174 71 281 AAM79931 Homo SapiensHYSE- Human protein 2166 68 SEQ ID NO

3577.

282 AAM93282 Homo SapiensHELI- Human polypeptide,2392 100 SEQ ID

NO: 2762.

282 AAU01338 Homo SapiensMILL- Human TANGO 351 2284 100 amino acid se uence.

282 AAB43919 Homo sapiensHUMA- Human cancer I 48 associated 121 rotein se uence SEQ
ID N0:1364.

283 AAY762I9 Homo SapiensHITMA- Human secreted 336 94 protein encoded by ene 96.

283 AAY30164 Homo SapiensASTR- Human dorsal 114 34 root receptor 6 hDRR6.

283 g119338918 Homo sa G protein-cou led receptor114 34 iens SNSR6 284 g11669370 Homo Sapienscoded for by human 348 100 cDNAs W37389 (NID:gI319205), (NID:g838529), 865794 (NID:g838432) and 865794 NID: 838432) 284 g115823303 Mus musculusODAG protein 345 98 284 g121430716 DrosophilaRH61522p 131 50 melano aster 285 AAM41438 Homo SapiensHYSE- Human polypeptide1537 60 SEQ

ID NO 6369.

285 AAM41436 Homo SapiensHYSE- Human polypeptide1537 60 SEQ

ID NO 6367.

285 AAM39652 Homo SapiensHYSE- Human polypeptide1537 60 SEQ

ID NO 2797.

286 AAB27956 Homo SapiensHL1MA- Human secreted 527 98 protein SEQ ID NO: 110.

286 g120799443 Pectinophoraacyl-CoA desaturase 71 30 goss iella 287 AA010250 Homo SapiensHYSE- Human polypeptide166 43 SEQ

ID NO 24142.

287 g11651212 Actinobacillus6-phosphogluconate 71 28 dehydrogenase actinomycetem comitans 287 gi~14768166~ref~Homo SapiensDIiFZP586C1324 protein578 100 XP 045876.1 288 AAU28254 Homo SapiensHYSE- Novel human secretory5243 99 rotein, SeqIDNo 611.

288 ~AAU28066 Homo SapiensHYSE- Novel human secretory5046 99 rotein, Se ID No 235.

288 AAB95567 Homo SapiensHELI- Human protein 527 33 ~ ~ ~ sequence ~ ~

Table 2B
SEQ Hit ID Species Description S Percent ID scoreidentit SEQ ID N0:18211.

289 g113592175 Leishmaniappg3 99 21 ma'or 289 12358287 Homo sa ALR 91 23 iens 289 g1995557 Rattus DRPLA 90 33 norve icus 290 g115150298 Homo sa lycogenin-interacting 371 27 iens rotein 1 290 g11488047 Xenopus RING finger protein 346 25 laevis 290 g1238611 Xenopus zinc forger nuclear 316 25 phosphoprotein laevis 291 g115150298 Homo sapiens1 co enin-interactin 348 26 protein 1 291 g11488047 Xenopus RING finger protein 336 24 laevis 291 g1238611 Xenopus zinc forger nuclear 304 23 phosphoprotein laevis 292 AAY36114 Homo sapiensLEST Extended human 396 80 secreted rotein sequence, SEQ
ID NO. 499.

292 AAY36113 Homo SapiensGEST Extended human 396 80 secreted rotein sequence, SEQ
ID NO. 498.

292 g1487805 Danio rerioLIM domain homeobox 76 31 rotein 293 AA012985 Homo SapiensHYSE- Human polypeptide148 75 SEQ

ID NO 26877.

293 AAU28320 Homo SapiensHYSE- Novel human secretory147 93 protein, Seq ID No 677.

293 AAU28132 Homo SapiensHYSE- Novel human secretory147 93 rotein, Seq ID No 301.

294 gi~18402635~ref~Arabidopsisexpressed protein 68 30 NP 566663.1 thaliana ~

295 g114714553 Homo SapiensSimilar to RIKEN cDNA 1261 97 1700006D24 gene 295 g118490773 Mus musculusRIKEN cDNA 1700006D24 834 67 ene 295 g18132056 Trypanosomaphosphatase 150 36 brucei 296 AAG68343 Homo SapiensBODA- Human TFIIS 2670 100 characteristic sequence fragment rotein SEQ ID N0:2.

296 g119343979 Homo sapienssimilar to ubiquitiii 1973 65 UBF-fl (H.

sa iens) 296 AAM78392 Homo SapiensHYSE- Human protein 1798 53 SEQ ID NO

1054.

297 121068656 Mus musculusapoA-I binding protein279 39 297 AAY79507 Homo Sapiens1NCY- Human carbohydrate-277 39 associated rotein CRBAP-3.

297 121068652 Homo SapiensapoA-I bindin rotein 277 39 298 g114211816 Homo SapiensHBV pX associated protein2739 98 8 large isoform 298 112001942 Homo sa M 001 rotein 2281 99 iens 298 g110803733 Homo SapiensHBV pX associated protein-8;1443 96 299 g19650982 Homo Sapienstestis-s ecific RING 336 56 Fin er rotein 299 15114351 Homo sa RING fm er rotein terf179 29 iens 299 AAB95586 Homo SapiensHELI- Human protein 173 33 sequence SEQ ID N0:18252.

~00 AAM73644 Homo SapiensMOLE- Human bone marrow303 100 ~

Table 2B
SEQ Hit ID Species Description S Percent ID scoreidentit expressed probe encoded protein SEQ ID NO: 33950.

300 AAM60951 Homo SapiensMOLE- Human brain expressed303 100 single exon probe encoded protein SEQ ID NO: 33056.

300 gi847953 ChoristoneuraIE2 68 37 fumiferana nucleopolyhed rovirus 301 11359886 Homo sa transmembrane xotein 712 95 iens 301 11407826 Homo sa rotein trafficking 712 95 iens rotein 301 g13288463 Homo Sapiensintegral membrane protein,712 95 Tmp21-302 gi~20835168~ref~Mus musculusRIKEN cDNA 1700008H02 84 35 XP 132164.1 302 gi~2193870~dbj~BMus musculusreverse transcriptase 83 35 AA20419.1 ~

302 gi~20346368~ref~Mus musculussimilar to reverse 83 35 transcriptase XP 110682.1 ~

Table 3 SEQ Database Description Results*

ID entr ID

152 BL00471 Small cytokines BL0047I 23.92 8.826e-40 (iiitercrine/chemokine) C-x-C

subfamily si nat.

152 PR00436 INTERLEUKIN-8 SIGNATUREPR00436C 10.51 3.475e-29 PR00436B 10.31 4.000e-29 PR00436D I2.23 2.406e-27 PR00436A 9.64 8.826e-27 152 PR00437 SMALL CXC CYTOKINE FAMILYPR00437B 14.81 5.500e-20 SIGNATURE PR00437C 14.85 1.281e-15 PR00437A 9.50 9.250e-12 152 BL00472 Small cytokines BL00472C 20.76 8.286e-11 (intercrine/chemokine) C-C subfamily signatur.

153 BL01187 Calcium-binding EGF-likeBL01187B 12.04 3.118e-15 domain 150-165 proteins pattern roteins.BL01187A 9.98 6.571e-10 153 BLOI I Clq domain proteins. BL01113A 17.99 3.323e-15 BL01113A 17.99 2,636e-14 BL01113A 17.99 3.182e-14 BL01113A 17.99 1,973e-13 BL01113A 17.99 2.615e-12 BL01113A 17.99 8.364e-11 BL01113A 17.99 1.000e-10 BL01113A 17.99 6.553e-10 BL01113A 17.99 6.936e-10 BLOI 1 I3A 17.99 2.558e-09 BL01113A 17.99 3.423e-09 BL01113A 17.99 4.115e-09 BL01113A 17.99 4.981e-09 BL01113A 17.99 5.500e-09 153 PR00010 TYPE II EGF-LIKE SIGNATUREPROOOlOC 11.16 7.857e-09 153 BL00420 Speract receptor repeatBL00420A 20.42 9.481e-I2 proteins 256-284 domain proteins. BL00420A 20.42 9.182e-11 BL00420A 20.42 6.902e-10 BL00420A 20.42 3.077e-09 BL00420A 20.42 5.431e-09 BL00420A 20.42 9.862e-09 154 BL00022 EGF-like domain proteins.BL00022B 7.54 3.250e-10 154 PR0001I TYPE III EGF-LIKE SIGNATUREPROOO11B 13.08 9.135e-15 PROOOI 1B 13.08 9.000e-14 PROOOIlA 14.06 1.000e-13 PROOO11D 14.03 1.340e-13 PROOO11B 13.08 1.458e-13 PROOO11D 14.03 5.755e-I3 PROOO11A 14.06 6.034e-13 PROOOl IB 13.08 7.254e-13 PROOO11B 13.08 3.468e-12 PROOO11A 14.06 3.613e-12 PROOO11D 14.03 3.842e-12 PROOOl ID 14.03 4.316e-IZ

PROOO11D 14.03 4.789e-12 PROOOl 1B 13.08 7.242e-12 PROOO11B 13.08 7.968e-12 PROOOlID 14.03 8.105e-12 PROOO11D 14.03 2.623e-11 PROOOl IB 13.08 4.046e-1 PROOO11D 14.03 5.721e-11 PROOO11A 14.06 5.985e-11 Table 3 SEQ Database Description ResuIts*

ID entr ID

PROOO11A 14.06 7.369e-I

PROOO11A 14.06 8,615e-11 PROOO11B 13.08 9.03Ie-11 PROOO11A 14.06 1.652e-10 PROOO11D 14.03 5.015e-10 PROOO11A 14.06 6.870e-10 154 BL00243 Integrins beta chain BL00243H 17.53 1.829e-09 cysteine-rich 44-69 domain proteins. BL00243H 17.53 3,013e-09 154 PR00907 THROMBOMODULIN SIGNATUREPR00907B 11.29 9.890e-09 155 PR00437 SMALL CXC CYTOKINE FAMTLYPR00437A 9.50 5.909e-10 SIGNATURE

156 PR00437 SMALL CXC CYTOKINE FAMILYPR00437C 14.85 4.789e-19 SIGNATURE PR004378 14.81 6.760e-12 PR00437A 9.50 5.909e-10 156 BL00471 Small cytokines BL00471 23.92 2.191e-12 (intercrine/chemokine) BL00471 23.92 4.375e-09 C-x-C 84-131 subfamily si nat.

157 PR00269 PLEIOTROPHIN/MIDKINE PR00269B 13.02 8.250e-33 FAMILY SIGNATURE PR00269A 13.91 2.000e-28 PR00269C 12.63 7.840e-20 I57 BL00619 PTN/MK heparin-binding BL00619B 17.94 1.000e-40 protein 61-105 family proteins. BL00619A 19.07 3.500e-30 BL00619A 19.07 2.636e-19 158 DM01077 SEX HORMONE-BINDING DM01077A 16.30 9.743e-11 GLOBULIN.

158 DM00516 186 DISCOIDIN I N-TERMINAL.DM00516 30.53 5390e-10 I58 PR00261 LOW DENSITY LIPOPROTEINPR00261F 11.57 9.772e-09 (LDL) RECEPTOR SIGNATURE

159 BL01209 LDL-receptor class A BL01209 9.31 6.063e-16 (LDLRA) 169-181 domain proteins. BL01209 9.31 8.313e-16 BL01209 9.3I 6.684e-I5 BL01209 9.31 9.526e-15 BL01209 9.31 1.375e-14 BL01209 9.31 1.000e-13 BL01209 9.31 4.300e-13 BL01209 9.31 5.500e-13 159 PR00261 LOW DENSITY LIPOPROTEINPR00261A I I.02 9.600e-2I

(LDL) RECEPTOR SIGNATUREPR0026IA 11.02 2.212e-19 PR00261D 12.47 6.824e-19 PR00261C 11.37 7.404e-19 PR00261D 12.47 7.882e-19 PR00261D 12.47 8.588e-19 PR00261D 12.47 1.667e-18 PR00261D 12.47 S.OOOe-18 PR00261A l I.02 6.833e-18 PR00261A 11.02 6.833e-18 PR00261F 11.57 7.980e-18 PR00261D 12.47 9.333e-18 PR00261B 14.12 9.368e-18 PR00261B 14.12 9.684e-18 PR00261C 11.37 2.086e-17 PR00261A 11.02 4.632e-17 PR00261B 14.12 5.050e-17 PR00261B 14.12 5.650e-17 PR00261A 1 I.02 7.316e-17 PR00261F 11.57 9.827e-17 PR00261B 14.12 9.850e-17 TahlP ~
SEQ Database Description Results*

ID entr ID

PR00261C 11.37 1.443e-16 PR00261C 11.37 4.246e-16 PR00261F 11.57 5.821e-16 PR00261E 11.08 8.333e-16 PR00261E 11.08 8.667e-16 PR00261C 11.37 1.857e-15 PR00261C 11.37 2.857e-15 PR00261E 11.08 4.000e-15 PR00261E 11.08 5.263e-15 ' PR00261C 11.37 6.571e-15 PR00261B 14.12 6.954e-15 PR00261E 11.08 1.300e-14 PR00261C 11.37 1.537e-14 PR00261D 12.47 1.703e-14 PR00261F 11.57 3.032e-14 PR00261B 14.12 3.912e-14 PR00261E 11.08 5.200e-14 PR00261F 11.57 5.355e-14 PR00261E 11.08 6.100e-14 PR00261A 11.02 7.092e-14 PR00261F 11.57 8.548e-14 PR00261E 11.08 9.550e-14 PR00261D 12.47 4.574e-13 PR00261B 14.12 7.845e-13 PR00261F 11.57 3.250e-12 PR00261F 11.57 3.515e-12 PR00261A 11.02 3.915e-12 159 PR00764 COMPLEMENT C9 SIGNATUREPR00764B 13.56 5.424e-15 PR00764B 13.56 5.714e-14 PR00764B 13.56 9.714e-14 PR00764B 13.56 5.500e-13 PR00764B 13.56 2.696e-12 PR00764B 13.56 1.500e-11 PR00764B 13.56 2.625e-11 159 PR00010 TYPE II EGF-LIKE SIGNATUREPROOOlOC 11.16 4.643e-09 159 BL01187 Calcium-binding EGF-likeBL01187B 12.04 1.257e-10 domain 496-511 proteins pattern proteins.BL01187B 12.04 5.950e-09 160 PR00659 CHROMOGRANIN SIGNATURE PR00659C 7.46 5.091e-22 PR00659A 12.29 6.538e-20 PR00659B 13.09 2.957e-19 160 PF00992 Troponin. PF00992A 16.67 1.947e-09 160 PR00761 BIND1N PRECURSOR SIGNATUREPR00761E 14.32 4.411e-09 160 BL00422 Granins proteins. BL00422E 26.86 2.929e-27 BL00422A 28.34 4.343e-23 BL00422C 16.18 6.727e-20 BL00422D 19.48 5.355e-19 BL00422B 12.83 4.724e-09 161 PR00907 THROMBOMODULIN SIGNATUREPR00907B 11.29 8.274e-11 161 BL00279 Membrane attack complexBL00279E 37.11 1.325e-10 components 68-115 / perform roteins.

161 PR00764 COMPLEMENT C9 SIGNATUREPR00764F 16.89 3.836e-10 161 DM00864 EGF-LIKE DOMAIN. DM00864B 11.34 6.910e-10 161 BL01187 Calcium-binding EGF-likeBL01187B 12.04 4.150e-14 domain 187-202 roteins attern proteins.BL01187B 12.04 2.575e-09 161 BL01177 Ana h latoxin domain BL01177E 20.64 7.632e-09 roteins. 185-211 161 BL00799 Granulins roteins. BL00799B 11.02 9.679e-09 161 PR00343 SELECTIN SUPERFAMILY PR00343C 16.85 9.727e-09 Tahla ~
SEQ Database Description Results*

ID entr ID

COMPLEMENT-BINDING REPEAT

SIGNATURE

161 PR00010 TYPE II EGF-LIKE SIGNATUREPROOOlOA 11.79 3.077e-11 PROOOlOC 11.16 7.545e-10 PROOOlOC 11.16 9.786e-09 162 BL01187 Calcium-binding EGF-likeBL01187B 12.04 4.343e-10 domain 156-171 roteins attern roteins.

162 PR00010 TYPE II EGF-LIKE SIGNATUREPROOOlOC 11.16 3.357e-09 162 PR00879 FISH ACETYLCHOLINESTERASEPR00879A 6.28 5.950e-09 SIGNATURE

162 PDO1101 INHIBITOR HEAVY CHAIN PDO1101B 21.53 8.543e-09 CHANNEL IN.

162 PR00453 VON WILLEBRAND FACTOR PR00453A 12.79 1.310e-14 TYPE A DOMAIN SIGNATUREPR00453B 14.65 6.087e-14 PR00453C 12.26 8.875e-11 PR00453C 12.26 9.000e-09 163 BL00472 Small cytokines BL00472C 20.76 4.717e-17 (intercrine/chemokine) C-C subfamily signatur.

163 PR00437 SMALL CXC CYTOKINE FAMILYPR00437C 14.85 5.661e-09 SIGNATURE

164 BL00472 Small cytokines BL00472C 20.76 4.717e-17 (intercrine/chemokine) C-C subfamily si natur.

164 PR00437 SMALL CXC CYTOKINE FAMILYPR00437C 14.85 5.661e-09 SIGNATURE

165 BL00261 Glycoprotein hormones BL00261B 25.64 1.000e-40 beta chain 95-138 proteins. BL00261A 23.97 3.500e-34 166 PR00009 TYPE I EGF SIGNATURE PR00009A 14.15 3.089e-09 167 PR00264 INTERLEUK1N-1 SIGNATUREPR00264B 20.98 4.234e-11 PR00264C 17.77 4.527e-10 168 PR00449 TRANSFORMING PROTEIN PR00449A 13.20 7.938e-11 RAS SIGNATURE

168 PR00326 GTPl/OBG GTP-BINDING PR00326A 8.75 8.200e-11 PROTEIN FAMILY SIGNATURE

168 BL00113 Adenylate kinase proteins.BL00113A 12.74 2.523e-09 173 BL01224 N-acetyl-gamma-glutamyl-phosphateBL01224A 15.62 7.171e-09 reductase proteins.

173 PR00308 TYPE I ANTIFREEZE PROTEINPR00308A 5.90 8.624e-09 SIGNATURE

175 PR00080 ALCOHOL DEHYDROGENASE PR00080C 17.16 9.250e-12 S UPERFAMILY SIGNATURE

175 PR00081 GLUCOSE/RIBITOL PR00081D 15.80 2.731e-13 DEHYDROGENASE FAMILY PR00081A 10.53 6.226e-13 SIGNATURE PR00081F 15.71 7.632e-12 PR00081C 15.13 9.609e-12 PR00081E 17.54 1.587e-10 175 BL00061 Short-chain dehydrogenases/reductasesBL00061B 25.79 2.588e-21 family proteins. BL00061C 7.86 2.588e-09 176 PF00777 Sialyltransferase family.PF00777C 18.60 9.786e-09 178 PR00705 PAPAW CYSTE1NE PROTEASEPR00705A 10.55 1.529e-16 (C1) FAMILY SIGNATURE PR00705B 10.22 4.115e-10 178 BL00139 Eukaryotic t11io1 (cysteine)BL00139D 9.24 9.182e-18 proteases 275-291 cysteine proteins. BL00139A 10.29 6.478e-14 BL00139B 10.19 6.400e-10 ~ R2 BL00166 Enoyl-CoA hydratase/isomeraseBL00166D 22.87 6.478e-27 Table 3 SEQ Database Description Results*

ID entr ID

proteins. BL00166C 18.93 7.261e-26 BL00166B 16.92 7.500e-19 183 BL01279 Protein-L-isoaspartate(D-aspartate)BL01279A 24.27 5.663e-12 meth ltransferase si na.

183 PR00850 GLYCOSYL HYDROLASE FAMILYPR00850D 13.37 3.227e-09 183 BL01131 Ribosomal RNA adenine BL02131A 26.62 8.200e-09 dimethylases 235-280 proteins.

189 PR00411 PYRIDINE NUCLEOTIDE PR00411A 15.95 7.828e-09 DISULPHIDE REDUCTASE
CLASS-I SIGNATURE

189 BL00623 GMC oxidoreductases BL00623A 12.60 9.100e-09 proteins. 45-63 191 PR00958 HOMOSERINE ICINASE PR00958E 10.56 9.135e-10 SIGNATURE

193 BL00911 Dihydroorotate dehydrogenaseBL00911G 19.64 3.739e-29 proteins. BL00911B 22.10 2.406e-24 BL00911E 9.42 2.421e-14 BL00911A 10.44 3.118e-12 BL0091 IF 10.03 8.200e-12 BL00911C 7.34 9.769e-11 BL00911D 9.08 1.000e-09 195 BL00612 Osteonectin domain proteins.BL00612B 11.35 1.000e-40 BL00612C 9.90 1.000e-40 BL00612D 10.06 1.000e-40 BL00612E I3.I2 1.000e-40 BL00612A 12.09 3.077e-20 197 PR00463 E-CLASS P450 GROUP I PR00463E 17.37 8.043e-23 SIGNATURE PR00463G 18.24 7.480e-19 PR00463F 17.63 2.895e-17 PR00463B 17.50 3.880e-16 PR00463A 11.40 7.750e-15 PR00463D 14.02 1.667e-14 PR00463C 12.85 2.552e-14 197 PR00385 P450 SUPERFAMILY SIGNATUREPR00385A 14.97 8.500e-15 PR00385B 10.22 8.500e-13 PR00385C 16.94 7.158e-10 _ 197 PR00464 E-CLASS P450 GROUP II PR00464D 17.40 8.636e-12 SIGNATURE PR00464E 18.28 5.018e-09 PR00464G 12.41 6.940e-09 PR00464C 18.84 7.904e-09 198 BL00086 Cytochrome P450 cysteineBL00086 20.87 6.294e-23 heme-iron 409-440 ligand roteins.

198 PR00385 P450 SUPERFAMILY SIGNATUREPR00385A 14.97 8.500e-15 PR00385B 10.22 B.SOOe-13 PR00385E 12.66 4.000e-12 PR00385G 16.94 7.158e-10 PR00385D 13.11 2.000e-09 198 PR00463 E-CLASS P450 GROUP I _ PR00463E 17.37 8.043e-23 SIGNATURE PR00463I 15.02 8.286e-21 PR00463G 18.24 7.480e-19 PR00463F 1?.63 2.895e-17 PR00463B 17.50 3.880e-16 PR00463A 11.40 7.750e-15 PR00463C 12.85 2.552e-14 PR00463H 12.41 1.000e-11 PR00463D 14.02 3.423e-09 198 PR00464 E-CLASS P450 GROUP II PR00464D 17.40 8.636e-12 Table 3 SEQ Database Description Results*

ID entr ID

SIGNATURE PR00464I 14.64 2.731 e-09 419-442 PR00464E 18.28 5.018e-09 PR00464G 12.41 6.940e-09 199 PR00211 GLUTELIN SIGNATURE PR00211B 0.86 7.750e-10 202 DM00179 w KINASE ALPHA ADHESIONDM00179 13.97 5.263e-10 CELL.

203 PD01066 PROTEIN ZINC FINGER PD01066 19.43 6.855e-14 FINGER METAL-BINDING
NU.

203 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 5.200e-14 BINDI. PD00066 13.92 4.115e-10 PD00066 13.92 5.500e-10 PD00066 13.92 8.962e-10 203 BL00028 Zinc forger, C2H2 type,BL00028 16.07 1.000e-11 domain 417-433 proteins. BL00028 16.07 2.500e-10 BL00028 16.07 4.900e-10 BL00028 16.07 6.100e-10 BL00028 16.07 4.343e-09 203 BL01030 RNA polymerases M / BL01030 23.44 6.241e-09 15 Kd subunits 389-426 roteins.

203 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 3.368e-11 SIGNATURE PR00048B 6.02 7.923e-11 PR00048A 10.52 4.522e-10 PR00048A 10.52 6.040e-09 PR00048A 10.52 6.040e-09 PR00048A 10.52 7.480e-09 PR00048A 10.52 9.640e-09 205 BL00422 Granins proteins. BL00422C 16.18 3.824e-09 205 BL00226 Intermediate filaments BL00226D 19.10 1.000e-40 proteins. 386-432 BL00226B 23.86 1.321e-31 BL00226C 13.23 1.000e-18 BL00226A 12.77 3.571e-15 BL00226B 23.86 5.709e-09 205 PF00992 Tro onin. PF00992A 16.67 6.211 e-09 499-533 205 BL00412 Neuromodulin (GAP-43) BL00412D 16.54 1.511e-11 proteins. 507-557 BL00412D 16.54 2.534e-11 . BL00412D 16.54 1.978e-10 BL00412D 16.54 4.228e-10 BL00412D 16.54 4.326e-10 BL00412D 16.54 9.315e-10 BL00412D 16.54 5.500e-09 BL00412D 16.54 5.776e-09 BL00412D 16.54 6.510e-09 BL00412D 16.54 9.724e-09 210 BL00518 Zinc finger, C3HC4 typeBL00518 12.23 8.200e-11 (RING 36-44 fin er), proteins.

213 DM00372 CARCINOEMBRYONIC ANTIGENDM00372B 20.31 3.148e-11 PRECURSOR AMINO-TERMINALDM00372C 23.69 5.440e-09 DOMAIN.

214 BL00290 Immunoglobulins and BL00290A 20.89 7.480e-10 major 160-182 histocompatibili com BL00290B 13.17 2.875e-09 lex roteins. 226-243 215 DM00031 IMMLJNOGLOBULIN V REGION.DM00031B 15.41 9.746e-11 2I6 PR00927 ADENINE NUCLEOTIDE PR00927A 7.98 9.667e-09 217 BL01241 Link domain roteins. BL01241 35.81 1.000e-40 217 PR00658 CD44 ANTIGEN PRECURSOR PR00658B 6.73 1.225e-28 SIGNATURE PR00658C 7.17 1.265e-28 Table 3 SEQ Database Description Results*

ID entr ID

_ PR00658A 17.21 1.000e-26 PR00658D 8.52 2.286e-25 219 PR00792 PEPSIN (A1) ASPARTIC PR00792A 11.54 2.929e-22 PROTEASE FAMILY SIGNATUREPR00792D 12.74 5.200e-18 PR00792B 12.78 7.750e-14 PR00792C 9.10 1.000e-12 219 BL00141 Eukaryotic and viral BL00141E 14.32 4.750e-22 aspartyl proteases 431-454 proteins. BL00141A 12.10 5.500e-20 BL00141B 12.14 9.000e-12 BL00141C 9.74 1.000e-11 BL00141D 6.28 3.700e-1 221 BL00282 Kazal serine protease BL00282 16.88 5.183e-10 inhibitors family 107-129 proteins.

221 BL00222 Insulin-like growth BL00222B 11.09 3.833e-09 factor binding 61-76 proteins.

221 DM01688 2 POLY-IG RECEPTOR. DM01688G 16.45 9.100e-09 222 BL00290 Immunoglobulins and BL00290A 20.89 8.200e-12 major 157-179 histocom atibility com lex roteins.

223 BL00290 Immunoglobulins and BL00290A 20.89 8.200e-12 major 157-179 histocom atibility com lex roteins.

226 DM00031 IMML1NOGLOBULIN V REGION.DM00031B 15.41 2.139e-09 227 DM00031 IMMUNOGLOBULIN V REGION.DM00031B 15.41 6.108e-10 229 PF00023 Ai~lc repeat proteins. PF00023A 16.03 1,750e-10 PF00023B 14.20 9.591e-09 230 BL00514 Fibrinogen beta and BL00514C 17.41 4.789e-34 gamma chains C- 271-307 terminal domain proteins.BL00514G 15.98 7.845e-16 BL00514H 14.95 7.218e-13 BL00514E 14.28 4,109e-11 BL00514D 15.35 8.737e-10 BL00514F 11.65 5.500e-09 231 DM0003I IMMUNOGLOBULIN V REGION.DM00031B 15.41 4.649e-10 232 DM00031 IMMUNOGLOBULIN V REGION.DM00031A 16.80 5.333e-26 DM00031B 15.41 7.980e-19 DM00031C 12.79 4.429e-10 233 PR00019 LEUCTNE-RICH REPEAT PR00019A 11.19 8,200e-12 SIGNATURE PR00019A 11.19 3,250e-11 PR00019A 11.19 6,870e-10 PR00019B 11.36 3.520e-09 236 BL01113 Clq domain proteins. BL01113A 17.99 I.865e-09 BL01113A 17.99 5.846e-09 236 BL00420 Speract receptor repeatBL00420A 20.42 8.442e-12 proteins 65-93 domain proteins. BL00420A 20.42 3.492e-09 BL00420A 20.42 4,738e-09 BL00420A 20.42 1.000e-08 240 BL00243 Integrins beta chain BL00243I 31.77 4.930e-09 cysteine-rich 94-136 domain roteins.

241 BL00790 Receptor tyrosine kinaseBL00790I 20.01 7.480e-11 class V 1156-1186 proteins. BL00790I 20.01 6.963e-10 BL00790120.01 8.988e-10 BL00790H 13.42 9.514e-10 241 DM00215 PROLINE-RICH PROTEIN DM0021S 19.43 1.305e-09 3. 1974-2006 241 PD02870 RECEPTOR INTERLEUK1N-1 PD02870B 18.83 8.024e-12 PRECURSOR. PD02870D 15.74 9.900e-10 PD02870B 18.83 7.415e-09 241 PR00014 FIBRONECT1N TYPE III PR00014A 8.22 3.864e-09 SIGNATURE PR00014D 12.04 7.750e-09 Table 3 SEQ Database Description Results*

ID entr ID

241 DM00179 w KINASE ALPHA ADHESIONDM00179 13.97 8.043e-09 CELL.

241 PD02327 GLYCOPROTEIN ANTIGEN PD02327B 19.84 9.591e-09 PRECURSOR IMMUNOGLO. PD02327B 19.84 9.591e-09 241 BL00240 Receptor tyrosine kinaseBL00240B 24.70 7.907e-10 class III 427-450 roteins. BL00240B 24.70 I.OOOe-08 242 PR00237 RHODOPSIN-LIKE GPCR PR00237C 15.69 6,143e-10 SUPERFAMILY SIGNATURE

242 PR00245 OLFACTORY RECEPTOR PR00245A 18.03 2.429e-12 SIGNATURE PR00245B 10.38 7,525e-11 PR00245E 12.40 2.019e-10 PR00245C 7.84 7.286e-10 242 BL00237 G-protein coupled rece BL00237A 27.68 2.837e-09 tors proteins. 72-111 243 PR00300 ATP-DEPENDENT CLP PROTEASEPR00300A 9.56 6.577e-10 SIGNATURE

243 PR00193 MYOSIN HEAVY CHAIN PR00193B 11.69 2.220e-16 SIGNATURE PR00193C 12.60 6.657e-14 PROOI93A 15.41 7.092e-09 247 PR00179 LIPOCALIN SIGNATURE PROOI79B 9.56 1.000e-12 PR00179C 19.02 1.000e-10 PR00179A 13.78 5.680e-10 247 BL00213 Lipocalin proteins. BL00213B 8.78 8.000e-10 BL00213A 12.95 9,526e-10 249 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 4.522e-10 SIGNATURE

249 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 3.571e-12 BINDI. PD00066 13.92 5.846e-10 24.9 BL00028 Zinc finger, C2H2 type,BL00028 16.07 7.429e-09 domain 175-191 roteins.

250 BL00674 AAA-protein family proteins.BL006748 4.46 7.500e-19 BL00674D 23.41 1.180e-18 250 PR00830 ENDOPEPTIDASE LA (LON) PR00830A 8.41 5.286e-11 SERINE PROTEASE (S 16) SIGNATURE

250 PR00300 ATP-DEPENDENT CLP PROTEASEPR00300A 9.56 6.591e-11 ATP-BINDING SUBUNIT

SIGNATURE

250 PR00819 CBXX/CFQX SUPERFAMILY PR00819B 10.83 7.632e-11 SIGNATURE

250 PR00918 CALICIVIRUS NON-STRUCTURALPR00918A 13.76 2.193e-10 POLYPROTEIN FAMILY

SIGNATURE

253 PR00449 TRANSFORMING PROTEIN PR00449A 13.20 6.586e-09 RAS SIGNATURE

253 BL00567 Phos horibulolcinase BL00567A 10.66 6.657e-09 proteins. 134-152 253 PR00326 GTPI/OBG GTP-BINDING PR00326A 8.75 9.100e-09 PROTEIN FAMILY SIGNATURE

254 PR00007 COMPLEMENT C1Q DOMAIN PR00007B 14.16 5.114e-15 SIGNATURE PR00007A 19.33 7.052e-10 254 PR00524 CHOLECYSTOK1NIN TYPE PR00524F 5.36 4.35Ie-09 RECEPTOR SIGNATURE

254 DM00250 kw ANNEX1N ANTIGEN PROLINEDM00250B 13.84 6,595e-09 TUMOR.

254 BL00415 Synapsins roteins. BL00415N 4.29 7.372e-09 254 BL01113 C1 domain roteins, BL01113B 18.26 3.786e-23 TahlP ~
SEQ Database Description Results*

ID entr ID

BL01113A 17.99 7.968e-15 BL01113A 17.99 5.091e-14 BL01113A 17.99 5.295e-11 BL01113A 17.99 8.568e-11 BL01113A 17.99 8.977e-11 BL01113A 17.99 4.635e-09 BL01113A 17.99 6.192e-09 BL01113A 17.99 7.750e-09 254 BL00420 Speract receptor repeatBL00420A 20.42 8.691e-11 proteins 73-101 domain proteins. BL00420A 20.42 9.673e-11 BL00420A 20.42 2.180e-10 BL00420A 20.42 8.062e-09 255 PR00007 COMPLEMENT C1Q DOMAIN PR00007B 14.16 5.114e-15 SIGNATURE PR00007C 15.60 5.875e-13 PR00007A 19.33 7.052e-10 255 PR00524 CHOLECYSTOKININ TYPE PR00524F 5.36 4.351e-09 RECEPTOR SIGNATURE

255 DM00250 kw ANNEXIN ANTIGEN PROLINEDM00250B 13.84 6.595e-09 TUMOR.

255 BL00415 Syna sins roteins. BL00415N 4.29 7.372e-09 255 BL01113 Clq domain proteins. BL01113B 18.26 3.786e-23 BL01113A 17.99 7.968e-15 BL01113A 17.99 5.091e-14 BL01113C 13.18 4.000e-12 BL01113A 17.99 5.295e-11 BL01113A 17.99 8.568e-11 BL01113A 17.99 8.977e-11 BL01113A 17.99 4.635e-09 BL01113A 17.99 6.192e-09 BL01113A 17.99 7.750e-09 BL01113D 7.47 7.750e-09 255 BL00420 Speract receptor repeatBL00420A 20.42 8.691e-11 proteins 73-101 domain proteins. BL00420A 20.42 9.673e-11 BL00420A 20.42 2.180e-10 BL00420A 20.42 8.062e-09 257 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 2.200e-14 BINDI. PD00066 13.92 2.800e-14 PD00066 13.92 2.800e-14 PD00066 13.92 2.800e-14 PD00066 13.92 9.000e-13 PD00066 13.92 9.000e-13 PD00066 13.92 9.000e-13 PD00066 13.92 1.857e-12 PD00066 13.92 2.714e-12 PD00066 13.92 5.714e-12 PD00066 13.92 5.714e-12 PD00066 13.92 6.087e-11 PD00066 13.92 7.261e-11 PD00066 13.92 4.808e-10 PD00066 13.92 5.154e-10 257 BL00028 Zinc finger, C2H2 type,BL00028 16.07 6.063e-15 domain 522-538 proteins. BL00028 16.07 4.150e-13 BL00028 16.07 9.609e-12 BL00028 16.07 2.385e-11 BL00028 16.07 3.423e-11 BL00028 16.07 4.808e-11 BL00028 16.07 5.154e-11 Table 3 SEQ Database Description Results*

ID entr ID

BL00028 16.07 5.154e-11 BL00028 16.07 5.500e-11 BL00028 16.07 6.538e-11 BL00028 16.07 7.300e-10 BL00028 16.07 8.800e-10 BL00028 16.07 9, IOOe-10 BL00028 16.07 6.400e-09 BL00028 16.07 7.686e-09 257 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 2.500e-17 SIGNATURE PR00048A 10.52 9.182e-15 PR00048A 10.52 4.750e-I4 PR00048A 20.52 2.059e-12 PR00048A 10.52 4.176e-12 PR00048A 10.52 5.765e-12 PR00048A 10.52 I.OOOe-11 PR00048B 6.02 1.692e-11 PR00048A 10.52 2.895e-11 PR00048A 10.52 3.842e-1 l 155-168 PR00048A 10.52 5.737e-11 PR00048B 6.02 5.846e-11 PR00048B 6.02 5.846e-11 PR00048B 6.02 9.308e-11 PR00048B 6.02 9.308e-11 PR00048B 6.02 9.308e-1 PR00048A 10.52 3.739e-10 PR00048B 6.02 5.500e-10 PR00048B 6.02 6.063e-10 PR00048A 10.52 I.OOOe-09 PR00048B 6.02 2.421e-09 PR00048A 10.52 4.960e-09 PR00048B 6.02 9.053e-09 PR00048B 6.02 9.526e-09 PR00048B 6.02 I.OOOe-08 268 PD01066 PROTEIN ZINC FINGER PD01066 19.43 7.000e-28 FINGER METAL-BINDING
NU.

268 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 5.500e-13 SIGNATURE PR00048A 10.52 5.500e-13 PR00048A 10.52 8.714e-13 PR00048A 10.52 9.357e-13 PR00048A 10.52 2.059e-12 PR00048A 10.52 2.588e-I2 PR00048A 10.52 4.176e-12 PR00048A 10.52 7.882e-12 PR00048A 10.52 4.789e-11 PR00048A 10.52 4.789e-11 PR00048A 10.52 7.632e-11 PR00048A 10.52 I.OOOe-10 PR00048A 10.52 1.000e-10 PR00048A 10.52 1.391 e-10 280-293 PR00048A 10.52 2.174e-10 PR00048B 6.02 7.750e-10 268 BL00028 Zinc finger, C2H2 type,BL00028 16.07 3.348e-12 domain 339-355 proteins. BL00028 16.07 8.435e-12 BL00028 16.07 3.077e-I

BL00028 16.07 3.769e-11 BL00028 16.07 5.500e-11 BL00028 16.07 7.577e-11 Table 3 SEQ Database Description Results*

ID entr ID

BL00028 16.07 1.000e-10 BL00028 16.07 1.300e-10 BL00028 16.07 4.000e-10 BL00028 16.07 6.400e-10 BL00028 16.07 8.800e-10 BL00028 16.07 1.771e-09 BL00028 16.07 5.886e-09 BL00028 16.07 7.686e-09 BL00028 16.07 8.457e-09 268 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 1.692e-15 BINDI. PD00066 13.92 3.077e-15 PD00066 13.92 3.077e-15 PD00066 13.92 3.077e-IS

PD00066 13.92 3.077e-15 PD00066 13.92 3.077e-15 PD00066 13.92 3.077e-15 PD00066 13.92 1.500e-13 PD00066 13.92 7.000e-09 PD00066 13.92 8.800e-09 PD00066 13.92 8.800e-09 271 BL00107 Protein kinases ATP-bindingBL00107A 18.39 1.931e-11 region 63-93 proteins.

27I PR00109 TYROSINE I~INASE CATALYTICPR00109B 12.27 3.898e-10 DOMAIN SIGNATURE PR00109D 17.04 4.203e-10 273 PD01066 PROTEIN ZINC FINGER PD01066 19.43 5.636e-36 FINGER METAL-BINDING
NU.

273 DM01970 0 kw ZK632.12 YDR313C DM01970A 8.50 8.313e-10 ENDOSOMAL III.

273 BL00028 Zinc finger, C2H2 type,BL00028 16.07 4.938e-15 domain 336-352 proteins. BL00028 16.07 4.I76e-I4 BL00028 16.07 7.882e-14 BL00028 16.07 8.435e-I2 BL00028 16.07 2.500e-10 BL00028 16.07 4.000e-10 BL00028 16.07 7.900e-10 BL00028 16.07 8.200e-IO

BL00028 16.07 1.514e-09 273 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 7.231 e-15 BINDT. PD00066 13.92 2.800e-14 PD00066 13.92 2.800e-14 PD00066 13.92 2.800e-14 PD00066 13.92 2.800e-14 PD00066 13.92 4.600e-14 PD00066 13.92 7.000e-13 PD00066 13.92 5.714e-12 PD00066 13.92 1.600e-09 273 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 1.000e-14 SIGNATURE PR00048A 10.52 4.857e-13 PR00048A 10.52 S.SOOe-13 PR00048A 10.52 7.429e-13 PR00048A 10.52 1.000e-12 PR00048A 10.52 3.118e-12 PR00048A 10.52 3.118e-12 PR00048B 6.02 3.769e-I1 PR00048B 6.02 6.538e-11 . PR00048B 6.02 6.538e-11 PR00048B 6.02 7.231e-11 Table 3 SEQ Database Description Results*

ID entr ID

PR00048B 6.02 9.308e-11 PR00048A 10.52 4.522e-10 PR00048B 6.02 7.750e-10 PR00048A 10.52 3.880e-09 PR00048B 6.02 9.053e-09 274 PD01066 PROTEIN ZINC FINGER PDOI066 19.43 4.316e-24 FINGER METAL-BINDING
NU.

274 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 7.231 e-15 BINDI. PD00066 13.92 7.000e-14 PD00066 13.92 1.000e-13 PD00066 13.92 1.000e-13 PD00066 13.92 9.000e-13 PD00066 13.92 2.286e-12 PD00066 13.92 4.429e-12 PD00066 13.92 4.857e-12 PD00066 13.92 5.714e-12 PD00066 13.92 1.391 e-11 PD00066 13.92 4.462e-10 274 PD02462 PROTEIN BOLA TRANSCRIPTIONPD02462A 22.48 6.354e-10 REGULATION AC.

274 BL00028 Zinc finger, C2H2 type,BL00028 16.07 9.357e-16 domain 307-323 proteins. BL00028 16.07 4.938e-15 BL00028 16.07 2.588e-14 BL00028 16.07 8.412e-14 BL00028 16.07 1.450e-13 BL00028 16.07 4.150e-13 BL00028 16.07 3.348e-12 BL00028 16.07 2.731e-11 BL00028 16.07 7.231e-11 BL00028 16.07 7.577e-11 BL00028 16.07 4.300e-10 BL00028 16.07 4.900e-10 BL00028 16.07 4.343e-09 274 PR00048 C2H2-TYPE ZINC FINGER _ PR00048A 10.52 5.500e-17 SIGNATURE PR00048A 10.52 1.000e-14 PR00048A 10.52 4.750e-14 PR00048A 10.52 6.250e-14 PR00048A 10.52 2.929e-13 PR00048A 10.52 7.429e-13 PR00048B 6.02 9.100e-13 PR00048A 10.52 9.357e-13 PR00048A 10.52 3.118e-12 PR00048A 10.52 3.647e-12 PR00048A 10.52 4.706e-12 PR00048A 10.52 5.765e-12 PR00048A 10.52 5.765e-12 PR00048B 6.02 6.538e-11 PR00048B 6.02 2.125e-10 PR00048B 6.02 5.500e-10 PR00048B 6.02 5.263e-09 PR00048B 6.02 1.000e-08 277 BL00028 Zinc finger, C2H2 type,BL00028 16.07 3.647e-14 domain 402-418 proteins. BL00028 16.07 6.850e-13 BL00028 16.07 2.957e-12 BL00028 16.07 5.696e-12 BL00028 16.07 1.346e-11 BL00028 16.07 1.346e-11 Table 3 SEQ Database Description Results ID entr ID

BL00028 16.07 4.808e-11458-474 BL00028 16.07 7.577e-11290-306 BL00028 16.07 7.577e-11 BL00028 16.07 4.000e-10 BL00028 16.07 7.600e-10 277 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 1.600e-14 BINDI. PD00066 13.92 2.800e-14 PD00066 13.92 2.800e-I4 PD00066 13.92 4.000e-14 PD00066 13.92 7.000e-14 PD00066 13.92 8.800e-14 PD00066 13.92 5.500e-13 PD00066 13.92 9.000e-13 PD00066 13.92 4.000e-12 PD00066 13.92 5.714e-12 PD00066 13.92 5.500e-09 277 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 1.818e-15 SIGNATURE PR00048A 10.52 8.500e-14 PR00048A 10.52 3.118e-12 PR00048A 10.52 3.118e-12 PR00048A 10.52 6.824e-12 PR00048A 10.52 2.895e-11 PR00048A 10.52 3.842e-11 PR00048A 10.52 4.316e-11 PR00048B 6.02 4.462e-11 PR00048A 10.52 5.263e-11 PR00048B 6.02 7.231e-11 PR00048B 6.02 7.231e-11 PR00048B 6.02 5.500e-10 PR00048B 6.02 5.500e-10 PR00048B 6.02 6.063e-10 PR00048B 6.02 9.438e-10 PR00048B 6.02 1.000e-09 PR00048A 10.52 2.440e-09 PR00048B 6.02 3.368e-09 PR00048B 6.02 7.632e-09 277 BL00479 Phorbol esters / diacylglycerolBL00479A 19.86 8.468e-09 binding 361-383 domain roteins.

277 PD01066 PROTEIN ZINC FINGER PD01066 19.43 2.929e-32 FINGER METAL-BINDING PD01066 19.43 9.743e-09 NU. 57-95 281 PD01066 PROTEIN ZINC FINGER PD01066 19.43 3.727e-36 FINGER METAL-BINDING
NU.

281 BL00028 Zinc forger, C2H2 type,BL00028 16.07 7.188e-I5 domain 343-359 proteins. BL00028 16.07 1.000e-14 BL00028 16.07 1.529e-I4 BL00028 16.07 4.176e-14 BL00028 16.07 1.450e-I3 BL00028 16.07 5.950e-13 BL00028 16.07 9.100e-13 BL00028 16.07 1.783e-12 BL00028 16.07 4.130e-12 BL00028 16.07 7.261e-12 BL00028 16.07 5.154e-11 BL00028 16.07 7,923e-11 BL00028 16.07 9.654e-I1 281 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 8.200e-14 BINDI. PD00066 13.92 8.200e-14 z63 Table 3 SEQ Database Description Results*

ID entr ID

PD00066 13.92 S.SOOe-13 PD00066 13.92 5.500e-13 PD00066 13.92 8.500e-13 PD00066 13.92 1.857e-12 PD00066 13.92 1.000e-11443-455 PD00066 13.92 1.000e-10 PD00066 13.92 1.000e-10 PD00066 13.92 1.346e-10 281 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 8.200e-18 SIGNATURE PR00048A 10.52 3.250e-16 PR00048A 10.52 8.875e-16 PR00048A 10.52 6.727e-15 PR00048A 10.52 6.727e-15 PR00048A 10.52 4.750e-14 PR00048A 10.52 1.000e-13 PR00048A 10.52 2.059e-12 PR00048A 10.52 8.941e-12 PR00048B 6.02 1.000e-10 PR00048B 6.02 1.000e-10 PR00048A 10.52 4.130e-10 PR00048A 10.52 4.522e-10 PR00048B 6.02 8.875e-10 PR00048B 6.02 3.368e-09 PR00048B 6.02 4.789e-09 PR00048B 6.02 6.211 e-09 PR00048A 10.52 8.560e-09 PR00048B 6.02 8.579e-09 PR00048B 6.02 9.S26e-09 285 PD01066 PROTEIN ZINC FINGER PD01066 19.43 2.500e-32 FINGER METAL-BINDING
NU.

285 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 3.077e-1S

BINDI. PD00066 13.92 7.231e-15 PD00066 13.92 5.800e-14 PD00066 13.92 8.200e-14 PD00066 13.92 8.800e-14 PD00066 13.92 4.500e-13 PD00066 13.92 7.500e-13 PD00066 13.92 3.143e-12 PD00066 13.92 1.391 e-11 285 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 4.750e-14 SIGNATURE PR00048A 10.52 2.929e-13 PR00048A 10.52 6.143e-I3 PR00048A 10.52 8.071e-13 PR00048A 10.52 1.529e-12 PR00048A 10.52 3.368e-11 PR00048A 10.52 5.263e-11 PR00048A 10.52 8.579e-11 PR00048A 10.52 2.174e-10 PR00048B 6.02 3.250e-10 PR00048B 6.02 6.625e-10 285 BL00028 Zinc fingex, C2H2 type,BL00028 16.07 7.300e-13 domain 363-379 proteins. BL00028 16.07 4.913e-12 BL00028 16.07 7.231e-11 BL00028 16.07 7.577e-11 BL00028 16.07 5.200e-10 BL00028 16.07 6.700e-10 BL00028 16.07 7.300e-10 Table 3 SEQ Database Description Results*

ID entr ID

BL00028 16.07 7.600e-10 BL00028 16.07 2.543e-09 BL00028 16.07 8.714e-09 287 BL00273 Heat-stable enterotoxinsBL00273 12.24 1.000e-08 roteins. 74-86 288 BL00412 Neuromodulin (GAP-43) BL00412D 16.54 6.870e-10 proteins. 93-I43 BL00412D 16,54 6.967e-10 288 PR00049 WILM'S TUMOUR PROTEIN PR00049D 0.00 7.500e-10 SIGNATURE

238 PR0035I MAS20 PROTEIN IMPORT PR00351C 7.03 4.974e-09 RECEPTOR SIGNATURE

288 BL00319 Amyloidogenic glycoproteinBL00319C 17.12 5.263e-09 extracellular domain BL003I9C 17.12 8.461e-09 proteins. 114-147 288 BL00422 Granins proteins. BL00422C 16.18 8.085e-10 BL00422C 16.18 7.706e-09 BL00422C 16.18 8.765e-09 290 BL00518 Zinc finger, C3HC4 typeBL00518 12,23 5.286e-10 (RING 27-35 fm er), roteiiis.

290 BL01282 BIR re eat roteins. BL01282B 30.49 4.214e-09 291 BL00518 Zinc finger, C3HC4 typeBL00518 12.23 5.286e-10 (RING 27-35 fin er), roteins.

291 BL01282 BIR repeat proteins. BL01282B 30.49 4.214e-09 295 PR00019 LEUCINE-RICH REPEAT PR00019A 11.19 8.826e-10 SIGNATURE

296 PD01066 PROTEIN ZINC FINGER PD01066 19.43 6.644e-23 FINGER METAL-BINDING
NU.

296 BL00028 Zinc finger, C2H2 type,BL00028 16.07 1.529e-14 domain 649-665 proteins. BL00028 16.07 3.250e-13 BL00028 16.07 3.700e-13 BL00028 16.07 5.500e-13 BL00028 16.07 8.650e-13 BL00028 16.07 8.435e-12 BL00028 16.07 1.692e-11 BL00028 16.07 1.692e-11 BL00028 16.07 4.808e-11 BL00028 16.07 6.538e-1 l 316-332 BL00028 16.07 4.900e-10 BL00028 16.07 5.800e-10 BL00028 16.07 2.029e-09 BL00028 16.07 2.029e-09 296 PR00048 C2H2-TYPE ZINC FINGER PR00048A 10.52 I.OOOe-16 SIGNATURE PR00048A 10.52 3.455e-15 PR00048A 10.52 6.727e-15 PR00048A 10.52 6.727e-15 PR00048A 10.52 2.500e-14 PR00048A 10.52 5.500e-14 PR00048A 10.52 5.500e-14 PR00048A 10.52 7.750e-14 PR00048A 10.52 8.500e-14 PR00048A 10.52 2.286e-13 PR00048A 10.52 3.571e-13 PR00048A 10.52 5.235e-12 PR00048A 10.52 8.941e-12 PR00048A 10.52 1.000e-11 PR00048B 6.02 6.538e-11 PR00048B 6.02 4.938e-10 PR00048B 6.02 7.188e-10 PR00048B 6.02 7.188e-10 Table 3 SEQ Database Description Results*

ID entr ID

PR00048B 6.02 8.875e-10 PR00048B 6.02 8.875e-10 PR00048B 6.02 1.474e-09 PR00048B 6.02 5.263e-09 PR00048B 6.02 7.632e-09 PR00048B 6.02 8.105e-09 296 PD00066 PROTEIN ZINC-FINGER PD00066 13.92 8.800e-14 BINDI. PD00066 13.92 8.800e-14 PD00066 13.92 8.800e-14 PD00066 13.92 1.500e-13 PD00066 13.92 2.000e-13 PD00066 13.92 2.000e-13 PD00066 13.92 2.000e-13 PD00066 13.92 3.500e-13 PD00066 13.92 9.000e-13 PD00066 13.92 2.286e-12 PD00066 13.92 2.286e-I2 PD00066 13.92 3.571e-12 PD00066 13.92 5.696e-11 PD00066 13.92 6.478e-11 PD00066 13.92 9.217e-11 PD00066 13.92 1.000e-08 299 BL00518 Zinc finger, C3HC4 typeBL00518 12.23 2.800e-1 (RING I 75-83 forger), proteins.

301 BL00254 Interleukin-6 / G-CSF BL00254A 10.66 7.750e-09 / MGF proteins. 24-37 301 BL00726 AP endonucleases familyBL00726C 19.90 9.735e-09 1 roteins. 78-103 302 PR00289 DISINTEGRIN SIGNATURE PR00289A 13.62 7.085e-09 *Results include iii order: accession number subtype; raw score; p-value;
position of signature in amino acid sequence.

Table 4A
SEQ Pfam Model Description E-value Score TD
NO:

152 IL8 Small cytokines (intecrine/chemokine),2.5e-34 118.8 interleukin-8 like 153 EGF EGF-like domain 2.3e-07 37.9 154 EGF EGF-like domain 2.2e-23 91.1 155 IL8 Small cytokines (intecrine/chemokine),1.7e-I6 59.4 interleukin-8 like 156 IL8 Small cytokines (intecrine/chemokine),1.5e-29 102.9 interleukin-8 like 157 PTN MK PTN/MK he arin-binding rotein 1.4e-95 330.9 family 158 F5 F8 a F5/8 type C domain 3e-49 177.0 C

159 ldl rece Low-densi 1i o rotein rece 4.3e-117402.4 t b for re eat class B

160 Granin Granin chromo anin or secretogranin)5.7e-173588.0 161 sushi Sushi domain (SCR repeat) 4e-22 53.7 162 vwa von Willebrand factor t a A 6e-73 255.8 domain I63 IL8 Small cytokines (intecrine/chemokine),2e-10 39.2 interleukin-8 like 164 IL8 Small cytokines (intecrine/chemokine),2e-10 39.2 interleukin-8 like 165 Cys knot Cystine-knot domain 3.3e-52 186.9 167 ILl Interleukin-1 / 18 6.9e-20 74.8 168 MMR HSRI GTPase of unknown function 0.0011 -45.5 175 adh short short chain dehydro enase 1.3e-50 181.6 176 cyclin Cyclin, N-terminal domain 0.077 -10.2 178 Peptidase Papain family cysteine rotease4.8e-101338.4 182 ECH Enoyl-CoA hydratase/isomerase 7.1e-85 295.4 family 183 RrnaAD Ribosomal RNA adenine dimethylase0.045 9.2 189 pyr redox P idine nucleotide-disul hide 0.0075 -46.1 oxidoreductase 191 QRPTase Quinolinate phosphoribosyl 1.5e-87 300.9 transferase, C-terminal domain 193 DHOdehase Dihydroorotate dehydro enase 1.3e-169576.9 195 kazal Kazal- a serine rotease inhibitor4e-15 63.7 domain 197 450 Cytochrome P450 9.4e-204690.3 198 p450 Cytochrome P450 1.3e-213723.1 202 i Immunoglobulin domain l.Ie-09 36.3 203 zf C2H2 Zinc finger, C2H2 a 6e-41 149.5 205 filament Intermediate filament rotein 1.4e-154526.9 ~

206 Armadillo Armadillo/beta-catenin-like 0.046 20.3 se repeat 210 zf C3HC4 Zinc finger, C3HC4 t a (RING 5.2e-11 39.9 fm er) 213 ig Immunoglobulin domain 5.1 e-1863.1 214 ig Immuno lobulin domain ' 1.9e-09 35.5 215 ig Immuno lobulin domain 7.2e-10 36.8 217 Xlink Extracellular link domain 3e-68 158.8 219 asp Eukaryotic aspartyl rotease 1.6e-204692.9 221 ig Immuno lobulin domain 5.4e-08 30.8 222 i Immuno lobulin domain 1.1e-18 65.2 223 ig Immuno lobulin domain 1.1e-18 65.2 226 ig Immuno lobulin domain 2.1e-07 28.9 227 i Immuno lobulin domain 8.5e-12 43.1 229 ank Ank re eat l.Ie-13 59.0 230 fibrinogen_CFibrinogen beta and gamma chains,1.3e-89 311.1 C-terminal lobular domain 231 i Immuno lobulin domain 6.6e-08 30.5 232 i Immuno lobulin domain 5e-07 27.7 Table 4A
SEQ Pfam Model Description E-value Score zD
NO:

233 LRR Leucine Rich Re eat 8.5e-28 105.8 236 lectin c Lectin C- a domain 1.7e-08 41.7 238 MAM33 Mitochondrial lyco rotein 1.4e-85 297.7 241 fn3 Fibronectin t a III domain 4.1e-102352.6 242 7tm 1 7 transmembrane rece for (rhodo3.5e-17 56.7 sin family) 243 myosin headMyosin head (motor domain) 5.6e-57 198.7 247 lipocalin Lipocalin / cytosolic fatty-acid3.9e-35 125.1 binding protein family 249 zf C2H2 Zinc finger, C2H2 type 3.9e-12 53.7 250 Peptidase Peptidase family M41 Se-44 159.7 254 Clq Cl domain 2.7e-13 55.1 255 C1 C1 domain 6.4e-29 109.5 257 zf C2H2 Zinc fin er, C2H2 type 1.2e-119410.9 268 zf C2H2 Zinc fin er, C2H2 a 1.7e-104360.5 269 PH PH domain 2.9e-06 27.3 271 kinase Protein ltinase domain 2.4e-25 95.3 272 ig Immuno lobulin domain 0.0015 16.5 273 zf C2H2 Zinc fm er, C2H2 a 1.7e-65 231.0 277 zf C2H2 Zinc fin er, C2H2 a 1,1e-84 294.8 281 zf C2H2 Zinc finger, C2H2 type 5,2e-101349.0 285 zf C2H2 Zinc finger, C2H2 type 1.4e-60 214.7 290 zf C3HC4 Zinc finger, C3HC4 type (RING 2,9e-14 50.5 ~ fin er) 291 zf C3HC4 Zinc finger, C3HC4 t a (RING 2.9e-14 50.5 fin er) 296 zf C2H2 Zinc finger, C2H2 type 2.6e-124426.4 299 zf C3HC4 Zinc finger, C3HC4 a (RING 7.8e-05 19.9 fin er) 301 EMP24 GP25Lemp24/ 2SL/ 24 family 5.2e-06 30.3 Table 4B
SEQ Model Description E-value Score RepeatsPosition ID

152 IL8 Small cytokines 2.6e-32 120.8 1 59-127 (intecrine/chemokine), inter 153 EGF EGF-like domain 2.3e-07 37.9 2 93-126:138-153 CollagenCollagen triple 0.0012 7.7 1 271-329 helix repeat (20 co ies 153 TIL Trypsin Inhibitor 1.8 -6.7 1 85-138 like cysteine rich domain 154 EGF EGF-like domain 2.2e-23 91.1 9 37-67:80-110:123-153:166-I96:209-239:252-282:295-325:338-368:381-411 154 DSL Delta serrate ligand1.9 -15.2 1 183-239 155 IL8 Small cytokines 0.0004 21.4 1 68-I08 (intecrinelchemokine), inter 156 IL8 Small cytokines 1.7e-27 104.8 1 68-133 (intecrinelchemokine), inter 157 PTN MK PTN/MK heparin-binding1.4e-95 330.9 1 1-146 protein family 157 tsp 1 Thrombospondin type1.5 -2.9 1 86-132 1 domain 158 FS_F8_tyF5/8 type C domain 3e-49 177.0 1 34-174 a C

158 laminin_GLaminin G domain 5.2e-43 156.3 4 212-344:398-527:821-943:1046-158 EGF EGF-like domain 4.6e-07 36.9 2 553-585:962-996 158 TSPN Thrombospondin N-terminal0.71 -42.4 1 981-1178 -like domain 158 Alpha Alphavirus lyco 8.9 -10.8 1 1165-1287 GJ rotein J

158 COLFI Fibrillar collagen 9.6 -131.11 581-786 C-terminal domain 159 ldl_receptLow-density lipoprotein4.3e-117402.4 10 567-receptor b repeat 608:610-651:653-695:697-738:740-780:872-913:915-956:958-1000:1002-1043:1044-159 ldl_receptLow-density lipoprotein1.5e-110380.7 8 I02-receptor a domain 147:148-186:187-224:225-263:268-306:316-TahlP 4R
SEQ Model Description E-value Score RepeatsPosition ID

354:355-393:397-438 159 EGF EGF-like domain 1.9e-06 34.8 9 150-184:189-222:227-261:318-352:357-391:445-480:486-520:789-823:1093-159 NHL NHL repeat 0.38 17.2 2 597-625:902-930 159 metalthioMetallothionein 4.7 -9.3 1 440-509 159 SMP-30 Senescence marker 7.5 -179.61 853-1092 protein-30 (SMP-30) 159 TILa TILa domain 9.6 -9.6 1 435-491 160 Granin Granin (chromogranin6.7e-173587.8 1 64-520 or secreto anin) 161 sushi Sushi domain (SCR 4e-12 53.7 1 24-77 re eat) 161 EGF EGF-like domain 2.6e-11 51.0 3 83-114:171-211:217-261 161 ranulin Granulin 6.3 -17.2 1 186-241 161 EB EB module 6.4 -10.5 1 50-114 161 TIL Trypsin Inhibitor 9.4 -15.2 1 105-171 like cysteine rich domain 162 vwa von Willebrand factor6e-73 255.8 1 193-365 type A

domain 162 EGF EGF-like domain 8.8e-07 36.0 1 145-180 162 DUF296 Domain of unknown 8.8 -42.8 1 20-208 - function (DUF296) 162 NTF2 Nuclear transport 8.9 -23.0 1 211-316 factor 2 (NTF2) domain 163 IL8 Small cytokines 7.3e-08 39.6 1 94-154 (intecrine/chemokine), int 163 TFIID_30Transcription initiation9.3 -21.0 1 82-117 factor kD TFIID 23-3 164 IL8 Small cytokines 7.3e-08 39.6 1 24-84 (intecrine/chemokine), int 164 TFIID Transcription initiation3.5 -17.7 1 13-47 30 factor -kD TFIID 23-3 165 Cys knotCystine-knot domain8.3e-53 188.9 1 38-148 166 EGF EGF-like domain 1.1 14.4 1 9-45 167 IL1 Interleukin-1 / 7.1e-18 72.8 1 54-200 168 MMR_HS GTPase of unknown 0.0011 -45.5 1 15-281 function 168 GTP EFT Elongation factor 0.066 -63.8 1 111-346 Tu GTP

U b indin domain 168 KH- KH domain 0.49 8.1 1 370-413 domain 168 ras Ras family 0.73 -127.11 115-402 172 thiored Tllioredoxin 0.03 -16.6 1 97-202 173 SemialdhySemialdehyde dehydrogenase,2.3 -47.8 1 52-165 de dh NAD bindin Table 4B
SEQ Model Description E-valueScore RepeatsPosition ID

I75 adh shortshortchain dehydro 3.7e-51183.4 1 31-240 enase 176 cyclin Cyclin, N-terminal 0.27 -11.1 1 242-381 domain 178 PeptidasePapain family cysteine3.9e-9S329.5 1 80-311 protease 182 ECH Enoyl-CoA hydratase/isomerase7.1e-85295.4 1 39-196 family 182 PeptidasePeptidase family 1 -59.4 1 104-216 183 PCMT Protein-L-isoaspartate(D-4.2 -131.71 165-374 aspartate) O-methyl 189 pyr redoxPyridine nucleotide-disulphide0.013 -45.3 1 45-327 oxidoreducta 191 QRPTase Quinolinate phosphoribosyl7.5e-86298.6 1 114-301 transferase, C-f 19I QRPTase Quinoliiiate phosphoribosyl1.2e-35131.8 1 27-112 N transferase, N-t 193 DHOdeha Dihydroorotate dehydrogenase1.9e-170579.7 1 177-477 se 194 Acyl- Cytosolic long-chain0.6 -31.8 1 120-237 acyl-CoA

CoA~hydrthioeste ~

195 kazal Kazal-type serine 4e-15 63.7 1 120-174 protease inhibitor domain 197 450 Cytochrome P450 4.3e-182618.3 1 33-471 198 p450 C ochrome P450 1.3e-195663.2 1 33-471 202 i Immuno lobulin domain2.7e-0737.6 1 51-126 203 zf C2H2 Zinc finger, C2H2 5.8e-41149.5 7 174-type 196:202-224:230-252:258-280:359-381:387-409:415-437 203 KRAB KR AB box 2.1e-0841.4 1 32-72 203 FYVE FYVE zinc fin er 0.38 -12.4 1 165-241 205 filamentIntermediate filament3.9e-155528.8 1 120-431 protein 205 Tektin Tektin family 4.2 -236.81 80-441 205 V- Vacuolar (H+)-ATPase5.9 -48.7 1 298-411 G

ATPase_ subunit G

205 ApolipoprApolipoprotein A1/A4/E7 -115.41 138-388 family otein 205 Trypan_glTrypanosome variant 7.9 -103.01 177-549 surface yco lycoprote 206 ArmadilloArmadillo/beta-catenin-like0.046 20.3 2 148-se repeat 189:231-269 210 SPRY SPRY domain l.le-IO48.9 1 310-432 Z10 zf C3HC4Zinc finger, C3HC4 1.6e-0841.7 1 _ type (RING 21-62 fin er 210 zf MIZ MIZ zinc fin er 0.37 -10.2 1 19-68 210 PHD PHD-fm er 9.7 -19.0 1 20-65 213 ig Immunoglobulin domain8.1e-1356.0 3 _ 44-120:155-212:247-296 214 ig Immunoglobulin domain1.6e-0635.1 _ 41-124:156-Table 4B
SEQ Model Description E-value Score RepeatsPosition ID

215 i Immuno lobulin domain2e-07 38.1 1 38-113 217 Xlink Extracellular link 2.6e-44 160.7 1 31-119 domain 219 as Eukar otic as amyl 1.6e-204692 1 I7-456 protease .9 220 UPAR_L u-PAR/Ly-6 domain 1.8 _ 1 21-96 -16.0 220 Zn_clusFungal Zn(2)-Cys(6) 5.1 -5.7 1 21-60 binuclear cluster domain 22I ig Immuno lobulin domain7e-06 33.0 1 169-245 221 kazal Kazal-type serine 7.5e-05 _ 1 91-151 protease 29.6 inhibitor do 221 IGFBP Insulin-like growth 0.39 -1.4 1 38-97 factor bindin rot 221 TransposaTransposase 5.1 -15.5 I 152-240 se 9 221 GYR GYR motif 5.7 I0.0 1 117-134 222 ig Immunoglobulin domain2.4e-17 71.1 2 31-110:153-223 ig Immunoglobulin domain2.4e-17 71.1 2 31-110:153-224 UPAR_L u-PAR/Ly-6 domain 1.8e-05 31.6 1 28-110 226 i Immuno lobulin domain0.00015 28.5 1 40-115 227 i Immunoglobulin domain9.6e-09 42.5 1 37-111 229 ank Ankyrin repeat 8.6e-14 59.3 2 345-383:384-417 230 fibrinogenFibrinogen beta and 4.1e-90 312.8 1 240-457 gamma C chains, C-term 231 i Immunoglobulin domain5.8e-06 33.2 1 31-115 232 ig Immuno lobulin domain7.5e-06 _ 1 34-117 2.9 233 LRR Leucine Rich Repeat 7.2e-28 _ 7 45-68:69-_ 106.0 92:93-115:117-140:141-164:165-188:189-212 236 lectin Lectin C-type domain1.7e-08 4 1 159-263 c 1.7 236 CollagenCollagen triple helix8.6e-08 _ 1 53-112 repeat (20 39.3 ' co ies) 238 MAM33 Mitochondrial lycoprotein1.4e-85 297.7 1 86-261 240 tsp Thrombos ondin t 0.22 4.3 1 I55-210 1 a I domain 240 PSI Plexin repeat 8.7 -5.6 1 102-149 253 MMR_HS GTPase of unknown 9.7 -103.51 41-268 function 254 Clq Clq domain 1e-05 13.2 1 111-230 254 CollagenCollagen triple helix0.0043 -3.0 1 50-107 repeat (20 co ies) 255 Clq Clq domain 1.8e-29 111.4 I 111-235 255 CollagenCollagen triple helix0.0043 -3.0 1 50-107 repeat (20 co ies 257 zf C2H2Zinc finger, C2H2 1.4e-119410.6 17 128-type 150:156-178:184-206:2I2-234:240-262:268-Table 4B
SEQ Model Description E-value Score RepeatsPosition ID

290:296-318:324-346:352-374:380-402:408-430:436-458:464-486:492-514:520-542:548-570:576-598 257 zf BED BED zinc finger 0.91 1.9 1 449-487 257 VSP Giardia variant-specific2 -247.61 270-556 surface protein 257 GATA GATA zinc fnger 4.6 -9.7 1 182-210 257 zf MIZ MIZ zinc fin er 7.9 -20.6 1 423-475 257 LIM LIM domain 8.4 -19.5 1 438-502 257 Yippee Yippee putative zinc-binding8.9 -69.4 1 338-422 rotein 264 RibosomaRibosomal protein 2.4 -18.1 1 105-162 1 L35p 268 zf C2H2Zinc finger, C2H2 1.8e-104360.5 15 103-type 125:141-163:169-191:197-219:225-247:253-275:281-303:309-331:337-359:365-387:393-415:421-443:449-471:477-499:505-527 268 KRAB KRAB box 2.6e-22 87.5 1 4-44 268 zf MIZ MIZ zinc finger 0.34 -9.9 1 179-236 268 LIM LIM domain 1.7 -13.6 1 227-291 268 zf BED BED zinc finger 3.8 -3.6 2 85-126:322-269 PH PH domain 0.0063 23.2 1 51-167 271 plcinaseProtein kinase domain8e-10 6.6 1 1-208 271 WD40 WD domain, G-beta 1.5 15.3 2 268-repeat :313-355 273 zf C2H2Zinc finger, C2H2 1.8e-65 230.9 12 _ type _ 191:223-244:250-272:278-300:306-328:334-356:362-384:390-412:418-440:446-Table 4B
SEQ Model Description E-value Score RepeatsPosition ID

468:474-496:502-521 273 KRAB DRAB box 1.3e-25 98.5 1 6-46 273 zf BED BED zinc fin er 2.9 -2.6 1 459-497 274 zF C2H2Zinc finger, C2H2 1.2e-96 334.5 13 249-type 271:277-299:305-327:333-355:361-383:389-411:417-439:445-467:473-495:501-523:529-551:557-579:585-607 274 KRAB KR.AB box 3.2e-23 90.6 1 13-53 274 zf BED BED zinc finger 0.081 11.3 1 514-552 274 LIM LIM domain 0.76 -10.7 1 307-371 274 BolA BolA-like protein 3.4 -22.8 1 212-289 274 TFIIS Transcription factor5.7 -5.1 1 308-343 S-II (TFIIS) 274 FYVE FYVE zinc finger 7.3 -24.9 1 266-344 277 zf C2H2Zinc forger, C2H2 1.2e-84 294.6 11 204-type 226:232-254:260-282:288-310:316-338:344-366:372-394:400-422:428-450:456-478:484-506 277 KRAB I~R_AB box 8.4e-24 92.5 1 14-54 277 FYVE FYVE zinc finger 1 -16.7 1 338-411 277 Yippee Yippee putative zinc-binding5.5 -67.4 1 209-320 protein 277 DM- DM DNA binding domain6.7 -11.7 1 454-503 domain 281 zf C2H2Zinc finger, C2H2 5.7e-101348.9 13 173-type 195:201-223:229-251:257-279:285-307:313-335:341-363:369-391:397-419:425-447:453-475:481-503:509-531 281 KRAB KR.AB box 5.3e-24 93.2 1 4-44 281 L1M LIM domain 1.2 -12.5 1 287-351 Table 4B
SEQ Model Description E-valueScore RepeatsPosition ID

281 TFIIS Transcri Lion factor3.7 -3.5 1 313-351 S-II (TFIIS) 281 PHD PHD-fin er 6 -17.1 1 202-265 281 UPAR_L u-PAR/Ly-6 domain 7.9 -22.8 1 113-185 285 zf C2H2 Zinc finger, C2H2 1.5e-60214.6 10 137-type 159:193-215:221-243:249-271:277-299:305-327:333-355:361-383:389-411:417-439 285 KR.AB KRAB box 3.7e-2493.7 1 1-41 285 zf BED BED zinc fin er 1.1 1.0 1 289-328 290 zf C3HC4Zinc finger, C3HC4 1.1e-1152.2 1 12-50 type (RING

forger) 290 SPRY SPRY domain 0.0001624.7 1 359-483 290 zf B B-box zinc fm er 0.0001728.3 1 90-130 box 291 zf C3HC4Zinc finger, C3HC4 1.1e-1152.2 1 12-50 type (RING

fin er) 291 SPRY SPRY domain 0.0001624.7 1 381-505 291 zf B B-box zinc finger 0.0001728.3 1 90-130 box 291 Herpes Herpesvirus leader 7.8 -88.5 1 248-415 L protein P

295 LRR Leucine Rich Repeat 0.09 19.3 2 42-63:64-87 296 zf C2H2 Zinc finger, C2H2 2.7e-124426.3 17 82-104:230-type 252:258-280:286-308:314-336:342-364:370-392:398-420:426-448:454-476:482-504:510-532:538-560:566-585:591-613:619-641:647-669 296 KRAB KRAB box 3.1e-1977.3 1 5-45 296 LIM LIM domain 2.2 -14.7 1 593-647 296 TFIIS Transcription factor6 -5.3 1 373-408 S-II (TFIIS) 299 zf C3HC4Zinc finger, C3HC4 0.0037 21.4 1 60-87 type (RING

fin er) 299 zf B B-box zinc fm er 0.21 6.7 1 130-168 box 301 EMP24 emp24/gp25L/p24 family5.4e-0630.0 1 16-162 G

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td t~7 DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:

Claims (26)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-151.

2. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.

3. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 99% sequence identity with the polynucleotide of claim 1.

4. The polynucleotide of claim 1 wherein said polynucleotide is DNA.

5. An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.

6. A vector comprising the polynucleotide of claim 1.

7. An expression vector comprising the polynucleotide of claim 1.

8. A host cell genetically engineered to comprise the polynucleotide of claim 1.

9. A host cell genetically engineered to comprise the polynucleotide of claim operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.

10. An isolated polypeptide, wherein the polypeptide is selected from the group consisting of:
(a) a polypeptide encoded by any one of the polynucleotides of claim 1;
and (b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-151.

11. A composition comprising the polypeptide of claim 10 and a carrier.

12. An antibody directed against the polypeptide of claim 10.

13. A method for detecting the polynucleotide of claim 1 in a sample, comprising:
a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.

14. A method for detecting the polynucleotide of claim 1 in a sample, comprising:
a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions;
b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.

15. The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA
polynucleotide.

16. A method for detecting the polypeptide of claim 10 in a sample, comprising:
a) 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 b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.

17. A method for identifying a compound that binds to the polypeptide of claim 10, comprising:

a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.

18. A method for identifying a compound that binds to the polypeptide of claim 10, comprising:
a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.

19. A method of producing the polypeptide of claim 10, comprising, a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of any of the polynucleotides from SEQ ID NO: 1-151, under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).

20. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of any one of the polypeptides SEQ ID NO: 152-302.

21. The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.

22. A collection of polynucleotides, wherein the collection comprising of at least one of SEQ ID NO: 1-151.

23. The collection of claim 22, wherein the collection is provided on a nucleic acid array.

24. The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.

25. The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.

26. The collection of claim 22, wherein the collection is provided in a computer-readable format.