Nucleic acids and polypeptides

Abstract

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

Description

1. BACKGROUND OF THE INVENTION

1.1 Technical Field

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

1.2 Background

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

2. SUMMARY OF THE INVENTION

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 ID NO:1-441 and are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A is adenine; C is cytosine; G is guanine; T is thymine; and N is any of the four bases. In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.

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

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

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

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

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

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

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

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 process 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., in 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 form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected. The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.

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 for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound the binds to a polypeptide of the invention is identified.

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

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

3. DETAILED DESCRIPTION OF THE INVENTION

3.1 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 some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules. The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.

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

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, C is cytosine, T is thymine, G is guanine and N is A, C, G or T (U). It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provided herein is substituted with U (uracil). Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.

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

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

The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NOs: 1-441. The sequence information can be a segment of any one of SEQ ID NOs: 1-441 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-441. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 4 20 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 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÷4 25 ) times the increased probability for mismatch at each nucleotide position (3×25). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.

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

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

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 totitpotent 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 resides 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, lipidation 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 insertion, deletions, and substitutions, created using, e.g., recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conversed regions) or by replacing amino acid 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 prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.

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, hydrophibicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. “Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertion, 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 terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological maromolecules, 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 normally present in a solution of the same. The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.

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

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 enhances, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

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 enhances. 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 hot cell. “Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. “Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum. “Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et al. (1998) Annu. Rev. Immunol. 16:27-55).

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 term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1X 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/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

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

3.2 NUCLEIC ACIDS OF THE INVENTION

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

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

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

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: 1-441 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of SEQ ID NO: 1-441 or a portion thereof as a probe. Alternatively, the polynucleotides of SEQ ID NO: 1-441 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.

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

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

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

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 charge 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 11 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 change amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983). A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.

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

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

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 polynucelotides 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: 1-441, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. Also included are the cDNA inserts of any of the clones identified herein.

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, N.Y.). Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phase derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organisms.

The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NOs: 1-441 or a fragment thereof or any other polynucleotides of the invention. In one embodiment, the recombinant constructs of the present invention comprises a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleoitde sequences of SEQ ID NOs: 1-441 or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (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. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

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

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

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.

3.3 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: 1-441, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprises a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 1-441 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-441 are additionally provided.

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 ID NO: 1-441, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is an oligonucleotide that is antisense by 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-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine, Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

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

3.4 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 Haselfhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of an mRNA. A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-441). For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a SECX-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, SECX mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

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

In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) 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 chimers, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 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) Bioorg Med Chem 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. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.

3.5 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 further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.

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

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

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

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

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

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

The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequence 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. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein its entirety.

3.6 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 ID NO: 1-441 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NOs: 1-441 or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in SEQ ID NOs: 1-441 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 1-441 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions. The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 1-441 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 1-441.

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.

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

Protein compositions of the present invention may further comprise an acceptable carrier, 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. As the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. The synthetically-constructed protein sequences, by virtue of sharping 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-exchanged chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Proteins Purification: Principles and Practice, Springer-Verlag (1994); Sambrook et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology. Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.

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

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

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

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 MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed”.

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

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 to 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, azathiprine, CD3 antibodies and steroids. Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.

3.6.1 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(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp. Biol., Vol 6, pp. 219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, Vol. 4, pp. 202-209, herein incorporated by reference), pFam software (Sonnhammer et al., Nucleic Acids Res., Vol. 26(1), pp. 320-322 (1998), herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference). The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda. Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).

3.7 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. In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention. Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other. The polypeptide can be used 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 extracellularly domain of a second protein. In other 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 in vivo. The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand. Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e.g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the imunoglobulin 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 ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphate 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.

3.8 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, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (19889); Verma, Scientific American: 68 -84 (1990); and Miller, Nature, 357: 455-460 (1992). Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease sates. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.

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

The present invention still further provides cell 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-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

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 even may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthin-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. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

3.9 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. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122, incorporated herein by reference.

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

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

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.

3.10 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.

3.10.1 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 kit 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, Berger, S. L. and A. R. Kimmel eds. 1987.

3.10.2 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 carbon source, use as a nitrogen source and use as 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.

3.10.3 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, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitations 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. 137:494-3400, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761, 1994. Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology, J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin-γ, Schrieber, 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 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto, 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin-6—Nordan, R. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

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

3.10.4 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 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).

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

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 disorder. 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: Principles of Tissue Engineering eds. Lanza et al., Academic Press (1997)). Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.

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 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).

3.10.5 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 York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I, Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

3.10.6 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 tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

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. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (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. Invest. Dermatol 71:382-84 (1978).

3.10.7 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 immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.

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 be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention. The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).

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. Immunosupression 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 erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

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 β 2 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); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect TH1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

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

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; Mecatonia 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 homeostatis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.

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.

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

3.10.9 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, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic compositions (e.g. proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune response 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. Suitably assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.

3.10.10 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. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.

3.10.11 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.

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

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 HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

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

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

3.10.12 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, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses. Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.

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.

3.10.13 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 eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.

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, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).

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

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.

3.10.14 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 response of the two cell populations to the addition of ligands(s) are then compared. Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s). As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.

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

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

3.10.16 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 myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

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

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).

3.10.18 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.

3.10.19 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 immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately. For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.

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

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.

3.10.20 Arthritis and Inflammation

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

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

3.11 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.

3.11.1 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 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight. For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin. The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.

3.12 Pharamaceutical 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, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF 2 , G-CSF, Meg-CSF, thombopoietin, stem cell factor, and erythropoietin. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.

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 hametopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents). A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.

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

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

3.12.1 Routes 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.

3.12.2 Compositions/Formulations

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

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, have due regard to pH, isotonicity, stability, and the like, as within the skill in the art. A preferred embodiment pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily to combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, 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 tables 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 of 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 capasules 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 unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the computer and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

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:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. Thus 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 deliveries 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 carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibody 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 carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.

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

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, polyactic acid, polyglycolic acid and polyanhyrides. 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 polyactic 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, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

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 formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example X-rays, histomorphometric determinations and tetracycline labeling.

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

3.12.3 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 subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 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 LD 50 (the dose lethal to 50% of the population) and the ED 50 (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 LD 50 and ED 50 . 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 ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration of 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 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.

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

3.12.4 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 carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

3.13 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, F ab , F ab′ and F (ab′)2 fragments, and an F ab 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 IgG 1 , IgG 2 and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO: 1-441, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of alpha-2-macroglobulin-like protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

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, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, full-length polypeptides of the invention. As with antibodies that are specific for full length polypeptides of the invention, antibodies of the invention that recognize fragments are those which can distinguish polypeptides from the same family of polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.

Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of a polypeptide of the invention), diagnostic purposes to detect or quantitate a polypeptide of the invention, as well as purification of a polypeptide of the invention. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific. The invention further provides a hybridoma that produces an antibody according to the invention. Antibodies of the invention are useful for detection and/or purification of the polypeptide 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 metastic 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 in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed. The antibodies may also be used directly in therapies or other diagnostics. The present invention further provides the above-described antibodies immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose®, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzy. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immuno-affinity purification of the proteins of the present invention.

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

3.13.1 Polyclonal Antibodies

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

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., Philadephia, Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

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

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 enzymatic hypoxanthine 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, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

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

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

3.13.3 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. Pat. No. 5,225,539). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR of 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)).

3.13.4 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. 77-76). 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. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al., ( Bio/Technology 10, 779-783 (1992)); Lonberg et al. ( Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, ( Nature Biotechnology 14, 845-51 (1996)); Neuberger ( Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar ( Intern. Rev. Immunol. 13 65-93 (1995)).

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

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

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

3.13.5 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. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specifically for a protein or derivatives, fragments, analogs or homologous 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 F ab fragment generated by reducing the disulfide bridges of an F (ab′)2 fragment; (iii) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.

3.13.6 Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the presence 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-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixing 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 chromatogaphy steps. Similar procedures are disclosed in WO 93/08829, published May 13, 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 first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

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

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

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. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers have 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. Immunol. 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 Fe reports for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antiben described herein and further binds tissue factor (TF).

3.13.7 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 cell (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

3.13.8 Effector Functional 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 residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

3.13.9 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 inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 13 I, 131 In, 90 Y, and 186 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), iminohiolane (11), 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 fluoride compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

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.

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

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

As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a “target sequence” can be any nucleic acid or amino acid sequences 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 of combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

3.15 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, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.

3.16 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 label.

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

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

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.

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

3.17 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 in vivo at the target site.

3.18 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 NOs: 1-441, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of:

(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 he 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,” In Synthetic Peptides, A User's Guide, W. H. Freeman, N.Y. (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 Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.

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.

3.19 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 NOs: 1-441. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NOs: 1-441 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.

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

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

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

3.20 Preparation of Support Bound Oligonucleotides

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

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

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 Technolgoies (Alameda, Calif.).

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

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

More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm 7 ), is then added to a final concentration of 10 mM 1-MeIm 7 . A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm 7 , is made fresh and 25 ul added per well. The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).

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

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

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

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

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

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

3.22 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 nl of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm 2 , depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones. Each of the subarrays may represent replica spotting of the same samples. In one example, a selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane. Subarrays may contain 64 samples, one from each patient. Where the 96 subarrays are identical, the dot span may be 1 mm 2 and there may be a 1 mm space between subarrays.

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

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.

4.0 EXAMPLES

4.1. Example 1

Novel Nucleic Acid Sequences 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 (ABI) sequencer to obtain the novel nucleic acid sequences. In some cases RACE (Random Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.

4.2 Example 2

Novel Nucleic Acids

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

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

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

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

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

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

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

TABLE 1
		HYSEQ
	LIBRARY/	LIBRARY
TISSUE ORIGIN	RNA SOURCE	NAME	SEQ ID NOS:
Adult brain	GIBCO	AB3001	76-77 91 106-107 115 134 163-164 178 203
			232 255 265 276 279 322-323
Adult brain	GIBCO	ABD003	16 19 24 77 80-81 85 89-90 92 96 98 105
			110 116 121-123 125 130-132 134-136 138
			142-143 151 153 158-159 163-164 184 191
			193 196 198 200 208-209 213-214 216 219-220
			223 229 232-234 236 239 241 243 257-259
			262 265 267 274-276 278 284 292 302
			317 321 324-325 327 337-338 340 348 359
			371 391-392 400
Adult brain	Clontech	ABR001	1 18-19 35 80 98 125 136 153 185 200 209
			221 228-229 239 243 274-275 302 399-400
Adult brain	Clontech	ABR006	5 7-8 18 32 35 52 57 85 91 96 111 113
			126 131 135 138-139 142 148 153-154 181
			188 192 199 209-211 217 221 224 226 229
			233 235 238 243 248 273 283-284 286 292
			316 322 348 357 361 367 376 378 399 407
			409 417 428
Adult brain	Clontech	ABR008	2 4 6-11 19-21 23-25 31 35-37 39-41 45-46
			72-73 76 80-81 85 88-90 94-95 97 102-105
			109 111-112 114-119 121-122 126-131
			134-135 138-139 144 146-150 152-153 156-157
			159 168-172 174-175 178 180 182 185-186
			189-190 194 196 198-201 203 205-210
			217 219 221-222 224 229-230 232-233 236-239
			243-244 248 253-256 260-261 263-265
			273 276 281-282 286-289 291-292 299-300
			302 304 315-317 319 321-322 324 326 329
			331-332 341 352-357 360 362 365 367-368
			370 376-377 379-380 383-384 387-389 391-392
			394 396-402 407-410 412-413 419 425-426
			433
Adult brain	Clontech	ABR011	85 90
Adult brain	BioChain	ABR012	148 213
Adult brain	Invitrogen	ABR013	85 322
Adult brain	Invitrogen	ABR014	9 23 85 146 200 233 282 321 330
Adult brain	Invitrogen	ABR015	14 31 69 121 124 163 209 216 224 291 377
Adult brain	Invitrogen	ABR016	92 136 219 279
Adult brain	Invitrogen	ABT004	2 7-8 20-21 33 85 90-91 95 97 102-103
			108 121 123 129-131 138-139 143 146 151
			153 157-158 172 178 180 209-210 213 219
			229-230 232 234 239 308 321 330 360 365
			370-373 375 401 412
cultured	Strategene	ADP001	3-4 23 36 79 81 106-107 116 129 133-134
preadipocytes			147 151 154 158 179 181 192 196 222 230
			256-257 287 292 297 313 329 359
adrenal gland	Clontech	ADR002	2 25 27 33 57 76 85-86 88 96 98 105-108
			114 121-122 125 129-130 134 147 164 178
			180 182 198-199 201 205 207-208 240-241
			244 246 253-254 257 261 276 280 292 320
			329 336 352 403
Adult heart	GIBCO	AHR001	3 17-21 27 32 74 76 85 89-91 95-96 102-103
			105-110 117 121 124-125 128 131 134-136
			139 141 148 151-153 155-156 161 163
			181-182 186 190 193 198 200-201 205 207
			211-213 215 222 225 229-230 234 251-254
			257-259 263 274-277 280 292-297 301 303-304
			315-316 319 329-331 345 359 384 417
			423-424
Adult kidney	GIBCO	AKD001	3-5 14 16 19-29 34-35 76 79 85 89-90 94-95
			97 101-103 106-107 115 117-118 121-122
			124-125 128-130 133-136 138 144 146
			158-159 163 166 171 177-178 182 185-186
			189 194 196-198 200-201 205 207 211-213
			216-217 223 225 228-232 243 247-248 253-255
			257 267 272 277-278 283 287 291 298-301
			315 326 358-359 364
Adult kidney	Invitrogen	AKT002	3 6 14 20-21 25-26 76 79 85 89 94 101
			111 114 118 121 124 126 130-131 138 146
			163 170 177-178 189 196 198 201 204 213
			231 253-254 256-259 271 273-275 277 298
			315 320 329 342
Adult lung	GIBCO	ALG001	4 29 74 79 85 90 96 105 111 119 132 134
			136 142 144 149 159 181 189 198 200 205-207
			226 255 257 263 283 294 300 302-303
			328 358-359 365 426
Lymph node	Clontech	ALN001	6 16 31 105 120 215 257 295 306 309 359
Young liver	GIBCO	ALV001	10-11 25-26 29 31 33 76 85 95 115 121-122
			124 126 130 143 146 156 158 164 178
			182 187 189 229 248 253-254 261 278 283
			304 342 375
Adult liver	Invitrogen	ALV002	10-12 23 26 31 33-34 38 53 56 90-92 94-95
			118 121 124 128-129 138 141 146 148
			153 156 161 171 178 198 216 232 248 253-254
			256-257 264 302 306 365 375 383 396
Adult liver	Clontech	ALV003	10-11 156 171 188
adult ovary	Invitrogen	AOV001	3-8 10-11 14 16 19-22 24 27-31 34 36 57
			73 75-76 81-82 85 89-91 94-98 104-109
			111 115-116 121-128 130-131 134 136 138-139
			141 143-144 146 149-150 152 155 157-160
			163-166 170-173 175 177-178 180 182
			184-187 189-190 193-194 196-197 200-201
			212-213 215 217 222 225-226 228 230-233
			235 241-243 245 248 253-259 261 266-267
			270 272-273 276-278 283-285 287 289 292
			297-299 305-306 315-317 319 323-325 329-331
			341 343-344 352 358-359 363-366 382-383
			386 389-390 412
adult placenta	Clontech	APL001	73 92 117 135 182 194 232 246 261 272
			282 359
placenta	Invitrogen	APL002	16 28 92 121 135 144 157 178 210 394
adult spleen	GIBCO	ASP001	3-4 16 32-33 35 90 96 99-100 123-125 128
			131 134 136 139 151 178 181 189 194 200
			210 218 229 251 253-255 257 276 283 307-309
			315 329 354-355 357 392 400
testis	GIBCO	ATS001	22 73 82 91 96-97 104-105 117 124 130
			134 164 173 200 209 222 233 241 253-254
			257 285 287-288 305 325 329 351-353 359
adult bladder	Invitrogen	BLD001	4 108 130 150 212 226 236 240 242 257
			276 287 305 395-396 415
bone marrow	Clontech	BMD001	1 4-5 22 29-30 34 72 85 88 90 92 94 98
			104-107 109 111 113 117 120 123-125 128-129
			132 135 140 142 144 146 152 163 165-166
			170-173 177 180 182 186 189-190 198-209
			215 222 225 232 240-246 251-252 260-261
			273-275 277-280 283-285 300 316 318
			346-347 359
bone marrow	Clontech	BMD002	1 4 7-8 10-11 16 19 25 31 49 61-62 72 74
			76 80 85 88 90 93-95 97-101 109-110 112
			114 116-117 121 126 129 132 135 141 144
			146 149-150 154 157 160 162-163 165-166
			170-172 175 178-180 182-183 186-190 192-194
			198-200 203 208 210-213 215 223 225
			234 242 245 247 251-254 256-257 265 270
			273 276-278 280 285 287 289 291 293-294
			299 302 307 309 315 322 324 337-338 353
			356-357 359 367 369 388 407 414 419 426
			434
bone marrow	Clontech	BMD007	144
adult colon	Invitrogen	CLN001	4 25 33 85 138 146 148 158-159 198 210
			229 301 360 384 397
Mixture of 16	Various	CTL021	23 359
tissues -	Vendors*
mRNAs*
Mixture of 16	Various	CTL028	172
tissues -	Vendors*
mRNAs*
adult cervix	BioChain	CVX001	3 5 10-11 18 20-21 24-25 29 36 41 47 57
			63 72 74 76 86 90 94 104 108-109 111 125
			127 130 134 138 144 147 162 174 178-179
			182 186 189 193 197 211 222 225-226 228
			232 241 243 257 261 267 270 273-275 278-281
			288-289 298 301-302 305 315 319 324-325
			329 331 337-338 359 391-392 395 420
endothelial	Strategene	EDT001	3-6 18-19 24 27-29 35 72 76 79-80 85 89
cells			96 98 104-107 111 117 119-121 124-131
			134 136 138-139 141 144 146-147 149 152
			158-159 166-167 170-173 178-179 182-183
			186-187 191 193-194 196-197 200 210-211
			222-224 226 231-232 236 241 243 246 248
			253-256 258-259 276 279 282 287 292 300
			302-303 315 329 337-338 358-362 382-383
			385-388
Genomic clones	Genomic DNA	EPM001	435
from the short	from
arm of	Genetic
chromosome 8	Research
esophagus	BioChain	ESO002	257
fetal brain	Clontech	FBR001	34
fetal brain	Clontech	FBR004	3 139 144 271 284 337-338
fetal brain	Clontech	FBR006	4 6-11 14 18-21 24 28 31 37-38 40 63 76
			85 87 89-90 94-95 97 105 108-109 112-113
			115 117-120 127-130 133 138 140 144-146
			148 170 172 175 180 182 186-188 190 192
			194 199 201 203 209-210 215 219 222 229-230
			232-233 240 243 245 253-255 270 273
			276 281 288-292 295 304 315 317 319
			324 330-331 356-357 359-360 364 367-368
			379-380 383 389 397 399-401 408-409 411
			413 419 421 423
fetal brain	Invitrogen	FBT002	2 14 19 23 28 31 90 94 105 121 124 126
			131 135 139 142 149 158 186 193 198 210
			214-215 232 239 242 248 255 267 326 332
			365 369 371 376-383 394 399
fetal heart	Invitrogen	FHR001	4 7-8 10-11 14 17-21 28-29 31-32 60 64-65
			73 85 87 92 95 102-103 105 108 111
			113 117 119 121 125 128-129 134-135 141
			152 154 156-157 160-161 172 176 178 194
			196 198-200 203 208 212 215 218 222 226
			229 233-234 253-257 261 265 272 276 281
			292-293 295 303 305 319 325 327 337-338
			341 345 349 354-355 367-368 389 395-396
			398 412 417 436
fetal kidney	Clontech	FKD001	1 14 22 94 110 115 132 134-135 146 178
			189 199 235-236 242 247 257 267 292 295
			359
fetal kidney	Clontech	FKD002	22 31 38 40 46 94 122 127 131 156 160 94
			198 229 253-254 270 292 303 319 354-355
			389 396
fetal kidney	Invitrogen	FKD007	303
fetal lung	Clontech	FLG001	85 89 98-100 111 175 271 281 369
fetal lung	Invitrogen	FLG003	84 88 106-107 122 135 140 146 160 181
			246 272 284 292 328 330 396 404 416 426
fetal liver-	Columbia	FLS001	1-3 6-12 14 19 23 28-31 33 57 59-60 72-76
spleen	University		78 80 83 85-138 140-141 143-144 146-155
			157-161 163-197 200 204 208 210-211
			223 225 230 232-233 235 241-243 245-266
			268-273 277 281 285-287 292 297 303 314
			329 343 346-347 357-359 369 397 399 407
			415
fetal liver-	Columbia	FLS002	1 3-4 6 10-12 23-24 29 31-33 35-37 53-54
spleen	University		74-76 79 81-82 86-89 91 94-95 99-104
			106-109 111-112 115 117-120 122 125-126
			128-129 132 134 136-138 141 146 149 153
			157-159 162-166 170 172 175 178-180 183
			185-191 194 196-197 205 207-212 222-225
			228 232-233 239-241 248 251-252 255-256
			258-259 261-262 264 266-267 270-271 273-275
			277-278 283 285 287 298 305 315 317-318
			322 330-332 337-338 341 343 349 357-360
			365 388 390-391 399 402 418 424
fetal liver-	Columbia	FLS003	12 29 91 98 111 119 156 163 165 178 186
spleen	University		193 210-211 276 286 315 322 346-347 357
			365 424
fetal liver	Invitrogen	FLV001	7-8 14 35 118 122-123 129 146 182 211
			230 232 248 251-252 264 287 304 337-338
			344 346-347 352 365 367-369
fetal liver	Clontech	FLV002	102-103 147 149 300
fetal liver	Clontech	FLV004	73 85 105 108 118 122 126 141 156-157
			161 165 170 178 180 182 194 215 218 225
			240 242 247 251-252 292 330 337-338 369
			407 411 440
fetal muscle	Invitrogen	FMS001	17 80 85 90 139 147 153 173 178 198 201
			220 229 236 293 296 303 323 334 341 345
			359 372
fetal muscle	Invitrogen	FMS002	5 9 17-18 20-21 29 38 85 88 97 106-107
			129 131 136 150-152 155 165 170 179 182
			192-193 212-213 229 234 242 258-259 270
			282 286 289 300 316 319 345 351 354-355
			360 389 396 408 410 437 439
fetal skin	Invitrogen	FSK001	2 4 7-8 29 33 42-43 49 51-52 58 74 82 85
			90 94 110-111 116 118 121 133 136 138-139
			145 151 154 156-157 161-162 172 181
			184 186 193 198 200 205 207 209-211 222
			227-230 232 235 240 246 253-257 266 270
			276 292 295 299 316 318 323 330 332 337-340
			343 357 369 389 394-395 412 422 427
fetal skin	Invitrogen	FSK002	4 9 42 44 51 66 72 81 85 89-90 95 98 105
			112-114 119 121 129 133 135 162 172 179-182
			197 200 208 210 231 243-244 272 304
			316 330 339 354-355 357 360 389 395 410
			417 437
fetal spleen	BioChain	FSP001	157 223
umbilical cord	BioChain	FUC001	4-6 20-21 25 29 73-74 83 87 89-91 94 101
			109 120 123 125 128 130-131 133 141 143-144
			147 149 154 161 165 173 175 179 184
			188 210-212 217 226 235 240 248 251-252
			257 262 267 270 277 293 305 307 316 319
			323 327 331 341 356 359 389 392 407 416
fetal brain	GIBCO	HFB001	2-4 16 20-21 74 77 85 89-91 96-98 104-105
			111 114 118 121-122 124-125 127-128
			131 134 137-140 142 144 146-148 151 153
			158-159 163-164 166 173 178 180 182 191
			194 196 200 203 209-214 216-232 234-236
			238-239 243 253-255 263 270 272-273 276
			281 292 310 316 319-321 332 348 357 359
			365 399
macrophage	Invitrogen	HMP001	2 247
infant brain	Columbia	IB2002	2-4 7-8 19-22 26-27 31-32 35 73-74 80 85
	University		89 91 96-98 106-107 110 112 118-119 121-122
			125 128-131 134-144 148 153 164 166
			172-173 177 180 186-187 191-194 196 202-203
			208-210 217 219 223-224 227 229 232-234
			236-237 239 241-243 245 248 253-259
			273-275 278-279 282 287 294 298 309 314
			317 322 327 330 333-334 341 348-350 360
			368 376 379-380 382 396 406 424
infant brain	Columbia	IB2003	3-4 20-21 26 28 31 35 73 85 95-96 110
	University		113 119 122-123 130-131 135 138 140 142-143
			143 146 153 155 170 172-173 186 191-193
			196 209 219 223 226 229 233-234 236 239
			245 248 253-254 256-257 273 279 291-292
			304 314 337-338 343 359 367 371 376 397
			413
infant brain	Columbia	IBM002	129 138 140 193 209 251-252 256 317 373
	University		397
infant brain	Columbia	IBS001	31 48 85 138 151 180 193 229 239 265 292
	University		326 337-338 371
lung,	Strategene	LFB001	3 6 31 72-73 90 92 105-107 124 126-127
fibroblast			133 136 139 144 146 172 189 198 204 233
			235 246 258-259 268 272 276 282 310 335
			359 434
lung tumor	Invitrogen	LGT002	4 19-21 28 33 35-36 49 72 79 81 85 88
			90-91 94-95 101 106-107 109 118 120-125
			127 130-131 133 135-138 141-142 144 147
			149 157 159-161 163 166 170-173 193-194
			196-197 212 216 218 221 223 226 228-229
			231 233 241 247-248 253-255 257 261 266-267
			270-275 277-278 282-283 292 298 301
			303 315 318 324 331 335 354-355 359 367
			369 381 392-393 398
lymphocytes	ATCC	LPC001	4 6 10-11 16 42 76 88 91 95-98 104 109
			114 120 122 133-134 144 146 148 157-158
			161 164 171 180 186-187 203 209 212 223
			231 242-243 248 251-252 255 258-259 271-273
			281-282 299 334 336 362 374 418
leukocyte	GIBCO	LUC001	1-5 15 19-21 28 30-33 37 72 74 91 94-95
			97-100 108-109 113 115 117 119-122 124-125
			127-128 134-138 141 144 146-148 150-151
			157-158 160 162-167 170-173 175-178
			180-181 187 189 192 194 197 200 212-213
			215-216 218-219 223 225 228-232 241-242
			245-246 251-254 261 272-276 278-282 284
			287-290 297-298 305 307 310-314 325 331
			336 340 358-359 372 399 414
leukocyte	Clontech	LUC003	1 5 124 171 176 204 225 248 253-254 283
			285 307 315
melanoma from	Clontech	MEL004	4-5 24 37 72-74 81 85 106-107 113 136
cell line ATCC			177 203 205-207 209 231 243 284-285 315-316
#CRL 1424			320 326 359 374 428
mammary gland	Invitrogen	MMG001	2 4-5 7-8 10-12 29 31 34-35 38 50 80-81
			85 89-90 92 94-97 105 108-109 119-124
			126 128-130 135 138-139 141-142 144 146-147
			153 155 157-159 163 178-179 181-182
			198 200 209-210 219 223 228 230 232-233
			235-236 239 242 248 253-255 257 260-261
			265-267 270 272 281 287 292 294 315-316
			318 324 327 330 337-340 354-355 357 369
			372 383 392-395 401 404
induced neuron	Strategene	NTD001	35 47 89-90 111 118 164 232 253-254 276
cells			324 331 382
retinoid acid	Strategene	NTR001	20-21 37 122 147-149 170 179 181 186 212
induced			226 258-259 265 276 369 436 438
neuronal cells
neuronal cells	Strategene	NTU001	7-8 37 55 80 85 112 118 126-127 133 138
			140-141 151 170 181 210 214 225-226 236
			243 287 328 330-331 357 383 400 436
pituitary	Clontech	PIT004	92 124 159 231
gland
placenta	Clontech	PLA003	34 46 88 126 128 159 182 186 197 201 267
			278 281-282 305 330 356 361 365 418
prostate	Clontech	PRT001	18 36 72 74 86 95 106-107 111 118 122
			144 161 179 211 218 233 286 297
rectum	Invitrogen	REC001	9 31 85 121 128 147 171 200 219 257 292
			340 394 398 407 412
salivary gland	Clontech	SAL001	3 24 38 80 122 136 147 189 241 282 296
			310 351 392 395 415
salivary gland	Clontech	SALs03	118
skin	ATCC	SFB003	257
fibroblast
small	Clontech	SIN001	12 16 25 82-83 89-90 93 95 98 105-109
intestine			111 122-123 125-128 133-134 137 139 142
			161 167 171 184 197 201 204 212 218 236
			242-243 248-249 253-254 257 267 276 284-285
			292 297 300 303 310 313 317-318 325
			340 343 352 354-355 359 383 391 416
skeletal	Clontech	SKM001	3 85 90 121 136 154 200 211-212 224 234-235
muscle			272 288 293 303 353 426
spinal cord	Clontech	SPC001	3 39 84 86 94 96 105 115 117 130-131 134
			136 141 143 148 155 176 190-191 203 213
			224 233-234 236 239 279 283 298 320-321
			332 336-338 356 359 365 404-406
adult spleen	Clontech	SPLc01	7-8 35 80 94 106-107 120 133 144 152 155
			241 253-254 263 329 407 428
stomach	Clontech	STO001	10-11 23 30 77 98 106-107 117 131 135
			139 144 257 285 343 367 382 394
thalamus	Clontech	THA002	2 20-21 23 74 81 85 105-106 116 121 131
			146 171 185 188 200 209 219 233 239 256
			258-259 273 276 362 399
thymus	Clontech	THM001	16 29 33 57 80 82 85 90 93-94 106-107
			120 126 128 134 141 161 176 194 223 228
			235 253-254 261 274-275 278 285 298 319
			332 336 343 353 359 425
thymus	Clontech	THMc02	1-2 7-9 14 26 34 44 73 75 82 85 87 94 98
			106-107 109-111 117 119-120 125-126 128-129
			139 141 144 147-148 151 154-155 162
			165 170-172 175-176 179 182 186 193-194
			199-200 208-209 213 218 233 235 240 242
			247 253-254 257 265 276 281 287 290 305
			307 312 319 336 342 354-356 359 364 367
			399 408 412-413 415 419 421 426 429-433
thyroid gland	Clontech	THR001	3 5 7-8 28 30-31 33 73-77 80 82 85 88
			90-92 94 96-98 105-107 109 113 117 121-122
			124-125 127-128 130 134 136 141 143
			146-148 152 161-163 166 175 177-178 181
			194 199 201 204 210 212 216 218 223-226
			228 230-231 234 236 241 243 246 253-257
			261 270 272-273 276-278 281-283 287 292
			295 298 303-304 308 315 323 329 335 352
			359 362 401 416-417
trachea	Clontech	TRC001	88 138 180 226 228 279 359 411 436
uterus	Clontech	UTR001	3 10-11 23 77 92 106-107 109 111 141
			197-198 218 241 257 270 274-275 302 315
			329 396 400 413
*The 16 tissue-mRNAs and their vendor source, are as follows: 1) Normal adult brain mRNA (Invitrogen), 2) normal adult kidney mRNA (Invitrogen), 3) normal adult liver mRNA (Invitrogen), 4) normal fetal brain mRNA (Invitrogen), 5) normal fetal kidney mRNA (Invitrogen), 6) normal fetal liver mRNA (Invitrogen), 7) normal fetal skin mRNA (Invitrogen), 8) human adrenal gland mRNA (Clontech), 9) human bone marrow mRNA (Clontech),
# 10) human leukemia lymphablastic mRNA (Clontech), 11) human thymus mRNA (Clontech), 12) human lymph node mRNA (Clontech), 13) human spinal cord mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA (BioChain), 16) human conceptional umbilical cord mRNA (BioChain).

TABLE 2
SEQ ID	ACCESSION		SMITH-WATERMAN
NO:	NUMBER	DESCRIPTION	SCORE	% IDENTITY
1	AF255303	Homo sapiens membrane-	2074	67
		associated nucleic acid
		binding protein
2	Y57911	Human transmembrane	865	94
		protein HTMPN-35
3	AL121961	Homo sapiens dJ104A17.1	1426	100
		(novel protein)
4	X90872	Homo sapiens associated	1096	98
		to Golgi apparatus
5	AL161659	Homo sapiens bA526K24.1	4348	99
		(A novel protein)
6	AF047185	Homo sapiens NADH-	505	100
		ubiquinone
		oxidoreductase subunit
		CI-B8
7	AL162458	Homo sapiens bA465L10.1	3765	100
		(novel protein similar
		to Drosophila CG11399)
8	AL162458	Homo sapiens bA465L10.1	3366	100
		(novel protein similar
		to Drosophila CG11399)
9	U28928	Caenorhabditis elegans	450	30
		similar to C. elegans
		protein F59B2.2
10	U66411	Drosophila melanogaster	336	57
		putative type III
		alcohol dehydrogenase
11	U66411	Drosophila melanogaster	425	52
		putative type III
		alcohol dehydrogenase
13	AL096710	Homo sapiens dJ61B2.2	733	99
		(similar to MACF
		cytoskeletal protein)
14	L05093	Homo sapiens ribosomal	887	93
		protein L18a
16	AF061961	Mus musculus putative	345	33
		zinc finger protein
		FLIZ1
17	U76373	Mus musculus skm-BOP1	2494	94
18	AJ272073	Torpedo marmorata male	2307	80
		sterility protein 2-like
		protein
19	Z82084	Caenorhabditis elegans	279	27
		contains similarity to
		Pfam domain: PF01769
		(Divalent cation
		transporter),
		Score = 211.5, E-
		value = 4.2e-60, N = 2
20	AF090989	Homo sapiens high-risk	3089	68
		human papilloma viruses
		E6 oncoproteins targeted
		protein E6TP1 alpha;
		putative GAP protein
		alpha
21	AF090989	Homo sapiens high-risk	3089	68
		human papilloma viruses
		E6 oncoproteins targeted
		protein E6TP1 alpha;
		putative GAP protein
		alpha
22	U20286	Rattus norvegicus lamina	739	59
		associated polypeptide
		1C
23	AF207829	Homo sapiens SCAN-	1104	100
		related protein RAZ1
24	X78817	Homo sapiens p115	1118	45
25	D86417	Bacillus subtilis YfmJ	359	43
26	L01083	Oryctolagus cuniculus	683	36
		UDP-
		glucuronosyltransferase
27	AF000240	Gallus gallus	385	37
		monocarboxylate
		transporter 3
28	Z36948	Caenorhabditis elegans	138	42
		contains 3 cysteine rich
		repeats
29	D17554	Homo sapiens TAXREB107	1207	98
30	L06505	Homo sapiens ribosomal	845	100
		protein L12
31	AL136538	Schizosaccharomyces	680	37
		pombe putative
		cysteinyl - trna
		synthetase
32	AB040900	Homo sapiens KIAA1467	2232	100
		protein
33	AL031685	Homo sapiens dJ963K23.2	521	46
		(novel protein)
34	AF263742	Homo sapiens golgin-like	297	45
		protein
35	AF030131	Mus musculus Plenty of	2951	88
		SH3s; POSH
36	Z22818	Canis familiaris Rab12	1071	99
		protein
37	AL110477	Caenorhabditis elegans	198	34
		Y113G7B.24
38	AC002505	Arabidopsis thaliana	157	28
		putative
		phosphatidylinositol-4-
		phosphate 5-kinase
39	AF099810	Homo sapiens neurexin	1255	99
		III-alpha
40	AL133215	Homo sapiens bA108L7.6	4406	99
		(semaphorin 4G (sema
		domain, immunoglobulin
		domain (Ig),
		transmembrane domain
		(TM) and short
		cytoplasmic domain))
41	AL109657	Homo sapiens dJ842G6.1	247	100
		(novel protein)
42	AB033744	Mus musculus type II	2419	91
		cytokeratin
43	M27685	Mus musculus ultra-high	702	60
		sulphur keratin
44	M94081	Homo sapiens putative	110	100
46	AJ278348	Homo sapiens pregnancy-	8966	99
		associated plasma
		protein-E
47	Z98762	Schizosaccharomyces	190	36
		pombe profilin.
48	AF026544	Ralstonia eutropha phbF	561	66
49	AB036882	Mus musculus midnolin	950	67
50	Z46967	Homo sapiens calicin	3076	99
51	AF204929	Sus scrofa thyroxine-	1060	52
		binding globulin
52	AL035694	Homo sapiens dJ33L1.1	2634	100
		(novel T-box (Brachyury)
		family protein)
54	AF079864	Rattus norvegicus	508	51
		putative G-protein
		coupled receptor RA1c
55	AL022373	Arabidopsis thaliana	200	35
		glycine-rich protein
56	AJ007767	Homo sapiens T-cell	566	97
		antigen receptor-alpha
57	AF041107	Rattus norvegicus tulip	1884	94
		2
58	L32179	Homo sapiens	1035	52
		arylacetamide
		deacetylase
59	AB010414	Homo sapiens G-protein	127	56
		gamma 7
60	AJ277486	Mus musculus T-box	1463	93
		transcription factor
61	AF164612	Homo sapiens Gag protein	131	29
62	S61069	Homo sapiens reverse	232	80
		transcriptase
		homolog = pol {retroviral
		element}
63	AF266172	Gillichthys mirabilis	127	81
		60S acidic ribosomal
		protein P1
64	M18184	Mus musculus lymphocyte	140	37
		differentiation antigen
65	AL035587	Homo sapiens dJ475N16.3	434	88
		(novel protein similar
		to RPL7A (60S ribosomal
		protein L7A))
66	M34225	Homo sapiens cytokeratin	866	64
		8
67	Y17166	Campylobacter jejuni	111	31
		ribosomal protein S6
68	AF252290	Mus musculus PAR6A	1492	76
69	Z46787	Caenorhabditis elegans	522	51
		contains similarity to
		Pfam domain: PF00097
		(Zinc finger, C3HC4 type
		(RING finger)),
		Score = 17.6, E-
		value = 0.00013, N = 1
70	AF205718	Homo sapiens	473	76
		hepatocellular
		carcinoma-related
		putative tumor
		suppressor
71	AJ006692	Homo sapiens ultra high	774	79
		sulfer keratin
72	AK001269	Homo sapiens unnamed	3518	100
		protein product
73	AE003453	Drosophila melanogaster	756	43
		CG9752 gene product
74	AK000725	Homo sapiens unnamed	3013	100
		protein product
75	AF231921	Homo sapiens unknown	1215	100
76	AK024664	Homo sapiens unnamed	2522	100
		protein product
77	AL133661	Homo sapiens	1612	100
		hypothetical protein
78	AK000765	Homo sapiens unnamed	1450	100
		protein product
79	AK023453	Homo sapiens unnamed	774	100
		protein product
80	AK000474	Homo sapiens unnamed	737	100
		protein product
81	W93948	Human regulatory	441	91
		molecule HRM-4 protein
82	AK024435	Homo sapiens FLJ00025	145	24
		protein
83	AK000820	Homo sapiens unnamed	652	100
		protein product
84	AL080154	Homo sapiens	838	100
		hypothetical protein
85	D45131	Homo sapiens basigin	1302	99
86	AL365412	Homo sapiens	580	100
		hypothetical protein,
		similar to (NP_006139.1)
		LASP-1, LIM and SH3
		domain protein 1
87	L03188	Saccharomyces cerevisiae	216	26
		putative
88	X74855	Mus musculus zinc finger	362	31
		protein 51
89	AF152344	Mus musculus F2 alpha	4195	89
		prostoglandin regulatory
		protein
90	AF020262	Bos taurus general	607	100
		protein transport factor
		p16
91	AL096882	Arabidopsis thaliana	146	24
		putative protein
92	M75099	Homo sapiens rapamycin-	740	100
		and FK506-binding
		protein
93	AF163151	Homo sapiens dentin	162	22
		sialophosphoprotein
		precursor
94	AL078593	Homo sapiens dJ210B1.1	745	40
		(KIAA0680)
95	AJ223802	Arabidopsis thaliana 2-	1463	42
		oxoglutarate
		dehydrogenase, E1
		subunit
96	AB020749	Arabidopsis thaliana	144	24
97	U06944	Mus musculus PRAJA1	1087	76
98	AL096678	Homo sapiens dJ122O8.4	10626	100
		(KIAA0301)
99	AL356276	Homo sapiens bA367J7.7	1533	100
		(novel Immunoglobulin
		domains containing
		protein)
100	AL356276	Homo sapiens bA367J7.7	1533	100
		(novel Immunoglobulin
		domains containing
		protein)
101	AF200691	Drosophila melanogaster	1031	38
		Dispatched
102	AF177144	Mus musculus mammalian	1027	50
		inositol
		hexakisphosphate kinase
		1
103	AF177144	Mus musculus mammalian	1039	51
		inositol
		hexakisphosphate kinase
		1
104	AL021918	Homo sapiens b3418.1	1624	48
		(Kruppel related Zinc
		Finger protein 184)
105	X69115	Homo sapiens ZNF37A	1371	99
106	AF142406	Babesia bigemina 200 kDa	325	24
		antigen p200
107	AF161358	Homo sapiens HSPC095	419	100
108	AL163852	Arabidopsis thaliana	142	30
		putative protein
109	Z99531	Schizosaccharomyces	346	31
		pombe caffeine-induced
		death protein 1
110	M29580	Homo sapiens zinc finger	510	57
		protein 7 (ZFP7)
111	L00923	Mus musculus myosin I	5389	96
112	AP001751	Homo sapiens gene	1527	50
		similar to rat protein
		kinase (KID2)
113	Y12781	Homo sapiens transducin	2431	86
		(beta) like 1 protein
114	AF215703	Drosophila melanogaster	2090	66
		KISMET-L long isoform
115	AL023828	Caenorhabditis elegans	445	48
		cDNA EST yk167h7.3 comes
		from this gene-cDNA EST
		yk167h7.5 comes from
		this gene-cDNA EST
		yk289g5.3 comes from
		this gene-cDNA EST
		yk332h9.3 comes from
		this gene-cDNA EST
		yk289g5.5 comes from
		this gene-cDNA EST
		yk332h9.5 comes from
		this gene-cDNA EST
		yk391h4.5 comes from
		this gene-cDNA EST
		yk653f1.5 comes from
		this gene
116	AF125960	Caenorhabditis elegans	475	35
		contains similarity to
		dual specificity
		phosphatase, catalyitic
		domain (Pfam:PF00782,
		Score = 16.8, E = 7.4e-05,
		N = 1)
117	AF151697	Homo sapiens sentrin-	3110	99
		specific protease
118	AF022655	Homo sapiens cep250	139	24
		centrosome associated
		protein
119	AK026098	Homo sapiens unnamed	1950	99
		protein product
120	X56203	Plasmodium falciparum	504	25
		liver stage antigen
121	U21324	Caenorhabditis elegans	1197	52
		similar to entire S.
		cerevisiae ABC1 protein
		(Swiss-Prot Acc: P27697)
122	AL138641	Arabidopsis thaliana	579	33
		putative protein
123	S73591	Homo sapiens brain-	674	38
		expressed HHCPA78
		homolog VDUP1
124	D00761	Homo sapiens proteasome	857	94
		subunit C5
125	AF000262	Caenorhabditis elegans	343	36
		similar to
		CCAAT/enhancer-binding
		protein
126	AF146075	Thermus thermophilus	144	31
		ribosomal protein L9
127	AC006585	Arabidopsis thaliana	1129	46
		putative SUDD-like
		protein
128	AL031804	Arabidopsis thaliana	186	25
		putative protein
129	AL035539	Arabidopsis thaliana	140	27
		putative protein
130	AF151816	Homo sapiens CGI-58	1023	54
		protein
131	AC024765	Caenorhabditis elegans	223	33
		contains similarity to
		O-linked G1cNAc
		transferases
132	M12623	Homo sapiens high	320	95
		mobility group protein
		17
133	AB017614	Mus musculus OASIS	2409	91
		protein
134	AL109827	Homo sapiens dJ309K20.4	4972	100
		(KIAA0765 (HRIHFB2091,
		RNA recognition motif
		(RNP, RRM or RBD domain)
		containing protein))
135	AL035661	Homo sapiens dJ568C11.3	2526	100
		(novel AMP-binding
		enzyme similar to
		acetyl-coenzyme A
		synthethase (acetate-coA
		ligase))
136	AL031804	Arabidopsis thaliana	237	35
		putative protein
137	AL161590	Arabidopsis thaliana	232	34
		putative protein
138	AB024032	Arabidopsis thaliana	372	55
		gene_id:K9P8.4˜
139	AL050367	Homo sapiens	4177	94
		hypothetical protein
140	AF081484	Homo sapiens alpha-	1195	97
		tubulin isoform 1
141	D14336	Mus musculus RNA	1694	77
		polymerase I associated
		factor (PAF53)
142	AF023657	Rattus norvegicus endo-	1415	67
		alpha-D-mannosidase
143	AB011541	Homo sapiens MEGF8	9785	100
144	AF112221	Homo sapiens rap2	1189	60
		interacting protein x
145	AF117382	Mus musculus	1333	91
		hypermethylated in
		cancer 2 protein; HIC2
146	AF151827	Homo sapiens CGI-69	1794	97
		protein
147	AC024792	Caenorhabditis elegans	424	35
		contains similarity to
		TR:P78316
148	AL022603	Arabidopsis thaliana	369	32
		putative protein
149	AF016430	Caenorhabditis elegans	370	43
		similar to Pan
		Troglodytes GOR
		(SP:P48778)
150	X15696	Homo sapiens translated	129	28
		region (partial)
151	AL022373	Arabidopsis thaliana	137	33
		putative protein
152	AB010077	Arabidopsis thaliana	881	56
		GTP-binding membrane
		protein LepA homolog
153	AF062655	Mus musculus plenty-of-	115	29
		prolines-101; POP101;
		SH3-philo-protein
154	AF159852	Drosophila melanogaster	243	48
		RNA-binding protein
		Smaug
155	AE003606	Drosophila melanogaster	465	32
		CG14646 gene product
156	AE004999	Halobacterium sp. NRC-1	335	35
		dihydrodipicolinate
		synthase; DapA
157	AL157953	Streptomyces coelicolor	143	28
		A3(2) putative
		nitroreductase
158	AF116238	Homo sapiens	1927	99
		pseudouridine synthase 1
159	AF263541	Homo sapiens protein	2844	100
		kinase DYRK4
160	Z81317	Schizosaccharomyces	148	37
		pombe putative ubiquitin
		carboxyl-terminal
		hydrolase
161	Y18208	Rattus norvegicus	1360	89
		serine - threonine
		specific protein
		phosphatase, glycogen-
		binding (GL) subunit
162	AC005970	Arabidopsis thaliana	926	58
		putative translation
		initiation factor eIF-2B
		alpha subunit
163	M83822	Homo sapiens beige-like	9733	99
		protein
164	AL080282	Arabidopsis thaliana	352	34
		putative protein
165	U61232	Homo sapiens cofactor E	292	27
166	AB014578	Homo sapiens KIAA0678	5255	100
		protein
167	X61123	Homo sapiens BTG1	813	96
168	Z92954	Bacillus subtilis	570	38
		product highly similar
		to metabolite transport
		proteins
169	AL031055	Homo sapiens dJ28H20.1	636	44
		(novel protein similar
		to membrane transport
		proteins)
170	AL021918	Homo sapiens b3418.1	1144	46
		(Kruppel related Zinc
		Finger protein 184)
171	U44731	Mus musculus purine	2260	70
		nucleotide binding
		protein
172	AF211859	Mus musculus cyclin	1052	92
		ania-6b
173	AF181252	Mus musculus TPR-	266	31
		containing protein
		involved in
		spermatogenesis TPIS
174	AF096864	Pseudomonas aeruginosa	173	33
		heat shock protein
175	AB042199	Homo sapiens APC-	1783	62
		stimulated guanine
		nucleotide exchange
		factor
176	Z11582	Saccharomyces cerevisiae	151	25
		nuf1
177	AJ235270	Rickettsia prowazekii	376	37
		PROBABLE O-
		SIALOGLYCOPROTEIN
		ENDOPEPTIDASE (gcp)
178	AK022710	Homo sapiens unnamed	527	100
		protein product
179	D50617	Saccharomyces cerevisiae	139	28
		YFL027C
180	AF065389	Homo sapiens tetraspan	867	59
		NET-4
181	D21261	Homo sapiens similar to	1048	100
		human 22kDa, SM22 mRNA
		(HUM22SM).
182	AL162971	Arabidopsis thaliana	529	38
		putative protein
183	AL080062	Homo sapiens	1530	99
		hypothetical protein
184	U93572	Homo sapiens putative	496	53
		p150
185	AF187064	Homo sapiens p75NTR-	227	47
		associated cell death
		executor; NADE
186	AL035413	Homo sapiens dJ657E11.5	1880	99
		(KIAA0090 PROTEIN)
187	AC073944	Arabidopsis thaliana	755	36
		ethylene-responsive RNA
		helicase, putative
188	AB017615	Mus musculus Eos protein	2750	95
189	Z99110	Bacillus subtilis	148	20
		similar to hexuronate
		transporter
190	AL138657	Arabidopsis thaliana	253	32
		putative protein
191	AJ002397	Trichoderma harzianum	177	30
		beta-1,3 exoglucanase
192	AY004877	Mus musculus cytoplasmic	5569	98
		dynein heavy chain
193	U02313	Mus musculus protein	6356	86
		kinase
194	AE003739	Drosophila melanogaster	596	43
		CG6949 gene product
195	U01317	Homo sapiens epsilon-	397	100
		globin
196	AF083116	Homo sapiens	770	46
		paraneoplastic cancer-
		testis-brain antigen
197	AL133156	Schizosaccharomyces	157	27
		pombe putative U4/U6
		small nuclear
		ribonucleoprotein
198	AF123653	Homo sapiens FEZ1	742	42
199	Z99162	Schizosaccharomyces	167	24
		pombe putative signal
		transduction pathway
		protein
200	U10406	Mus musculus capping	1440	98
		protein beta-subunit,
		isoform 1
201	AL365409	Homo sapiens similar to	1653	100
		(NP_034322.1|) sex-
		determination protein
		homolog Femla
202	AF116656	Homo sapiens PRO1167	389	100
203	AL161578	Arabidopsis thaliana	142	27
		putative protein
204	AF148805	Kaposi's sarcoma-	172	21
		associated herpesvirus
		latent nuclear antigen
205	AF102853	Rattus norvegicus	1500	62
		membrane-associated
		guanylate kinase-
		interacting protein 1
		Maguin-1
206	AF102853	Rattus norvegicus	779	69
		membrane-associated
		guanylate kinase-
		interacting protein 1
		Maguin-1
207	AF102853	Rattus norvegicus	933	71
		membrane-associated
		guanylate kinase-
		interacting protein 1
		Maguin-1
208	AF096896	Drosophila melanogaster	2089	50
		pushover
209	AF178941	Homo sapiens ATP-binding	9833	99
		cassette sub-family A
		member 2
210	AC004780	Homo sapiens F17127_1	1262	95
211	AL121845	Homo sapiens	1774	99
		dJ583P15.5.1 (novel
		protein (isoform 1))
212	AF170562	Homo sapiens ubiquitin-	1248	49
		specific processing
		protease
213	U00060	Saccharomyces cerevisiae	637	46
		Yhr088wp
214	AJ289133	Mus musculus chondroitin	565	42
		4-O-sulfotransferase
215	M63577	Saccharomyces cerevisiae	131	59
		SFP1
216	AB046635	Macaca fascicularis	300	46
		hypothetical protein
217	Z98884	Homo sapiens dJ467L1.1	3640	99
		(KIAA0833)
218	AB009883	Nicotiana tabacum KED	203	22
219	D86491	Xenopus laevis Nfrl	2416	75
220	U66707	Rattus norvegicus	3640	93
		densin-180
221	AJ006701	Homo sapiens putative	1457	84
		serine/threonine protein
		kinase
222	X61045	Hydra sp. mini-collagen	132	62
223	AF138883	Bos taurus type II	170	38
		collogen cyanogen
		bromide fragment CB10
224	U22816	Homo sapiens LAR-	2039	57
		interacting protein 1b
225	AL109865	Homo sapiens	2298	98
		bG120K12.3.1 (novel
		protein similar to
		archaeal, yeast and worm
		N2,N2-dimethylguanosine
		tRNA methyltransferase
		(isoform 1))
226	AJ011654	Homo sapiens triple LIM	557	41
		domain protein
227	AB030198	Mus musculus contains	318	50
		transmembrane (TM)
		region
229	AF233223	Homo sapiens F-box	1386	87
		protein FBG2
230	D88734	Equine herpesvirus 1	333	28
		membrane glycoprotein
231	X94699	Mesocricetus auratus	1306	87
		glucosamine-6-phosphate
		isomerase
232	AL358732	Arabidopsis thaliana	385	44
		putative protein
233	AB004306	Homo sapiens osteoblast	574	100
		stimulating factor-1
234	Z99281	Caenorhabditis elegans	154	22
		Y57G11C.17
235	U23181	Caenorhabditis elegans	173	26
		final exon in repeat
		region; similar to long
		tandem repeat region of
		sialidase
		(SP:TCNA_TRYCR, P23253)
		and neurofilament H
		protein
236	X99384	Mus musculus paladin	3563	80
237	AB020755	Arabidopsis thaliana	144	32
238	AF168122	Oryctolagus cuniculus	4053	96
		hyperpolarization-
		activated cyclic
		nucleotide-gated channel
		1
239	AF177909	Homo sapiens TTYH1	1055	100
240	AF265296	Drosophila melanogaster	647	48
		putative multipass
		transmembrane
241	AC024760	Caenorhabditis elegans	212	19
		contains similarity to
		TR:Q10466
242	AB034991	Mus musculus stress	614	37
		protein Herp
243	AF030558	Rattus norvegicus	2090	95
		phosphatidylinositol 5-
		phosphate 4-kinase gamma
244	M80537	Drosophila melanogaster	1610	30
		fat protein
245	U80741	Homo sapiens CAGH44	373	56
246	L11586	Rattus norvegicus	271	25
		leukocyte common
		antigen-related
		phosphatase
247	AF265555	Homo sapiens ubiquitin-	330	37
		conjugating BIR-domain
		enzyme APOLLON
248	AC024810	Caenorhabditis elegans	450	35
		contains similarity to
		Pfam family PF00560
		(Leucine Rich Repeat-2
		copies)
249	U13831	Homo sapiens cellular	715	99
		retinol binding protein
		II
250	J03799	Homo sapiens laminin-	209	87
		binding protein
251	AL109840	Homo sapiens dJ543J19.4	1447	100
		(A novel protein
		(ortholog of chicken
		tubulin beta-6 chain
		(beta-tubulin class-
		VI)))
252	AL109840	Homo sapiens dJ543J19.4	1842	100
		(A novel protein
		(ortholog of chicken
		tubulin beta-6 chain
		(beta-tubulin class-
		VI)))
253	D14710	Homo sapiens inport	1566	99
		precursor of human ATP
		synthase alpha subunit
254	Y07895	Strongylocentrotus	815	78
		purpuratus mitochondrial
		ATP synthase alpha
		subunit precursor
255	AJ243936	Homo sapiens protein G16	592	60
256	AL034380	Homo sapiens dJ50024.1	1262	100
		(novel protein similar
		to C. elegans K07B1.7
		(Tr:O01886))
257	D17409	Homo sapiens SM22 alpha	937	95
258	AL080276	Homo sapiens dJ101K10.1	1940	99
		(novel protein similar
		to Prokaryotic-type
		class I peptide chain
		release factors)
259	AL080276	Homo sapiens dJ101K10.1	1048	99
		(novel protein similar
		to Prokaryotic-type
		class I peptide chain
		release factors)
260	D50617	Saccharomyces cerevisiae	352	32
		YFL044C
261	D63378	Rattus norvegicus ER-60	174	28
		protease
262	AJ249900	Homo sapiens secreted	2346	99
		modular calcium-binding
		protein
263	U41557	Caenorhabditis elegans	281	33
		proline and glycine-rich
264	AE001751	Thermotoga maritima dnaJ	172	52
		protein
265	AC004144	Homo sapiens R34001_1	1699	89
266	AF221759	Homo sapiens Mam1	342	31
267	U33007	Saccharomyces cerevisiae	370	35
		Ydr438wp; CAI: 0.11
268	X15940	Homo sapiens ribosomal	546	92
		protein L31 (AA 1-125)
269	X00955	Homo sapiens prepro-apo	478	96
		AII
270	AL035460	Homo sapiens dJ860F19.3	3571	100
		(novel protein (ortholog
		of mouse
		metallocarboxypeptidase
		CPX-1))
271	U35730	Mus musculus jerky	758	33
272	X16576	Homo sapiens KUP protein	567	37
273	AK021815	Homo sapiens unnamed	1408	97
		protein product
274	AL391148	Arabidopsis thaliana	521	43
		putative protein
275	AL391148	Arabidopsis thaliana	521	43
		putative protein
276	U33335	Saccharomyces cerevisiae	376	29
		Lpa8p
277	AB046381	Homo sapiens testis-	2739	100
		abundant finger protein
278	Z19092	Oryctolagus cuniculus	217	25
		trichohyalin
279	U65079	Mus musculus actin-	516	26
		binding protein
280	M27878	Homo sapiens DNA binding	1491	56
		protein
281	U62907	Mus musculus zinc finger	675	36
		protein 95
282	AF052298	Drosophila silvestris Y	140	34
		box protein
283	AB020970	Homo sapiens up-	637	100
		regulated by BCG-CWS
284	X61585	Bos taurus	2194	60
		polynucleotide
		adenylyltransferase
285	AJ249978	Xenopus laevis p33 ringo	95	43
286	U96166	Streptococcus cristatus	194	17
		srpA
287	AE003701	Drosophila melanogaster	286	29
		CG9591 gene product
288	AL117337	Homo sapiens bA393J16.3	2015	100
		(novel KRAB box
		containing zinc finger
		gene)
289	AC004076	Homo sapiens R30217_1	1793	56
290	M27337	Homo sapiens T cell	399	91
		receptor gamma chain
291	V00507	Homo sapiens coding	886	93
		sequence of DHFR (1 is
		1st base in codon) (561
		is 3rd base in codon)
292	AE005053	Halobacterium sp. NRC-1	1062	37
		Vng1303c
293	AF061258	Homo sapiens LIM protein	510	88
294	AE003543	Drosophila melanogaster	142	60
		CG6938 gene product
295	AK024429	Homo sapiens FLJ00018	5938	100
		protein
296	AF151851	Homo sapiens CGI-93	557	49
		protein
297	AK025429	Homo sapiens unnamed	668	63
		protein product
298	Z19092	Oryctolagus cuniculus	188	21
		trichohyalin
299	AF195056	Mus musculus SorCSb	2188	47
		splice variant of the
		VPS10 domain receptor
		SorCS
300	L32978	Mesocricetus auratus	167	22
		synaptonemal complex
		protein
301	AC002130	Arabidopsis thaliana	237	30
		F1N21.22
302	U80736	Homo sapiens CAGF9	290	87
303	AF178428	Bos taurus submaxillary	183	26
		mucin
304	AJ243936	Homo sapiens protein G16	380	68
305	AF147791	Homo sapiens mucin 11	106	22
306	AF071010	Mouse mammary tumor	177	46
		virus putative integrase
307	X71997	Rattus norvegicus myosin	1654	62
		I
308	AF000560	Homo sapiens TTF-I	2081	99
		interacting peptide 20;
		TIP20; Transcription
		Termination Factor I
		Interacting Peptide 20
309	AF142780	Mus musculus	895	69
		butyrophilin-like
		protein
310	AF161384	Homo sapiens HSPC266	119	44
311	A08695	Homo sapiens rap2	204	31
312	X04956	Homo sapiens T-cell	590	100
		receptor alpha-chain
		(404 is 2nd base in
		codon)
313	X60489	Homo sapiens elongation	1066	92
		factor-1-beta
314	AB026190	Homo sapiens Kelch motif	1187	44
		containing protein
315	U66561	Homo sapiens kruppel-	916	45
		related zinc finger
		protein
316	AF038007	Homo sapiens FIC1	1499	54
317	AJ225124	Mus musculus	3554	96
		hyperpolarization-
		activated cation
		channel, HAC3
318	AL031788	Schizosaccharomyces	290	40
		pombe putative
		mitochondrial membrane
		protease subunit 2
319	AC003682	Homo sapiens F18547_1	814	49
320	AF188507	Mus musculus LAB300	1347	46
		isoform gamma
321	AL136125	Homo sapiens dJ304B14.1	374	32
		(novel protein)
322	U70312	Homo sapiens integrin	447	100
		binding protein Del-1
323	D13302	Megabalanus rosa lectin	155	30
		BRA-3
324	U59185	Homo sapiens putative	519	29
		monocarboxylate
		transporter
325	AF205599	Mus musculus	1016	35
		transposase-like protein
326	Z34465	Zea mays extensin-like	218	27
		protein
327	AL049553	Homo sapiens dJ402N21.2	1474	100
		(novel protein with MAM
		domain)
328	AF027505	Mus musculus putative	1118	83
		membrane-associated
		guanylate kinase 1
329	U37376	Xenopus laevis MAM	2448	63
		domain protein
330	AF030430	Mus musculus semaphorin	2958	94
		VIa
331	Z30174	Mus musculus domesticus	2087	71
		zinc finger protein 30
332	U55020	Saccharomyces cerevisiae	282	33
		Smp3p
333	Y07783	Rattus norvegicus ER	285	32
		transmembrane protein
334	AF291437	Rattus norvegicus	432	27
		neuronal leucine-rich
		repeat protein-3
335	W56097	Homo sapiens amino acid	2470	84
		sequence of the ODD4b5.3
		enzyme
336	AB029333	Halocynthia roretzi	810	44
		HrPET-1
337	AB030186	Mus musculus unnamed	1634	86
		protein product
338	AB030186	Mus musculus unnamed	1671	89
		protein product
339	AF128113	Mus musculus prominin-	1021	77
		like protein
340	AB007407	Mus musculus myeloid	209	43
		zinc finger protein-2
341	AF176665	Xenopus laevis F-box	249	29
		protein 27
342	AL079335	Homo sapiens dJ132F21.2	352	56
		(Contains a novel
		protein similar to the
		L82E from Drosophila)
343	AF177377	Homo sapiens cytoplasmic	628	62
		protein
344	AF002109	Arabidopsis thaliana	371	31
		putative ABC transporter
345	AJ010482	Homo sapiens Myopodin	579	29
		protein
346	X01831	Cairina moschata alpha	398	53
		D-globin
347	X01831	Cairina moschata alpha	314	54
		D-globin
348	AL163852	Arabidopsis thaliana	184	53
		putative protein
349	AF176532	Mus musculus F-box	706	59
		protein FBX17
350	D83674	Mus musculus MesP1	564	65
351	AC006539	Homo sapiens BC39498_3	387	100
352	AF229643	Mus musculus ubiquitin	2025	73
		specific protease
353	X17025	Homo sapiens homologue	808	64
		of yeast IPP isomerase
354	D79983	Homo sapiens There is a	659	52
		C3HC4 zinc-finger in the
		C-terminal region.
355	D79983	Homo sapiens There is a	434	44
		C3HC4 zinc-finger in the
		C-terminal region
356	AF109674	Rattus norvegicus late	968	86
		gestation lung protein 1
357	AC004780	Homo sapiens F17127_1	1258	87
358	X76116	Caenorhabditis elegans	567	48
		carrier protein (c2)
359	X77953	Rattus norvegicus	671	100
		ribosomal protein S15a
360	U67594	Methanococcus jannaschii	171	27
		Yeast KTI12 Protein
361	AF229643	Mus musculus ubiquitin	2711	95
		specific protease
362	AF083340	Homo sapiens double-	270	54
		stranded RNA-binding
		zinc finger protein JAZ
363	AE000161	Escherichia coli K12	296	59
		bacteriophage lambda
		lysozyme homolog
364	AL023705	Schizosaccharomyces	241	33
		pombe phenylalanyl-trna
		synthetase,
		mitochondrial precursor
365	J02870	Mus musculus laminin	321	64
		receptor
366	AL137391	Homo sapiens	1620	100
		hypothetical protein
367	AB012247	Arabidopsis thaliana	732	42
		contains similarity to
		zinc finger
		proteins˜gene_id:MSL1.13
368	AF167441	Mus musculus E-cadherin	2768	97
		binding protein E7
369	AF196576	Chlamydomonas	260	34
		reinhardtii flagellar
		protofilament ribbon
		protein
370	AF075050	Homo sapiens similar to	592	100
		(AJ223828) small
		glutamine-rich
		tetratricopeptide (SGT)
371	S79410	Mus musculus nuclear	107	51
		localization signal
		binding protein
372	AC015446	Arabidopsis thaliana	311	34
		Similar to AIG1 protein
373	AF217227	Homo sapiens zinc finger	1187	52
		protein ZNF287
374	AB013390	Arabidopsis thaliana	605	66
		ADP-ribosylation factor-
		like protein
375	AB026842	Mus musculus sialidase	732	51
376	D78174	Mus musculus Zic4	1490	86
		protein
377	X51394	Xenopus laevis APEG	186	26
		precursor protein
378	AJ001616	Mus musculus myeloid	1106	86
		associated
		differentiation protein
379	AF248651	Homo sapiens RNA-binding	869	100
		protein BRUNOL4
380	U16800	Xenopus laevis	764	72
		ribonucleoprotein
381	AE003503	Drosophila melanogaster	249	32
		CG4768 gene product
383	AB026192	Xenopus laevis Kielin	413	36
384	Y16430	Mus musculus ribosomal	201	54
		protein L35a
385	Z46259	Saccharomyces cerevisiae	169	40
		NO381
386	AF136010	Solanum chacoense SPP30	437	51
387	AK024432	Homo sapiens FLJ00022	1346	93
		protein
388	AB004665	Mus musculus Rab33B	1138	94
389	AF053768	Rattus norvegicus brain	589	36
		specific cortactin-
		binding protein CBP90
390	M20030	Homo sapiens small	434	94
		proline rich protein
391	AF182443	Rattus norvegicus F-box	1449	99
		protein FBL2
392	U53449	Rattus norvegicus jun	800	96
		dimerization protein 2
393	AB046381	Homo sapiens testis-	270	36
		abundant finger protein
394	AL031588	Homo sapiens dJ1163J1.2	863	100
		(novel protein similar
		to C. elegans B0035.16
		and bacterial tRNA (5-
		Methylaminomethyl-2-
		thiouridylate)
		Methyltransferases)
395	AF117814	Mus musculus odd-skipped	1413	97
		related 1 protein
396	AJ010228	Homo sapiens RFPL1L	889	58
397	U65079	Mus musculus actin-	2402	74
		binding protein
398	M64488	Rattus norvegicus	2128	95
		synaptotagmin II
399	AB017026	Mus musculus oxysterol-	548	99
		binding protein
400	U42580	Paramecium bursaria	213	33
		Chlorella virus 1
		contains 10 ankyrin-like
		repeats; similar to
		human ankyrin,
		corresponds to Swiss-
		Prot Accession Number
		P16157
401	Y00204	Xenopus laevis	464	49
		nucleoplasmin
402	U80741	Homo sapiens CAGH44	446	65
403	D16100	Rattus norvegicus 90kDa-	4055	95
		diacylglycerol kinase
404	AF132611	Homo sapiens	268	49
		monocarboxylate
		transporter MCT3
405	AB030505	Mus musculus UBE-1c2	661	50
406	AK024422	Homo sapiens FLJ00011	1450	97
		protein
407	AC007228	Homo sapiens BC37295_1	576	49
408	AB017059	Arabidopsis thaliana FH	183	43
		protein interacting
		protein FIP2
409	L06434	Rattus sp.	3709	96
		neurotransmitter
		transporter
410	AB017139	Rattus norvegicus Kilon	667	94
411	AF088982	Homo sapiens heat shock	478	49
		protein hsp40-3
412	L04948	Saccharomyces cerevisiae	467	37
		mitochondrial
		transporter protein
413	U09413	Homo sapiens zinc finger	1241	52
		protein ZNF135
414	X58079	Homo sapiens S100 alpha	274	57
		protein
415	X61617	Drosophila melanogaster	183	38
		neuralized protein
416	AF176694	Mus musculus neighbor of	344	41
		Punc ell protein
417	AC002086	Homo sapiens similar to	346	41
		zinc finger 5 protein
		from Gallus gallus ,
		U51640 (PID.g1399185)
418	AB046381	Homo sapiens testis-	564	43
		abundant finger protein
419	AF086824	Mus musculus rho/rac-	1506	89
		interacting citron
		kinase
420	AE000663	Mus musculus trypsinogen	898	65
		1
421	L20468	Rattus norvegicus	1132	87
		cerebroglycan
422	X73462	Ovis aries hair keratin	198	39
		cysteine rich protein
423	AC002394	Homo sapiens Gene	3043	100
		product with similarity
		to dynein beta subunit
424	AF118566	Mus musculus	722	48
		hematopoietic zinc
		finger protein
425	AJ010100	Homo sapiens NKp44RG2	163	30
426	AF059576	Mus musculus myosin	264	26
		binding protein-C
427	M37760	Mus musculus serine 2	461	41
		ultra high sulfur
		protein
428	AL359916	Homo sapiens bA550O8.2	1734	99
		(A novel protein similar
		to cell division protein
		kinase 4 (CDK4))
429	X68527	Homo sapiens TCR Vbeta	613	94
		5.5
430	AJ006078	Homo sapiens beta-1,3-	373	34
		galactosyltransferase
431	AF059493	Sus scrofa proteasome	618	53
		subunit LMP7
432	AL096770	Homo sapiens bA150A6.2	816	52
		(novel 7 transmembrane
		receptor (rhodopsin
		family) (olfactory
		receptor like) protein
		(hs6M1-21))
433	U38896	Homo sapiens zinc finger	501	62
		protein C2H2-171
434	AF093680	Homo sapiens	157	26
		transcription factor IIB
435	AJ237660	Bacteriophage 21 NinG	392	43
		protein
436	L10912	Oryctolagus cuniculus	1317	51
		cytochrome P-450 2B-Bx
437	AL031055	Homo sapiens dJ28H20.1	2624	99
		(novel protein similar
		to membrane transport
		proteins)
438	AJ007421	Homo sapiens spalt-like	6070	99
		zinc finger protein
439	X94744	Gallus gallus olfactory	443	45
		receptor 4
440	AB037973	Homo sapiens FGF-23	1360	100
441	X13986	Mus musculus minopontin	1521	100
		precursor (AA-66 to
		272)

TABLE 3
SEQ ID	ACCESSION
NO:	NO.	DESCRIPTION	RESULTS*
1	PF00642	Zinc finger C-x8-C-	PF00642 11.59 9.700e-12 426-437
		x5-C-x3-H type (and
		similar)
2	BL00291	Prion protein.	BL00291A 4.49 8.759e-09 185-220
3	PR00405	HIV REV INTERACTING	PR00405B 11.83 7.000e-21 49-67
		PROTEIN SIGNATURE	PR00405A 17.71 6.943e-13 30-50
			PR00405C 19.41 4.906e-12 70-92
4	PF01105	emp24/gp25L/p24	PF011058 25.12 1.000e-40 178-230
		family.
5	BL00307	Legume lectins beta-	BL00307G 9.91 8.531e-10 678-689
		chain proteins.
7	BL01159	ww/rsp5/WWP domain	BL01159 13.85 6.073e-09 61-76
		proteins.
8	BL01159	WW/rsp5/WWP domain	BL01159 13.85 6.073e-09 61-76
		proteins.
10	BL00913	Iron-containing	BL00913D 24.20 8.981e-17 170-204
		alcohol	BL00913C 7.62 4.375e-11 136-146
		dehydrogenases	BL00913B 10.94 7.706e-11 86-102
		proteins.
11	BL00913	Iron-containing	BL00913D 24.20 8.981e-17 218-252
		alcohol	BL00913C 7.62 4.375e-11 184-194
		dehydrogenases	BL00913B 10.94 7.706e-11 134-150
		proteins.
12	BL50062	BCL2-like apoptosis	BL50062C 6.66 8.500e-11 349-358
		inhibitors (spans
		part of BH3, BH1 and
		BH.
15	BL01144	Ribosomal protein	BL01144 25.07 9.069e-26 78-130
		L31e proteins.
16	PF00642	Zinc finger C-x8-C-	PF00642 11.59 6.694e-10 355-366
		x5-C-x3-H type (and
		similar).
18	PR00773	GRPE PROTEIN	PR00773D 16.14 5.922e-09 215-235
		SIGNATURE
24	PD00930	PROTEIN GTPASE	PD00930B 33.72 7.300e-26 600-641
		DOMAIN ACTIVATION.	PD00930A 25.62 1.514e-16 497-523
26	BL00375	UDP-	BL00375F 16.99 7.061e-35 291-336
		glycosyltransferases	BL00375C 18.27 2.615e-19 126-150
		proteins.	BL00375D 14.56 9.000e-17 192-220
			BL00375B 21.22 8.627e-16 67-108
			BL00375G 13.01 4.577e-13 390-430
29	BL01170	Ribosomal protein	BL01170A 12.34 9.143e-40 139-175
		L6e proteins.
30	BL00359	Ribosomal protein	BL00359B 23.07 4.231e-24 56-97
		L11 proteins.	BL00359C 22.18 6.586e-22 111-145
			BL00359A 20.66 4.000e-21 20-56
31	PR00983	CYSTEINYL-TRNA	PR00983D 14.16 3.647e-23 270-292
		SYNTHETASE SIGNATURE	PR00983C 11.27 3.415e-21 239-258
			PR00983A 11.10 1.878e-12 75-87
32	PR00718	PHOSPHOLIPASE D	PR00718E 8.61 1.000e-08 327-351
		SIGNATURE
33	BL00518	Zinc finger, C3HC4	BL00518 12.23 6.571e-10 49-58
		type (RING finger),
		proteins.
34	PF00992	Troponin.	PF00992A 16.67 7.972e-10 10-45
			PF00992A 16.67 5.145e-09 17-52
			PF00992A 16.67 6.684e-09 56-91
35	PR00452	SH3 DOMAIN SIGNATURE	PR00452D 17.02 8.000e-11 252-265
36	BL01019	ADP-ribosylation	BL01019A 13.20 8.000e-11 68-108
		factors family
		proteins.
39	PR00764	COMPLEMENT C9	PR00764F 16.89 7.783e-11 204-225
		SIGNATURE
40	PR00832	PAXILLIN SIGNATURE	PR00832B 9.87 6.284e-10 768-792
42	BL00226	Intermediate	BL00226D 19.10 3.172e-34 397-444
		filaments proteins.	BL00226B 23.86 5.929e-23 230-278
			BL00226C 13.23 4.808e-21 296-327
			BL00226A 12.77 5.065e-13 129-144
			BL00226B 23.86 6.400e-10 181-229
43	BL00243	Integrins beta chain	BL00243I 31.77 2.014e-09 156-199
		cysteine-rich domain	BL00243I 31.77 5.437e-09 159-202
		proteins.	BL00243I 31.77 5.690e-09 30-73
45	BL00291	Prion protein.	BL00291A 4.49 4.414e-09 47-82
46	PF00084	Sushi domain	PF00084B 9.45 7.188e-10 1549-1561
		proteins (SCR repeat
		proteins.
47	BL00414	Profilin proteins.	BL00414D 15.59 9.182e-10 81-108
50	PR00837	ALLERGEN V5/TPX-1	PR00837D 11.12 6.023e-09 22-36
		FAMILY SIGNATURE
51	BL00284	Serpins proteins.	BL00284A 15.64 2.350e-20 85-109
			BL00284D 16.34 4.240e-19 323-350
			BL00284C 28.56 5.600e-17 216-258
			BL00284E 19.15 7.500e-14 408-433
			BL00284B 17.99 9.379e-13 189-210
52	BL01283	T-box domain	BL01283A 24.15 2.125e-39 148-196
		proteins.	BL01283B 23.17 9.438e-34 208-250
			BL01283D 11.70 7.868e-31 298-331
			BL01283C 13.05 8.448e-16 260-274
53	PD01270	RECEPTOR FC	PD01270D 24.66 8.054e-09 50-86
		IMMUNOGLOBULIN
		AFFIN.
54	BL00237	G-protein coupled	BL00237A 27.68 2.543e-13 181-221
		receptors proteins.
55	PR00050	COLD SHOCK PROTEIN	PR00050A 11.28 3.143e-12 42-58
		SIGNATURE	PR00050C 9.82 9.151e-11 85-104
58	BL01173	Lipolytic enzymes G-	BL01173B 13.27 4.462e-17 140-167
		D-X-G family,	BL01173C 8.98 4.349e-14 182-196
		histidine.	BL01173A 9.41 1.818e-13 454-467
			BL01173C 8.98 6.553e-13 495-509
			BL01173A 9.41 8.364e-13 107-120
59	PR00321	GAMMA G-PROTEIN	PR00321C 15.39 2.473e-12 123-141
		(TRANSDUCIN)
		SIGNATURE
60	PR00937	T-BOX DOMAIN	PR00937A 15.25 1.000e-24 117-142
		SIGNATURE	PR00937D 13.41 5.500e-18 220-235
			PR00937B 14.58 5.235e-13 184-198
			PR00937F 12.53 1.450e-12 293-302
			PR00937E 11.86 1.918e-12 259-273
			PR00937C 10.51 3.571e-11 201-211
61	PD02059	CORE POLYPROTEIN	PD02059A 28.10 2.694e-09 116-157
		PROTEIN GAG
		CONTAINS: P.
65	BL00634	Ribosomal protein	BL00634 34.38 3.250e-15 46-97
		L30 proteins.
66	BL00226	Intermediate	BL00226B 23.86 1.643e-31 264-312
		filaments proteins.
70	DM00892	3 RETROVIRAL	DM00892C 23.55 6.727e-13 33-67
		PROTEINASE.
71	PR00874	FUNGI-IV	PR00874C 4.37 7.652e-10 68-83
		METALLOTHIONEIN
		SIGNATURE
72	PR00102	ORNITHINE	PR00102C 15.77 1.988e-07 42-57
		CARBAMOYLTRANSFERASE
		SIGNATURE
73	PR00359	B-CLASS P450	PR00359D 16.13 6.824e-08 291-314
		SIGNATURE
74	PR00066	XERODERMA	PR00066A 10.61 6.418e-08 77-95
		PIGMENTOSUM GROUP G
		PROTEIN SIGNATURE
75	PR00873	ECHINOIDEA (SEA	PR00873D 8.43 8.237e-08 13-32
		URCHIN)
		METALLOTHIONEIN
		SIGNATURE
76	PR00623	HISTONE H4 SIGNATURE	PR00623B 13.68 8.888e-09 364-384
77	BL00873	Sodium:alanine	BL00873E 24.91 2.800e-07 216-257
		symporter family
		proteins.
78	PR00478	PHOSPHORIBULOKINASE	PR00478C 12.84 5.012e-07 102-120
		FAMILY SIGNATURE
79	PF00878	Cation-independent	PF00878T 17.51 5.457e-08 52-79
		mannose-6-phosphate
		receptor repeat
		proteins.
80	PR00715	CATION-DEPENDENT	PR00715I 10.58 3.603e-06 112-131
		MANNOSE-6-PHOSPHATE
		RECEPTOR SIGNATURE
81	PR00350	VITAMIN D RECEPTOR	PR00350E 11.55 6.780e-07 45-65
		SIGNATURE
82	BL01131	Ribosomal RNA	BL01131A 26.62 2.376e-08 460-506
		adenine dimethylases
		proteins.
83	PR00629	SHC PHOSPHOTYROSINE	PR00629F 10.95 9.457e-07 96-123
		INTERACTION DOMAIN
		SIGNATURE
84	BL00111	Phosphoglycerate	BL00111A 9.65 7.783e-07 73-87
		kinase proteins.
88	PD00066	PROTEIN ZINC-FINGER	PD00066 13.92 7.000e-13 200-213
		METAL-BINDI.
89	PD02870	RECEPTOR	PD02870D 15.74 8.468e-09 358-393
		INTERLEUKIN-1
		PRECURSOR.
90	BL00048	Protamine P1	BL00048 6.39 6.815e-13 64-91 BL00048
		proteins.	6.39 4.750e-11 56-83 BL00048 6.39
			4.316e-10 55-82 BL00048 6.39 5.500e-
			10 70-97 BL00048 6.39 2.350e-09 62-89
			BL00048 6.39 3.700e-09 60-87
			BL00048 6.39 5.050e-09 63-90 BL00048
			6.39 6.288e-09 61-88 BL00048 6.39
			9.438e-09 71-98
91	PR00320	G-PROTEIN BETA WD-40	PR00320C 13.01 8.920e-10 202-217
		REPEAT SIGNATURE	PR00320B 12.19 9.486e-10 202-217
			PR00320C 13.01 7.900e-09 292-307
			PR00320A 16.74 8.902e-09 202-217
92	BL00453	FKBP-type peptidyl-	BL00453B 23.86 3.864e-28 106-140
		prolyl cis-trans	BL00453A 15.57 1.000e-15 81-96
		isomerase proteins.	BL00453C 9.72 1.000e-12 147-160
94	PR00299	ALPHA CRYSTALLIN	PR00299B 17.53 7.211e-09 324-337
		SIGNATURE
95	PF00676	Dehydrogenase E1	PF00676D 14.40 4.857e-13 421-441
		component.	PF00676C 16.88 1.931e-10 389-413
			PF00676B 24.71 5.433e-10 192-230
98	BL00824	Elongation factor 1	BL00824B 9.21 3.919e-09 1472-1492
		beta/beta′/delta
		chain proteins.
101	PR00417	PROKARYOTIC DNA	PR00417A 12.66 5.415e-09 866-880
		TOPOISOMERASE I
		SIGNATURE
104	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 6.936e-29 17-56
		ZINC-FINGER METAL-
		BINDING NU.
105	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 9.438e-37 10-49
		ZINC-FINGER METAL-
		BINDING NU.
106	PD01781	PROTEASE	PD01781B 27.55 8.680e-09 325-369
		IMMUNOGLOBULIN
		PRECURSO.
107	PD01781	PROTEASE	PD01781B 27.55 8.680e-09 379-423
		IMMUNOGLOBULIN
		PRECURSO.
109	PR00939	C2HC-TYPE ZINC-	PR00939B 13.27 3.647e-09 1302-1311
		FINGER SIGNATURE
110	PD00066	PROTEIN ZINC-FINGER	PD00066 13.92 2.800e-14 279-292
		METAL-BINDI.	PD00066 13.92 4.600e-14 307-320
			PD00066 13.92 1.000e-13 335-348
			PD00066 13.92 7.500e-13 363-376
111	PR00193	MYOSIN HEAVY CHAIN	PR00193D 14.36 5.680e-33 391-420
		SIGNATURE	PR00193C 12.60 4.789e-32 156-184
			PR00193B 11.69 1.692e-26 110-136
			PR00193E 19.47 5.500e-21 445-474
			PR00193A 15.41 4.130e-20 54-74
			PR00193E 19.47 5.091e-12 444-473
112	BL00239	Receptor tyrosine	BL00239B 25.15 2.985e-16 67-115
		kinase class II
		proteins.
113	BL00678	Trp-Asp (WD) repeat	BL00678 9.67 2.800e-10 366-377
		proteins proteins.	BL00678 9.67 5.263e-09 417-428
			BL00678 9.67 6.211e-09 186-197
114	DM00547	1 kw CHROMO	DM00547F 23.43 2.350e-35 384-431
		BROMODOMAIN SHADOW	DM00547C 17.30 7.000e-19 23-45
		GLOBAL.	DM00547E 13.94 5.592e-17 135-158
			DM00547D 11.60 2.750e-13 105-119
116	PR00700	PROTEIN TYROSINE	PR00700D 12.47 8.788e-11 237-256
		PHOSPHATASE
		SIGNATURE
118	PR00884	RIBOSOMAL PROTEIN	PR00884E 8.32 4.750e-09 449-466
		HS6 SIGNATURE
119	PD02890	ISOMERASE CHALCONE--	PD02890C 16.14 8.457e-09 200-235
		FLAVONONE FLAV.
120	BL00226	Intermediate	BL00226B 23.86 6.513e-10 401-449
		filaments proteins.
121	PD01823	PROTEIN INTERGENIC	PD01823C 16.13 7.000e-14 352-373
		REGION ABC1	PD01823B 14.96 3.782e-13 328-348
		PRECURSOR	PD01823D 16.66 6.857e-10 430-451
		MITOCHONDRION T.
124	BL00854	Proteasome B-type	BL00854C 29.92 8.435e-19 114-143
		subunits proteins.
126	BL00651	Ribosomal protein L9	BL00651A 23.25 8.477e-17 94-134
		proteins.
127	BL01245	RIO1/ZK632.3/MJ0444	BL01245F 18.75 2.373e-23 334-371
		family proteins.	BL01245A 14.04 8.342e-23 206-231
			BL01245C 13.31 6.564e-15 262-282
			BL01245E 15.28 1.000e-12 320-330
			BL01245B 11.91 9.809e-10 245-255
130	PR00793	PROLYL	PR00793C 12.24 1.333e-09 168-183
		AMINOPEPTIDASE (S33)
		FAMILY SIGNATURE
131	BL01160	Kinesin light chain	BL01160D 10.17 7.077e-09 505-534
		repeat proteins.
132	BL00355	HMG14 and HMG17	BL00355 5.97 8.412e-32 18-49
		proteins.
133	PR00041	CAMP RESPONSE	PR00041E 7.20 2.976e-13 305-326
		ELEMENT BINDING
		(CREB) PROTEIN
		SIGNATURE
134	PR00211	GLUTELIN SIGNATURE	PR00211B 0.86 1.750e-09 205-226
			PR00211B 0.86 8.750e-09 199-220
135	BL00455	Putative AMP-binding	BL00455 13.31 5.125e-11 293-309
		domain proteins.
138	PD00015	GLYCOPROTEIN	PD00015A 8.90 6.400e-09 243-251
		PRECURSOR CELL SI.
140	BL00227	Tubulin subunits	BL00227B 19.29 1.000e-40 52-107
		alpha, beta, and	BL00227C 25.48 1.000e-40 113-165
		gamma proteins.	BL00227A 24.55 8.200e-36 1-35
142	PR00049	WILM'S TUMOUR	PR00049D 0.00 8.377e-13 60-75
		PROTEIN SIGNATURE	PR00049D 0.00 7.500e-10 63-78
			PR00049D 0.00 8.071e-10 61-76
143	PR00049	WILM'S TUMOUR	PR00049D 0.00 6.438e-12 1613-1628
		PROTEIN SIGNATURE
144	DM01970	0 kw ZK632.12	DM01970B 8.60 4.750e-17 552-565
		YDR313C ENDOSOMAL
		III.
145	PD00066	PROTEIN ZINC-FINGER	PD00066 13.92 5.846e-15 579-592
		METAL-BINDI.	PD00066 13.92 9.217e-11 551-564
			PD00066 13.92 6.700e-09 523-536
146	PR00926	MITOCHONDRIAL	PR00926F 17.75 3.672e-10 262-285
		CARRIER PROTEIN
		SIGNATURE
149	DM01417	6 kw INDUCING XPMC2	DM01417C 12.93 3.250e-11 267-279
		MUSHROOM	DM01417D 11.08 2.200e-10 306-322
		SPAC22G7.04.
150	BL01160	Kinesin light chain	BL01160B 19.54 8.378e-10 349-403
		repeat proteins.
153	PR00049	WILM'S TUMOUR	PR00049D 0.00 7.807e-11 419-434
		PROTEIN SIGNATURE	PR00049D 0.00 8.563e-11 1284-1299
			PR00049D 0.00 3.929e-10 1283-1298
			PR00049D 0.00 3.288e-09 417-432
156	BL00665	Dihydrodipicolinate	BL00665D 14.76 1.000e-11 109-132
		synthetase proteins.	BL00665C 25.58 5.832e-11 50-101
158	PD02906	SYNTHASE I	PD02906C 24.17 9.115e-15 171-206
		PSEUDOURIDYLATE	PD02906B 15.35 4.886e-13 142-155
		PSEUDOURIDINE LYASE	PD02906D 12.27 1.000e-09 239-249
		TR.	PD02906A 10.84 8.333e-09 92-105
159	BL00107	Protein kinases ATP-	BL00107B 13.31 2.286e-11 396-412
		binding region	BL00107A 18.39 6.586e-11 332-363
		proteins.
162	PF01008	Initiation factor 2	PF01008B 25.59 9.609e-36 366-409
		subunit.	PF01008A 20.14 8.676e-12 315-336
			PF01008C 12.25 7.382e-10 449-469
163	BL00591	Glycosyl hydrolases	BL00591D 8.33 6.167e-09 2099-2112
		family 10 proteins.
165	PR00019	LEUCINE-RICH REPEAT	PR000198 11.36 7.120e-09 99-113
		SIGNATURE	PR000198 11.36 7.840e-09 73-87
166	BL00636	Nt-dnaJ domain	BL00636A 8.07 3.000e-14 143-160
		proteins.
167	PR00310	ANTI-PROLIFERATIVE	PR00310B 10.59 4.000e-39 41-71
		PROTEIN BTG1 FAMILY	PR00310C 12.74 2.256e-33 71-101
		SIGNATURE	PR00310D 9.10 9.820e-33 101-131
			PR00310A 11.17 7.000e-27 16-41
168	BL00216	Sugar transport	BL00216B 27.64 2.688e-21 124-174
		proteins.
169	BL00216	Sugar transport	BL00216B 27.64 5.636e-20 124-174
		proteins.
170	PD01066	PROTETN ZINC FINGER	PD01066 19.43 5.929e-32 59-98
		ZINC-FINGER METAL-
		BINDING NU.
172	PR00456	RIBOSOMAL PROTEIN P2	PR00456E 3.06 2.820e-11 6-21
		SIGNATURE	PR00456E 3.06 7.563e-10 3-18
173	PD00126	PROTEIN REPEAT	PD00126A 22.53 4.706e-14 140-161
		DOMAIN TPR NUCLEA.	PD00126A 22.53 6.824e-14 289-310
175	BL00741	Guanine-nucleotide	BL007418 14.27 3.418e-11 294-317
		dissociation
		stimulators CDC24
		family sign.
177	BL01016	Glycoprotease family	BL01016C 22.84 5.292e-19 60-105
		proteins.	BL01016H 13.71 6.595e-12 307-317
			BL01016E 14.88 3.182e-11 141-169
			BL01016D 8.86 6.741e-09 118-131
178	PR00850	GLYCOSYL HYDR0LASE	PR0085OB 6.67 5.455e-09 148-173
		FAMILY 59 SIGNATURE
180	PR00259	TRANSMEMBRANE FOUR	PR00259A 9.27 8.676e-20 17-41
		FAMILY SIGNATURE	PR00259C 16.40 4.750e-17 85-114
			PR00259B 14.81 8.615e-12 58-85
			PR00259D 13.50 2.528e-11 235-262
181	BL01052	Calponin family	BL01052C 18.51 6.806e-40 87-127
		repeat proteins.	BL01052A 16.12 7.618e-32 3-35
			BL01052B 15.31 8.031e-26 52-78
			BL01052D 10.26 1.000e-24 174-194
182	BL00875	Bacterial type II	BL00875A 25.57 6.447e-09 367-399
		secretion system
		protein D proteins.
183	PD01351	PROTEIN REPEAT	PD01351B 13.72 5.355e-09 238-264
		NEUROFILAMENT TRIPL.
184	DM01354	kw TRANSCRIPTASE	DM01354H 18.00 8.826e-27 109-149
		REVERSE II ORF2.	DM01354G 11.57 2.143e-25 78-109
			DM01354F 14.56 1.414e-15 42-78
			DM01354E 18.69 8.650e-14 17-47
187	BL00039	DEAD-box subfamily	BL00039A 18.44 4.000e-25 222-261
		ATP-dependent	BL00039D 21.67 4.529e-23 498-544
		helicases proteins.	BL00039C 15.63 4.300e-16 347-371
			BL00039B 19.19 9.379e-15 262-288
188	PD00066	PROTEIN ZINC-FINGER	PD00066 13.92 5.714e-12 152-165
		METAL-BINDI.	PD00066 13.92 6.143e-12 124-137
189	BL01022	PTR2 family	BL01022B 22.19 4.240e-10 308-354
		proton/oligopeptide
		symporters proteins.
192	PR00830	ENDOPEPTIDASE LA	PR00830A 8.41 3.342e-09 881-901
		(LON) SERINE
		PROTEASE (S16)
		SIGNATURE
193	PR00109	TYROSINE KINASE	PR00109B 12.27 9.234e-13 261-280
		CATALYTIC DOMAIN
		SIGNATURE
195	BL01033	Globins profile.	BL01033A 16.94 2.385e-18 25-47
197	PR00320	G-PROTEIN BETA WD-40	PR00320B 12.19 6.226e-11 140-155
		REPEAT SIGNATURE	PR00320A 16.74 4.971e-10 140-155
			PR00320C 13.01 9.280e-10 140-155
198	PR00832	PAXILLIN SIGNATURE	PR00832B 9.87 9.174e-10 309-333
199	PR00674	LIGHT HARVESTING	PR00674A 20.10 7.391e-09 134-155
		PROTEIN B CHAIN
		SIGNATURE
200	PR00192	F-ACTIN CAPPING	PR00192C 6.65 2.500e-36 57-84
		PROTEIN BETA SUBUNIT	PR00192D 8.23 4.462e-36 97-125
		SIGNATURE	PR00192E 8.85 7.000e-33 212-239
			PR00192A 8.23 1.474e-27 5-26
			PR00192B 6.20 3.000e-26 26-48
201	PF00023	Ank repeat proteins.	PF00023A 16.03 4.750e-10 45-61
203	PR00239	MOLLUSCAN RHODOPSIN	PR00239E 1.58 6.114e-09 183-195
		C-TERMINAL TAIL
		SIGNATURE
204	BL00412	Neuromodulin (GAP-	BL00412D 16.54 4.033e-10 319-370
		43) proteins.
205	BL00790	Receptor tyrosine	BL00790R 16.20 7.677e-09 29-73
		kinase class V
		proteins.
206	BL00790	Receptor tyrosine	BL00790R 16.20 7.677e-09 29-73
		kinase class V
		proteins.
207	BL00790	Receptor tyrosine	BL00790R 16.20 7.677e-09 29-73
		kinase class V
		proteins.
209	BL00211	ABC transporters	BL00211B 13.37 3.077e-17 573-605
		family proteins.	BL00211B 13.37 7.577e-17 1642-1674
			BL00211A 12.23 1.900e-09 472-484
211	PR00049	WILM'S TUMOUR	PR00049D 0.00 1.786e-10 288-303
		PROTEIN SIGNATURE
212	BL00972	Ubiquitin carboxyl-	BL00972D 22.55 3.348e-11 388-413
		terminal hydrolases	BL00972E 20.72 4.343e-09 415-437
		family 2 proteins.
216	PD00469	PROTEIN PRECURSOR	PD00469A 13.95 6.400e-09 73-86
		SIGNAL HYDROLA.
217	PF00023	Ank repeat proteins.	PF00023A 16.03 8.875e-10 839-855
			PF00023A 16.03 2.286e-09 884-900
219	BL00982	Bacterial-type	BL00982A 18.41 8.013e-12 328-360
		phytoene
		dehydrogenase
		proteins.
220	PF00595	PDZ domain proteins	PF00595 13.40 4.600e-09 688-699
		(Also known as DHR
		or GLGF).
221	BL00107	Protein kinases ATP-	BL00107A 18.39 7.000e-23 65-96
		binding region	BL00107B 13.31 4.214e-10 130-146
		proteins.
222	PR00239	MOLLUSCAN RHODOPSIN	PR00239E 1.58 3.045e-09 38-50
		C-TERMINAL TAIL
		SIGNATURE
224	BL00326	Tropomyosins	BL00326A 14.01 5.337e-10 825-859
		proteins.
226	BL00478	LIM domain proteins.	BL00478B 14.79 8.527e-09 143-158
227	DM00179	w KINASE ALPHA	DM00179 13.97 9.526e-10 135-145
		ADHESION T-CELL.
228	BL00048	Protamine P1	BL00048 6.39 6.063e-09 199-226
		proteins.
230	BL00115	Eukaryotic RNA	BL00115Z 3.12 5.744e-10 113-162
		polymerase II	BL00115Z 3.12 3.449e-09 120-169
		heptapeptide repeat
		proteins.
231	BL01161	Glucosamine/galactos	BL01161A 19.47 1.000e-40 37-77
		amine-6-phosphate	BL01161D 28.14 1.000e-40 199-244
		isomerases proteins.	BL01161B 21.37 5.091e-39 117-160
			BL01161C 18.47 1.500e-23 170-199
233	PR00269	PLEIOTROPHIN/MIDKINE	PR00269A 13.91 3.571e-30 88-113
		FAMILY SIGNATURE
238	BL00888	Cyclic nucleotide-	BL00888B 14.79 9.069e-13 499-523
		binding domain
		proteins.
240	BL01188	GNS1/SUR4 family	BL01188B 13.46 4.115e-26 120-151
		proteins.	BL01188C 22.65 4.136e-26 151-202
			BL01188D 8.62 1.290e-11 238-255
			BL01188A 18.82 6.718e-10 55-87
241	PR00929	AT-HOOK-LIKE DOMAIN	PR00929B 4.38 8.875e-09 571-583
		SIGNATURE	PR00929C 5.26 8.914e-09 571-582
244	BL00232	Cadherins	BL00232B 32.79 2.765e-25 541-589
		extracellular repeat	BL00232B 32.79 8.263e-22 766-814
		proteins domain	BL00232B 32.79 2.397e-21 67-115
		proteins.	BL00232B 32.79 4.57le-19 1481-1529
			BL00232B 32.79 1.000e-18 1371-1419
			BL00232B 32.79 2.662e-18 1691-1739
			BL00232B 32.79 5.292e-18 1287-1335
			BL00232B 32.79 9.585e-18 1586-1634
			BL00232B 32.79 1.265e-17 980-1028
			BL00232B 32.79 1.529e-17 426-474
			BL00232B 32.79 2.588e-17 1084-1132
			BL00232B 32.79 1.386e-16 1184-1232
			BL00232C 10.65 5.390e-12 1369-1387
			BL00232C 10.65 1.391e-11 642-660
			BL00232C 10.65 2.174e-11 1584-1602
			BL00232C 10.65 4.522e-11 1689-1707
			BL00232C 10.65 1.000e-10 65-83
			BL00232C 10.65 4.115e-10 1285-1303
			BL00232B 32.79 7.200e-10 649-697
			BL00232C 10.65 9.827e-10 978-996
			BL00232C 10.65 1.947e-09 170-188
			BL00232B 32.79 2.137e-09 172-220
			BL00232C 10.65 4.474e-09 11B2-1200
			BL00232C 10.65 8.737e-09 539-557
245	BL00795	Involucrin proteins.	BL00795C 17.06 4.977e-10 64-109
			BL00795C 17.06 6.300e-09 55-100
246	BL00790	Receptor tyrosine	BL00790I 20.01 7.823e-15 23-54
		kinase class V	BL00790I 20.01 9.400e-11 310-341
		proteins.	BL00790I 20.01 1.900e-10 117-148
			BL00790I 20.01 3.893e-09 215-246
247	BL00183	Ubiquitin-	BL00183 28.97 7.037e-10 140-188
		conjugating enzymes
		proteins.
248	PR00019	LEUCINE-RICH REPEAT	PR00019A 11.19 8.800e-12 205-219
		SIGNATURE	PR00019B 11.36 2.000e-11 202-216
249	BL00214	Cytosolic fatty-acid	BL00214B 26.51 7.618e-24 206-251
		binding proteins.	BL00214A 21.17 6.250e-22 165-191
250	PR00395	RIBOSOMAL PROTEIN S2	PR00395C 16.17 2.047e-13 46-64
		SIGNATURE
251	BL00227	Tubulin subunits	BL00227D 18.46 1.000e-40 74-128
		alpha, beta, and	BL00227F 21.16 1.529e-33 226-280
		gamma proteins.	BL00227E 24.15 1.409e-26 178-213
252	BL00227	Tubulin subunits	BL00227C 25.48 1.000e-40 39-91
		alpha, beta, and	BL00227D 18.46 1.000e-40 148-202
		gamma proteins.	BL00227F 21.16 1.529e-33 300-354
			BL00227E 24.15 1.409e-26 252-287
253	BL00152	ATP synthase alpha	BL00152B 21.40 1.900e-31 191-229
		and beta subunits	BL00152A 15.38 5.154e-21 134-160
		proteins.	BL00152C 11.41 6.250e-12 291-303
254	BL00152	ATP synthase alpha	BL00152E 22.68 1.000e-32 285-323
		and beta subunits	BL00152A 15.38 5.154e-21 134-160
		proteins.	BL00152C 11.41 6.250e-12 247-259
255	BL00518	Zinc finger, C3HC4	BL00518 12.23 2.200e-11 54-63
		type (RING finger),
		proteins.
256	DM00892	3 RETROVIRAL	DM00892C 23.55 9.739e-12 417-451
		PROTEINASE.
257	BL01052	Calponin family	BL01052C 18.51 1.000e-40 88-128
		repeat proteins.	BL01052A 16.12 2.875e-35 3-35
			BL01052B 15.31 5.219e-26 52-78
258	BL00745	Prokaryotic-type	BL00745C 13.66 1.000e-40 238-285
		class I peptide	BL00745B 22.56 8.683e-33 184-227
		chain release	BL00745D 14.90 8.435e-23 316-339
		factors signat.	BL00745A 26.45 1.818e-19 123-162
259	BL00745	Prokaryotic-type	BL00745C 13.66 1.000e-40 202-249
		class I peptide	BL00745B 22.56 8.683e-33 148-191
		chain release	BL00745D 14.90 8.435e-23 280-303
		factors signat.
261	BL00194	Thioredoxin family	BL00194 12.16 7.429e-10 6B4-697
		proteins.
262	BL00612	Osteonectin domain	BL00612E 13.12 3.948e-10 391-436
		proteins.
264	PR00625	DNAJ PROTEIN FAMILY	PR00625A 12.84 2.375e-09 288-308
		SIGNATURE
265	PR00320	G-PROTEIN BETA WD-40	PR00320B 12.19 2.125e-09 207-222
		REPEAT SIGNATURE
268	BL01144	Ribosomal protein	BL01144 25.07 1.000e-40 21-73
		L31e proteins.
270	DM00516	186 DISCOIDIN I N-	DM00516 30.53 8.606e-13 153-198
		TERMINAL.
271	BL00622	Bacterial regulatory	BL00622 32.69 9.780e-09 11-58
		proteins, luxR
		family proteins.
272	PR00048	C2H2-TYPE ZINC	PR00048A 10.52 1.000e-11 447-461
		FINGER SIGNATURE	PR00048A 10.52 4.316e-11 389-403
			PR00048A 10.52 6.684e-11 362-376
276	DM00303	6 LEA 11-MER REPEAT	DM00303A 13.20 3.310e-09 467-517
		REPEAT.
277	PF00622	Domain in SP1a and	PF00622B 21.00 9.357e-14 374-396
		the RYanodine	PF00622C 12.62 1.857e-12 458-472
		Receptor.
279	PF00651	BTB (also known as	PF00651 15.00 9.571e-10 65-78
		BR-C/Ttk) domain
		proteins.
280	PD00066	PROTEIN ZINC-FINGER	PD00066 13.92 8.200e-16 295-308
		METAL-BINDI.	PD00066 13.92 8.200e-16 519-532
			PD00066 13.92 1.692e-15 351-364
			PD00066 13.92 4.462e-15 547-560
			PD00066 13.92 4.600e-14 323-336
			PD00066 13.92 4.600e-14 435-448
			PD00066 13.92 7.000e-14 463-476
			PD00066 13.92 1.500e-13 239-252
			PD00066 13.92 3.143e-12 267-280
			PD00066 13.92 3.143e-12 407-420
			PD00066 13.92 8.826e-11 211-224
			PD00066 13.92 2.038e-10 491-504
			PD00066 13.92 2.385e-10 379-392
281	PD00066	PROTEIN ZINC-FINGER	PD00066 13.92 6.400e-16 449-462
		METAL-BINDI.	PD00066 13.92 6.538e-15 504-517
			PD00066 13.92 9.308e-15 421-434
			PD00066 13.92 7.000e-14 476-489
			PD00066 13.92 6.087e-11 393-406
282	BL00291	Prion protein.	BL00291A 4.49 5.229e-10 429-464
287	BL00276	Channel forming	BL00276A 8.87 6.500e-09 257-269
		colicins proteins.
288	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 2.000e-30 10-49
		ZINC-FINGER METAL-
		BINDING NU.
289	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 7.407e-23 3-42
		ZINC-FINGER METAL-
		BINDING NU.
291	PR00070	DIHYDROFOLATE	PR00070C 13.09 6.143e-16 51-63
		REDUCTASE SIGNATURE	PR00070D 11.63 2.929e-15 112-127
294	PR00250	FUNGAL PHEROMONE	PR00250D 14.62 9.163e-09 254-278
		MATING FACTOR STE2
		GPCR SIGNATURE
296	PR00081	GLUCOSE/RIBITOL	PR00081A 10.53 2.731e-09 39-57
		DEHYDR0GENASE FAMILY
		SIGNATURE
297	PR00806	VINCULIN SIGNATURE	PR00806B 4.28 8.920e-09 276-290
			PR00806B 4.28 9.640e-09 275-289
298	PF00992	Troponin.	PF00992A 16.67 3.789e-10 553-588
300	PR00511	TEKTIN SIGNATURE	PR00511C 7.86 4.214e-09 371-388
302	BL00353	HMG1/2 proteins.	BL00353B 11.47 9.609e-19 228-278
303	PR00240	ALPHA-1A ADRENERGIC	PR00240C 8.38 3.941e-10 316-336
		RECEPTOR SIGNATURE
304	BL00518	Zinc finger, C3HC4	BL00518 12.23 2.200e-11 54-63
		type (RING finger),
		proteins.
307	PR00193	MYOSIN HEAVY CHAIN	PR00193D 14.36 1.545e-31 390-419
		SIGNATURE	PR00193C 12.60 1.209e-25 143-171
			PR00193B 11.69 2.543e-24 95-121
			PR00193A 15.41 6.885e-19 39-59
			PR00193E 19.47 3.291e-12 444-473
308	PR00239	MOLLUSCAN RHODOPSIN	PR00239E 1.58 5.920e-11 47-59
		C-TERMINAL TAIL
		SIGNATURE
309	PD00015	GLYCOPROTEIN	PD00015A 8.90 6.400e-09 35-43
		PRECURSOR CELL SI.
312	DM00031	IMMUNOGLOBULIN V	DM00031B 15.41 3.662e-11 80-114
		REGION.
313	BL00824	Elongation factor 1	BL00824C 14.58 1.000e-40 129-167
		beta/beta′/delta	BL00824D 14.04 6.192e-39 167-202
		chain proteins.	BL00824B 9.21 2.080e-21 96-116
			BL00824E 12.49 3.333e-19 210-226
314	DM00099	4 kw A55R REDUCTASE	DM00099B 14.73 8.364e-11 525-535
		TERMINAL	DM00099B 14.73 9.438e-09 478-488
		DIHYDROPTERIDINE.
315	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 6.200e-30 43-82
		ZINC-FINGER METAL-
		BINDING NU.
316	PR00121	SODIUM/POTASSIUM-	PR00121D 16.72 1.577e-13 210-232
		TRANSPORTING ATPASE
		SIGNATURE
317	BL00888	Cyclic nucleotide-	BL00888B 14.79 1.692e-10 396-420
		binding domain
		proteins.
318	PR00727	BACTERIAL LEADER	PR00727C 13.04 9.063e-16 108-128
		PEPTIDASE 1 (S26)	PR00727B 12.51 7.848e-11 81-94
		FAMILY SIGNATURE
319	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 9.471e-27 13-52
		ZINC-FINGER METAL-
		BINDING NU.
321	PR00004	ANAPHYLATOXIN DOMAIN	PR00004C 12.46 8.579e-09 91-103
		SIGNATURE
322	DM00060	338 kw NEUREXIN	DM00060 6.92 6.500e-11 28-38
		ALPHA III CYSTEINE.
327	PR00020	MAM DOMAIN SIGNATURE	PR00020A 18.17 5.776e-12 344-363
			PR00020C 13.66 6.932e-10 417-429
328	BL00048	Protamine P1	BL00048 6.39 6.566e-10 167-194
		proteins.
329	PR00020	MAM DOMAIN SIGNATURE	PR00020C 13.66 2.615e-11 581-593
			PR00020B 15.52 5.059e-10 52-69
			PR00020B 15.52 1.789e-09 553-570
331	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 9.357e-32 8-47
		ZINC-FINGER METAL-
		BINDING NU.
334	PD02870	RECEPTOR	PD02870B 18.83 5.871e-11 468-501
		INTERLEUKIN-1
		PRECURSOR.
335	BL00738	S-adenosyl-L-	BL00738J 18.61 1.000e-40 592-642
		homocysteine	BL00738H 23.08 5.320e-36 468-521
		hydrolase proteins.	BL00738F 12.23 7.261e-29 387-419
			BL00738A 16.27 9.660e-27 216-256
			BL00738C 16.53 7.923e-25 281-319
			BL00738G 14.29 6.268e-23 446-468
			BL00738B 12.28 8.085e-21 256-281
			BL00738E 14.18 9.200e-19 361-384
			BL00738I 14.57 5.135e-17 545-583
			BL00738D 7.16 5.109e-13 335-350
339	PR00425	BRADYKININ RECEPTOR	PR00425C 13.23 3.586e-09 80-100
		SIGNATURE
344	PD01B23	PROTEIN INTERGENIC	PD01823E 9.30 6.824e-12 108-121
		REGION ABC1	PD01823D 16.66 1.265e-09 46-67
		PRECURSOR
		MITOCHONDRION T.
345	PR00976	RIBOSOMAL PROTEIN	PR00976C 10.41 2.837e-09 396-407
		S21 FAMILY
		SIGNATURE.
346	PR00613	MYOGLOBIN SIGNATURE	PR00613B 9.02 2.581e-10 25-49
347	PR00814	BETA HAEMOGLOBIN	PR00814C 9.20 6.523e-10 104-122
		SIGNATURE
350	BL00038	Myc-type1, ‘helix-	BL00038B 16.97 7.568e-10 117-138
		loop-helix’
		dimerization domain
		proteins.
351	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 6.571e-32 6-45
		ZINC-FINGER METAL-
		BINDING NU.
352	BL00972	Ubiquitin carboxyl-	BL00972A 11.93 6.318e-19 364-382
		terminal hydrolases	BL00972D 22.55 7.968e-16 648-673
		family 2 proteins.	BL009728 9.45 1.600e-12 445-455
354	BL00518	Zinc finger, C3HC4	BL00518 12.23 4.429e-10 214-223
		type (RING finger),
		proteins.
355	BL00518	Zinc finger, C3HC4	BL00518 12.23 4.429e-10 179-188
		type (RING finger),
		proteins.
356	BL01009	Extracellular	BL01009D 14.19 9.341e-17 160-181
		proteins SCP/Tpx-	BL01009A 13.75 3.769e-14 80-98
		1/Ag5/PR-1/Sc7	BL01009E 13.50 5.333e-14 194-210
		proteins.	BL01009C 10.54 2.667e-11 127-141
358	BL00215	Mitochondrial energy	BL00215A 15.82 8.500e-17 16-41
		transfer proteins.	BL00215B 10.44 4.900e-09 177-190
			BL00215A 15.82 6.786e-09 133-158
			BL00215B 10.44 7.300e-09 278-291
359	BL00053	Ribosomal protein S8	BL00053C 16.71 5.500e-26 107-140
		proteins.	BL00053B 14.56 4.789e-14 67-85
			BL00053A 8.83 5.320e-12 14-27
360	PR00326	GTP1/OBG GTP-BINDING	PR00326A 8.75 7.150e-11 21-42
		PROTEIN FAMILY
		SIGNATURE
361	BL00972	Ubiquitin carboxyl-	BL00972A 11.93 6.318e-19 324-342
		terminal hydrolases	BL00972D 22.55 3.903e-16 608-633
		family 2 proteins.	BL00972B 9.45 1.600e-12 405-415
364	PR00759	BASIC PROTEASE	PR00759C 14.15 7.750e-10 123-139
		(KUNITZ-TYPE)
		INHIBITOR FAMILY
		SIGNATURE
367	DM00215	PROLINE-RICH PROTEIN	DM00215 19.43 1.482e-10 355-388
		3.
368	BL00518	Zinc finger, C3HC4	BL00518 12.23 2.800e-11 125-134
		type (RING finger),
		proteins.
370	PD00126	PROTEIN REPEAT	PD00126A 22.53 8.448e-09 2-23
		DOMAIN TPR NUCLEA.
373	BL00028	Zinc finger, C2H2	BL00028 16.07 7.353e-14 157-174
		type, domain	BL00028 16.07 1.000e-13 269-286
		proteins.	BL00028 16.07 8.200e-13 493-510
			BL00028 16.07 3.739e-12 213-230
			BL00028 16.07 6.478e-12 381-398
			BL00028 16.07 1.346e-11 185-202
			BL00028 16.07 2.385e-11 129-146
			BL00028 16.07 2.385e-11 325-342
			BL00028 16.07 5.154e-11 241-258
			BL00028 16.07 9.654e-11 437-454
			BL00028 16.07 1.300e-10 297-314
			BL00028 16.07 9.100e-10 409-426
			BL00028 16.07 9.100e-10 465-482
374	BL01020	SAR1 family	BL01020C 15.35 8.063e-20 79-130
		proteins.
376	BL00028	Zinc finger, C2H2	BL0002B 16.07 4.522e-12 208-225
		type, domain
		proteins.
377	PR00308	TYPE I ANTIFREEZE	PR00308A 5.90 7.288e-11 533-548
		PROTEIN SIGNATURE	PR00308A 5.90 8.835e-09 534-549
380	PD02784	PROTEIN NUCLEAR	PD02784B 26.46 7.538e-09 147-190
		RIBONUCLEOPROTEIN.
381	PD01351	PROTEIN REPEAT	PD01351A 8.69 7.469e-09 155-166
		NEUROFILAMENT TRIPL.
383	PF00094	von Willebrand	PF00094C 12.88 1.918e-09 43-53
		factor type D domain
		proteins.
384	BL01105	Ribosomal protein	BL011058 12.95 7.930e-13 43-83
		L35Ae proteins.
387	PR00049	WILM'S TUMOUR	PR00049D 0.00 9.643e-10 10-25
		PROTEIN SIGNATURE	PR00049D 0.00 1.915e-09 9-24
388	BL01115	GTP-binding nuclear	BL01115A 10.22 8.909e-13 34-78
		protein ran
		proteins.
389	BL00115	Eukaryotic RNA	BL00115Z 3.12 7.977e-10 397-446
		polymerase II
		heptapeptide repeat
		proteins.
390	PR00021	SMALL PROLINE-RICH	PR00021A 4.31 2.125e-19 4-17
		PROTEIN SIGNATURE	PR00021D 6.09 3.323e-13 21-30
			PR00021D 6.09 3.323e-13 30-39
			PR00021E 6.82 7.545e-13 57-67
			PR00021B 7.29 9.333e-13 18-28
			PR00021B 7.29 3.302e-12 27-37
			PR00021C 5.55 1.225e-10 21-28
			PR00021C 5.55 1.225e-10 30-37
			PR00021D 6.09 2.770e-09 39-48
391	PF00646	F-box domain	PF00646A 14.37 9.036e-10 28-42
		proteins.
392	BL00036	bZIP transcription	BL00036 9.02 6.294e-12 81-94
		factors basic domain
		proteins.
393	PF00622	Domain in SP1a and	PF00622B 21.00 2.500e-13 85-107
		the RYanodine
		Receptor.
394	BL00564	Argininosuccinate	BL00564A 19.93 6.114e-09 7-44
		synthase proteins.
395	PR00048	C2H2-TYPE ZINC	PR00048A 10.52 7.750e-14 230-244
		FINGER SIGNATURE	PR00048A 10.52 4.316e-11 202-216
396	PF00622	Domain in SP1a and	PF00622B 21.00 1.391e-16 132-154
		the RYanodine
		Receptor.
397	PR00501	KELCH REPEAT	PR00501A 8.25 1.409e-09 537-551
		SIGNATURE
398	PR00399	SYNAPTOTAGMIN	PR00399A 9.52 3.571e-19 146-162
		SIGNATURE	PR00399C 12.82 8.200e-17 222-238
			PR00399B 14.27 7.750e-16 161-175
			PR00399D 14.48 4.000e-14 242-253
399	BL01013	Oxysterol-binding	BL01013A 25.14 7.231e-21 558-594
		protein family	BL01013B 11.33 1.000e-11 623-634
		proteins.
400	PF00023	Ank repeat proteins.	PF00023A 16.03 1.750e-10 55-71
401	BL00422	Granins proteins.	BL00422C 16.18 5.787e-10 134-162
402	PR00029	OCTAMER-BINDING	PR00029C 6.10 5.708e-09 140-153
		TRANSCRIPTION FACTOR
		SIGNATURE
403	PR00450	RECOVERIN FAMILY	PR00450D 16.58 8.986e-11 161-181
		SIGNATURE
404	PR00049	WILM'S TUMOUR	PR00049D 0.00 7.407e-09 325-340
		PROTEIN SIGNATURE
405	DM01117	2 kw TRANSPOSASE	DM01117A 11.17 7.750e-09 364-382
		WITHIN TRANSPOSITION
		VASOTOCIN.
406	DM01206	CORONAVIRUS	DM01206B 10.69 9.286e-12 724-744
		NUCLEOCAPSID	DM01206B 10.69 3.466e-10 726-746
		PROTEIN.	DM01206B 10.69 9.630e-10 722-742
			DM01206B 10.69 7.152e-09 718-738
			DM01206B 10.69 8.861e-09 728-748
407	PD01066	PROTEIN ZINC FINGER	PD01066 19.43 1.353e-27 31-70
		ZINC-FINGER METAL-
		BINDING NU.
408	PR00169	POTASSIUM CHANNEL	PR00169A 16.77 1.711e-09 94-114
		SIGNATURE
409	BL00610	Sodium:neurotransmitter	BL00610A 17.73 1.000e-40 68-118
		symporter family	BL00610B 23.65 1.000e-40 132-182
		proteins.	BL00610C 12.94 1.000e-40 225-277
			BL00610D 20.97 1.000e-40 291-344
			BL00610F 29.02 6.143e-36 540-595
			BL00610E 20.34 3.647e-35 448-491
			BL00610G 12.89 2.200e-15 611-634
410	DM00179	w KINASE ALPHA	DM00179 13.97 5.304e-09 111-121
		ADHESION T-CELL.
411	PR00625	DNAJ PROTEIN FAMILY	PR00625B 13.48 1.800e-16 45-66
		SIGNATURE	PR00625A 12.84 6.700e-12 15-35
412	PR00927	ADENINE NUCLEOTIDE	PR00927E 14.93 4.136e-11 246-268
		TRANSLOCATOR 1
		SIGNATURE
413	PD00066	PROTEIN ZINC-FINGER	PD00066 13.92 6.400e-17 411-424
		METAL-BINDI.	PD00066 13.92 8.200e-17 327-340
			PD00066 13.92 5.154e-15 271-284
			PD00066 13.92 2.800e-14 215-228
			PD00066 13.92 9.000e-13 355-368
			PD00066 13.92 6.143e-12 439-452
			PD00066 13.92 6.478e-11 187-200
			PD00066 13.92 9.217e-11 243-256
414	BL00303	S-100/ICaBP type	BL00303B 26.15 2.552e-23 51-88
		calcium binding	BL00303A 21.77 5.846e-20 4-41
		protein.
416	PR00014	FIBRONECTIN TYPE III	PR00014C 15.44 4.600e-10 73-92
		REPEAT SIGNATURE
417	PR00806	VINCULIN SIGNATURE	PR00806A 6.63 1.493e-09 785-796
418	PF00622	Domain in SP1a and	PF00622B 21.00 1.000e-13 331-353
		the RYanodine	PF00622C 12.62 3.160e-11 415-429
		Receptor.
419	PF00780	Domain found in	PF00780B 23.03 5.929e-33 442-485
		NIK1-like kinases,
		mouse citron and
		yeast ROM.
420	BL01253	Type I fibronectin	BL01253H 13.15 8.452e-13 203-238
		domain proteins.	BL01253D 4.84 2.016e-12 41-55
421	BL01207	Glypicans proteins.	BL01207B 23.69 9.122e-28 191-237
			BL01207A 12.21 1.000e-16 62-78
426	PD02870	RECEPTOR	PD02870D 15.74 4.351e-09 693-728
		INTERLEUKIN-1
		PRECURSOR.
427	BL00203	Vertebrate	BL00203 13.94 5.041e-09 13-59
		metallothioneins
		proteins.
428	BL00107	Protein kinases ATP-	BL00107A 18.39 8.579e-18 217-248
		binding region
		proteins.
431	PR00141	PROTEASOME COMPONENT	PR00141C 11.15 6.333e-12 234-246
		SIGNATURE	PR00141D 12.45 8.615e-12 259-271
			PR00141B 11.15 9.561e-12 223-235
			PR00141A 11.36 2.050e-11 102-118
432	PR00245	OLFACTORY RECEPTOR	PR00245A 18.03 9.413e-17 59-81
		SIGNATURE	PR00245C 7.84 7.500e-16 238-254
			PR00245E 12.40 2.500e-12 291-306
			PR00245B 10.38 9.550e-11 177-192
433	PF00651	BTB (also known as	PF00651 15.00 1.000e-11 87-100
		BR-C/Ttk) domain
		proteins.
436	BL00086	Cytochrome P450	BL00086 20.87 3.647e-23 430-462
		cysteine heme-iron
		ligand proteins.
437	BL00216	Sugar transport	BL00216B 27.64 7.943e-19 101-151
		proteins.
438	PR00049	WILM'S TUMOUR	PR00049D 0.00 8.429e-10 10-25
		PROTEIN SIGNATURE
439	PR00245	OLFACTORY RECEPTOR	PR00245A 18.03 2.667e-23 100-122
		SIGNATURE	PR00245C 7.84 1.783e-14 232-248
			PR00245D 10.47 7.070e-10 268-280
440	PR00262	IL1/HBGF FAMILY	PR00262A 28.26 1.000e-08 80-108
		SIGNATURE
441	BL00884	Osteopontin	BL00884B 12.47 1.000e-40 50-94
		proteins.	BL00884C 22.45 6.625e-39 131-173
			BL00884A 11.35 5.846e-32 1-31
			BL00884E 11.04 8.364e-23 273-295
			BL00884D 8.79 3.323e-18 255-272
*Results include in order: accession number subtype; raw score; p-value; postion of signature in amino acid sequence.

TABLE 4
SEQ ID
NO:	pFAM NAME	DESCRIPTION	p-value	pFAM SCORE
1	zf-CCCH	Zinc finger C-x8-C-x5-C-	2.5e-09	38.4
		x3-H type (and similar).
3	ArfGap	Putative GTP-ase	1.3e-52	188.3
		activating protein for
		Arf
4	EMP24_GP25L	emp24/gp25L/p24 family	4.1e-105	362.6
7	WW	WW domain	1.2e-05	32.2
8	WW	WW domain	1.2e-05	32.2
9	Aa_trans	Transmembrane amino acid	9.6e-64	225.2
		transporter protein
10	Fe-ADH	Iron-containing alcohol	9.9e-35	124.5
		dehydrogenases
11	Fe-ADH	Iron-containing alcohol	9.9e-35	124.5
		dehydrogenases
12	Bcl-2	Apoptosis regulator	0.016	−2.1
		proteins, Bcl-2 family
13	spectrin	Spectrin repeat	1.3e-10	43.6
14	Ribosomal_L18ae	Ribosomal L18ae protein	1.9e-128	440.1
		family
15	Ribosomal_L31e	Ribosomal protein L31e	2.4e-47	170.7
17	zf-MYND	MYND finger	1.4e-13	58.5
19	MgtE	Divalent cation	8.6e-39	142.3
		transporter
20	Rap_GAP	Rap/ran-GAP	2e-124	426.7
21	Rap_GAP	Rap/ran-GAP	2e-124	426.7
23	SCAN	SCAN domain	1.3e-24	95.2
24	RhoGAP	RhoGAP domain	3e-58	206.9
25	adh_zinc	Zinc-binding	1.5e-05	−25.4
		dehydrogenases
26	UDPGT	UDP-glucoronosyl and UDP-	1.6e-84	294.3
		glucosyl transferases
27	MCT	Monocarboxylate	3.3e-49	176.9
		transporter
29	Ribosomal_L6e	Ribosomal protein L6e	9.5e-78	271.7
30	Ribosomal_L11	Ribosomal protein L11	4.9e-64	226.2
31	tRNA-synt_1e	tRNA synthetases class I	1.6e-137	470.2
		(C)
33	zf-C3HC4	Zinc finger, C3HC4 type	0.00041	17.6
		(RING finger)
35	SH3	SH3 domain	8.7e-32	119.0
36	ras	Ras_family	2.6e-78	273.6
38	SET	SET domain	3.2e-05	10.0
39	laminin_G	Laminin G domain	1.5e-11	44.7
40	Sema	Sema domain	5e-127	435.4
42	filament	Intermediate filament	1.6e-138	473.6
		proteins
43	Keratin_B2	Keratin, high sulfur B2	1.8e-18	74.8
		protein
46	sushi	Sushi domain (SCR repeat)	3.8e-06	33.9
47	profilin	Profilins	4.1e-13	51.7
49	ubiquitin	Ubiquitin family	0.069	10.3
50	BTB	BTB/POZ domain	2.6e-21	84.2
51	serpin	Serpins (serine protease	2.4e-178	605.4
		inhibitors
52	T-box	T-box	3.6e-125	429.2
54	7tm_1	7 transmembrane receptor	1.2e-17	58.3
		(rhodopsin family)
55	CSD	‘Cold-shock’ DNA-binding	1.8e-16	63.6
		domain
56	ig	Immunoglobulin domain	2.5e-07	28.7
57	Rap_GAP	Rap/ran-GAP	5e-18	73.3
59	G-gamma	GGL domain	1.1e-11	45.4
60	T-box	T-box	8.9e-114	391.4
63	60s_ribosomal	60s Acidic ribosomal	0.0089	12.0
		protein
64	UPAR_LY6	u-PAR/Ly-6 domain	5.4e-05	22.3
65	Ribosomal_L30	Ribosomal protein	0.00042	18.5
		L30p/L7e
66	filament	Intermediate filament	1.1e-78	274.8
		proteins
67	Ribosomal_S6	Ribosomal protein S6	0.00082	7.5
68	PDZ	PDZ domain (Also known as	5.1e-09	43.4
		DHR or GLGF).
69	zf-C3HC4	Zinc finger, C3HC4 type	0.005	14.0
		(RING finger)
70	G-patch	G-patch domain	0.00051	26.8
71	Keratin B2	Keratin, high sulfur B2	0.037	−45.9
		protein
85	ig	Immunoglobulin domain	8.5e-09	33.4
88	zf-C2H2	Zinc finger, C2H2 type	7.1e-71	248.9
89	ig	Immunoglobulin domain	2.7e-35	118.7
91	WD40	WD domain, G-beta repeat	7.5e-10	46.2
92	FKBP	FKBP-type peptidyl-prolyl	1.3e-53	173.3
		cis-trans isomerases
95	E1_dehydrog	Dehydrogenase E1	8.7e-23	89.1
		component
97	zf-C3HC4	Zinc finger, C3HC4 type	8.7e-09	32.7
		(RING finger)
99	ig	Immunoglobulin domain	1.8e-20	71.0
100	ig	Immunoglobulin domain	1.8e-20	71.0
104	zf-C2H2	Zinc finger, C2H2 type	2.4e-94	326.8
105	zf-C2H2	Zinc finger, C2H2 type	1.6e-55	197.9
109	zf-CCHC	Zinc finger, CCHC class	2.6e-12	54.4
110	zf-C2H2	Zinc finger, C2H2 type	2.2e-42	154.3
111	myosin_head	Myosin head (motor	0	1267.5
		domain)
112	pkinase	Eukaryotic protein kinase	1.2e-96	334.5
		domain
113	WD40	WD domain, G-beta repeat	5.9e-65	229.2
114	SNF2_N	SNF2 and others N-	4.2e-78	272.9
		terminal domain
115	DUF15	Domain of unknown	0.0011	−65.2
		function DUF15
116	DSPC	Dual specificity	0.00056	3.7
		phosphatase, catalytic
		domain
119	Rhodanese	Rhodanese-like domain	1e-05	32.5
124	proteasome	Proteasome A-type and B-	7.4e-43	155.8
		type
126	Ribosomal_L9	Ribosomal protein L9	3.1e-05	−3.4
127	RIO1	RIO1/ZK632.3/MJ0444	7.8e-80	278.6
		family
130	abhydrolase	alpha/beta hydrolase fold	9e-20	79.1
131	TPR	TPR Domain	5.7e-27	103.0
132	HMG14_17	HMG14 and HMG17	1.9e-15	64.7
133	bZIP	bZIP transcription factor	8.3e-19	71.7
134	rrm	RNA recognition motif.	1.9e-31	117.9
		(a.k.a. RRM, RBD, or RNP
		domain)
135	AMP-binding	AMP-binding enzyme	6.3e-98	338.7
140	tubulin	Tubulin/FtsZ family	2.1e-151	516.4
143	laminin_EGF	Laminin EGF-like (Domains	7.6e-12	52.8
		III and V)
144	FYVE	FYVE zinc finger	2.3e-29	109.1
145	zf-C2H2	Zinc finger, C2H2 type	2.4e-33	124.2
146	mito_carr	Mitochondrial carrier	3e-54	188.9
		proteins
148	DAGKc	Diacylglycerol kinase	0.00015	26.0
		catalytic domain
		(presumed)
149	Exonuclease	Exonuclease	2.1e-17	71.3
152	GTP_EFTU	Elongation factor Tu	8.5e-09	32.3
		family
153	WH2	Wiskott Aldrich syndrome	6.5e-20	79.6
		homology region 2
154	SAM	SAM domain (Sterile alpha	0.032	11.4
		motif)
156	DHDPS	Dihydrodipicolinate	1.3e-26	101.9
		synthetase family
158	PseudoU_synth_1	tRNA pseudouridine	1e-30	115.4
		synthase
159	pkinase	Eukaryotic protein kinase	2.3e-59	210.6
		domain
162	IF-28	Initiation factor 2	1.7e-98	340.7
		subunit family
163	Beach	Beige/BEACH domain	1.1e-224	759.8
166	DnaJ	DnaJ domain	8.1e-11	49.4
167	Anti_proliferat	BTG1 family	7.4e-85	295.3
168	sugar_tr	Sugar (and other)	3.1e-80	280.0
		transporter
169	sugar_tr	Sugar (and other)	2.9e-52	187.0
		transporter
170	zf-C2H2	Zinc finger, C2H2 type	2.2e-93	323.6
171	GBP	Guanylate-binding protein	4.3e-255	860.8
173	TPR	TPR Domain	1.1e-42	155.2
175	RhoGEF	RhoGEF domain	3.3e-40	147.0
176	zf-C3HC4	Zinc finger, C3HC4 type	0.00011	19.4
		(RING finger)
177	Peptidase_M22	Glycoprotease family	2.3e-73	257.2
179	TBC	TBC domain	4.7e-08	10.1
180	transmembrane4	Transmembrane 4 family	1.9e-49	161.1
181	CH	Calponin homology (CH)	1.2e-25	98.6
		domain
184	AP_endonucleas1	AP endonuclease family 1	0.088	−90.2
186	Bacterial_PQQ	PQQ enzyme repeat	9.3e-05	29.2
187	DEAD	DEAD/DEAH box helicase	1.6e-60	194.3
188	zf-C2H2	Zinc finger, C2H2 type	4.2e-24	93.5
189	sugar_tr	Sugar (and other)	0.0011	−88.8
		transporter
190	tRNA_int_endo	tRNA intron endonuclease	0.0012	−32.1
191	WSC	WSC domain	1e-35	132.1
193	pkinase	Eukaryotic protein kinase	5.1e-75	262.6
		domain
195	globin	Globin	1.9e-26	96.6
197	WD40	WD domain, G-beta repeat	1.8e-07	38.2
200	F_actin_cap_B	F-actin capping protein,	1.7e-224	759.2
		beta subunit
201	ank	Ank repeat	3.1e-59	210.2
205	PDZ	PDZ domain (Also known as	4.2e-07	37.0
		DHR or GLGF).
206	SAM	SAM domain (Sterile alpha	2.2e-05	31.3
		motif)
207	SAM	SAM domain (Sterile alpha	2.2e-05	31.3
		motif)
208	zf-UBR1	Putative zinc finger in	3.9e-26	100.3
		N-recognin
209	ABC_tran	ABC transporter	2.4e-112	386.6
211	zf-C2H2	Zinc finger, C2H2 type	0.00031	27.5
212	UCH-2	Ubiquitin carboxyl-	1.5e-19	78.4
		terminal hydrolase family
		2
213	IMP4	Domain of unknown	2.2e-33	124.3
		function
215	zf-C2H2	Zinc finger, C2H2 type	3.5e-08	40.6
216	PG_binding_2	Putative peptidoglycan	2.1e-11	51.3
		binding domain
219	pyr_redox	Pyridine nucleotide-	1.2e-70	241.7
		disulphide oxidoreductase
220	PDZ	PDZ domain (Also known as	8.5e-19	75.9
		DHR or GLGF).
221	pkinase	Eukaryotic protein kinase	8.1e-67	235.4
		domain
222	dsrm	Double-stranded RNA	0.095	7.5
		binding motif
223	PHD	PHD-finger	4.7e-05	29.9
225	TRM	N2,N2-dimethylguanosine	7.3e-22	86.1
		tRNA methyltransferase
226	LIM	LIM domain containing	8.6e-08	32.7
		proteins
227	ig	Immunoglobulin domain	1.1e-07	29.8
229	F-box	F-box domain.	2.2e-07	38.0
231	Glucosamine_iso	Glucosamine-6-phosphate	1.8e-146	500.0
		isomerase
233	PTN_MK	PTN/MK heparin-binding	1.9e-44	161.1
		protein family
238	CNG_membrane	Transmembrane region	5.8e-31	116.3
		cyclic Nucleotide Gated
		Channel
240	GNS1_SUR4	GNS1/SUR4 family	4.1e-46	166.6
243	PIP5K	Phosphatidylinositol-4-	6.2e-142	484.9
		phosphate 5-Kinase
244	cadherin	Cadherin domain	0	1298.9
246	fn3	Fibronectin type III	1.2e-31	118.6
		domain
247	UQ_con	Ubiquitin-conjugating	1.4e-16	68.5
		enzyme
248	LRR	Leucine Rich Repeat	3.4e-14	60.6
249	lipocalin	Lipocalin/cytosolic	1.2e-28	102.8
		fatty-acid binding
		protein family
250	Ribosomal_S2	Ribosomal protein S2	2.9e-11	43.7
251	tubulin	Tubulin/FtsZ family	8.5e-163	554.2
252	tubulin	Tubulin/FtsZ family	2.4e-212	718.8
253	ATP-synt_ab	ATP synthase alpha/beta	6.3e-108	372.0
		family
254	ATP-synt_A-c	ATP synthase Alpha chain,	4.3e-106	313.5
		C terminal
255	zf-C3HC4	Zinc finger, C3HC4 type	5e-12	43.2
		(RING finger)
256	G-patch	G-patch domain	1e-07	39.1
257	CH	Calponin homology (CH)	1.6e-11	51.7
		domain
258	RF-1	Peptidyl-tRNA hydrolase	5.9e-66	232.5
		domain
259	RF-1	Peptidyl-tRNA hydrolase	5.9e-66	232.5
		domain
261	thiored	Thioredoxin	2e-09	35.7
262	thyroglobulin_1	Thyroglobulin type-1	3.1e-34	127.2
		repeat
264	DnaJ	DnaJ domain	7.9e-13	56.0
267	DUF6	Integral membrane protein	0.083	9.1
		DUF6
268	Ribosomal_L31e	Ribosomal protein L31e	1.7e-61	217.7
270	Zn_carbOpept	Zinc carboxypeptidase	3.5e-50	180.1
272	BTB	BTB/POZ domain	7.7e-18	72.7
273	Glycos_transf_1	Glycosyl transferases	0.027	12.8
		group 1
277	SPRY	SPRY domain	2.5e-28	107.6
279	BTB	BTB/POZ domain	6e-27	103.0
280	zf-C2H2	Zinc finger, C2H2 type	3.7e-116	399.3
281	SCAN	SCAN domain	2.4e-52	187.3
284	NTP_transf_2	Nucleotidyltransferase	8.5e-13	55.9
		domain
288	zf-C2H2	Zinc finger, C2H2 type	5.4e-93	322.4
289	zf-C2H2	Zinc finger, C2H2 type	4.5e-124	425.6
291	DiHfolate_red	Dihydrofolate reductase	1.5e-63	221.6
293	PDZ	PDZ domain (Also known as	7.4e-17	69.4
		DHR or GLGF).
295	PH	PH domain	1.6e-08	35.4
296	adh_short	short chain dehydrogenase	4.5e-32	120.0
299	BNR	BNR repeat	3.1e-06	34.1
302	HMG_box	HMG (high mobility group)	5.4e-05	20.0
		box
303	ig	Immunoglobulin domain	0.05	11.6
304	zf-C3HC4	Zinc finger, C3HC4 type	5e-12	43.2
		(RING finger)
305	START	START domain	0.013	4.8
306	integrase	Integrase DNA binding	7.2e-06	32.9
		domain
307	myosin_head	Myosin head (motor	7.6e-279	939.7
		domain)
308	zf-C2H2	Zinc finger, C2H2 type	1.5e-53	191.4
309	ig	Immunoglobulin domain	0.00023	19.1
311	ras	Ras family	0.00079	−93.3
312	ig	Immunoglobulin domain	2.1e-06	25.7
313	EF1BD	EF-1 guanine nucleotide	4.3e-56	199.8
		exchange domain
314	Kelch	Kelch motif	3.6e-94	326.2
315	zf-C2H2	Zinc finger, C2H2 type	6.8e-59	209.1
317	CNG_membrane	Transmembrane region	1.5e-28	108.3
		cyclic Nucleotide Gated
		Channel
318	Peptidase_S26	Signal peptidase I	2.8e-16	56.3
319	zf-C2H2	Zinc finger, C2H2 type	4.6e-56	199.7
322	EGF	EGF-like domain	4.7e-08	40.2
323	lectin_c	Lectin C-type domain	6.6e-13	56.3
324	MCT	Monocarboxylate	1.9e-50	181.0
		transporter
327	MAM	MAM domain.	1.8e-51	184.4
329	MAM	MAM domain.	2e-179	609.5
330	Sema	Sema domain	9.9e-211	713.5
331	zf-C2H2	Zinc finger, C2H2 type	1.5e-84	294.3
333	PAP2	PAP2 superfamily	9.8e-10	45.8
334	LRR	Leucine Rich Repeat	2.8e-36	134.0
335	AdoHcyase	S-adenosyl-L-homocysteine	1.2e-238	806.2
		hydrolase
336	TBC	TBC domain	9.4e-38	138.9
343	WD40	WD domain, G-beta repeat	8.7e-08	39.3
346	globin	Globin	3e-45	162.2
347	globin	Globin	7.5e-39	139.9
349	F-box	F-box domain.	8.5e-07	36.0
350	HLH	Helix-loop-helix DNA-	2e-08	41.4
		binding domain
351	KRAB	KRAB box	2.7e-39	144.0
352	UCH-2	Ubiquitin carboxyl-	1.7e-19	78.2
		terminal hydrolase family
		2
353	IPP_isomerase	Isopentenyl-diphosphate	9.5e-94	324.9
		delta-isomerase
354	IBR	IBR domain	1.6e-12	55.0
355	IBR	IBR domain	1.6e-12	55.0
356	SCP	SCP-like extracellular	1.4e-34	128.3
		protein
358	mito_carr	Mitochondrial carrier	3.9e-74	257.0
		proteins
359	Ribosomal_S8	Ribosomal protein S8	6e-58	192.1
361	UCH-2	Ubiquitin carboxyl-	6.7e-23	89.5
		terminal hydrolase family
		2
363	Phage_lysozyme	Lysozyme	9.3e-05	23.5
365	Ribosomal_S2	Ribosomal protein S2	3.3e-08	32.9
367	zf-C3HC4	Zinc finger, C3HC4 type	5.3e-09	33.4
		(RING finger)
368	zf-C3HC4	Zinc finger, C3HC4 type	0.0096	13.1
		(RING finger)
370	TPR	TPR Domain	0.041	20.5
373	zf-C2H2	Zinc finger, C2H2 type	5.6e-109	375.5
374	arf	ADP-ribosylation factor	4.9e-39	143.1
		family
375	BNR	BNR repeat	0.033	20.8
376	zf-C2H2	Zinc finger, C2H2 type	1.5e-24	95.0
379	rrm	RNA recognition motif.	0.00019	28.2
		(a.k.a. RRM, RBD, or RNP
		domain)
380	rrm	RNA recognition motif.	2.2e-19	77.9
		(a.k.a. RRM, RBD, or RNP
		domain)
383	vwc	von Willebrand factor	1.6e-31	118.2
		type C domain
384	Ribosomal_L35Ae	Ribosomal protein L35Ae	0.00013	7.0
388	ras	Ras family	3.9e-63	223.2
391	F-box	F-box domain.	2.3e-05	31.3
392	bZIP	bZIP transcription factor	6.3e-09	36.9
393	SPRY	SPRY domain	0.092	−9.4
395	zf-C2H2	Zinc finger, C2H2 type	3.1e-17	70.7
396	SCAN	SCAN domain	3.1e-39	143.8
397	Kelch	Kelch motif	1.1e-55	198.4
398	C2	C2 domain	2.2e-80	280.4
399	ank	Ank repeat	1.8e-29	111.3
400	ank	Ank repeat	2.4e-19	77.7
403	DAGKa	Diacylglycerol kinase	1.9e-124	426.8
		accessory domain
		(presumed)
404	MCT	Monocarboxylate	0.012	−34.1
		transporter
406	PDZ	PDZ domain (Also known as	7.7e-46	165.7
		DHR or GLGF).
407	zf-C2H2	Zinc finger, C2H2 type	3.9e-48	173.3
408	K_tetra	K+ channel	2.6e-23	90.9
		tetramerisation domain
409	SNF	Sodium:neurotransmitter	0	1268.7
		symporter family
410	ig	Immunoglobulin domain	1.1e-06	26.5
411	DnaJ	DnaJ domain	9.9e-28	105.6
412	mito_carr	Mitochondrial carrier	7.6e-66	228.6
		proteins
413	zf-C2H2	Zinc finger, C2H2 type	6e-97	335.5
414	S_100	S-100/ICaBP type calcium	9.7e-13	55.8
		binding domain
416	fn3	Fibronectin type III	8.6e-14	59.3
		domain
417	zf-C2H2	Zinc finger, C2H2 type	3.8e-27	103.6
418	zf-C3HC4	Zinc finger, C3HC4 type	4.4e-14	49.9
		(RING finger)
419	pkinase	Eukaryotic protein kinase	1.2e-54	195.0
		domain
420	trypsin	Trypsin	4.6e-38	122.5
421	Glypican	Glypican	5.7e-131	448.5
422	Keratin_B2	Keratin, high sulfur B2	0.0013	−23.4
		protein
424	zf-C2H2	Zinc finger, C2H2 type	0.00021	28.1
425	ig	Immunoglobulin domain	0.00074	17.5
426	ig	Immunoglobulin domain	2.9e-23	80.0
427	Keratin_B2	Keratin, high sulfur B2	0.0023	−27.1
		protein
428	pkinase	Eukaryotic protein kinase	2.3e-55	197.3
		domain
429	ig	Immunoglobulin domain	4.1e-09	34.4
430	Galactosyl_T	Galactosyltransferase	1.8e-36	134.6
431	proteasome	Proteasome A-type and B-	5.5e-28	106.4
		type
432	7tm_1	7 transmembrane receptor	3.4e-38	123.5
		(rhodopsin family)
433	BTB	BTB/POZ domain	8.1e-23	89.2
436	p450	Cytochrome P450	6.9e-175	594.4
437	sugar_tr	Sugar (and other)	5.9e-65	229.2
		transporter
438	zf-C2H2	Zinc finger, C2H2 type	2.1e-52	187.5
439	7tm_1	7 transmembrane receptor	2.2e-40	130.4
		(rhodopsin family)
440	FGF	Fibroblast growth factor	4.6e-14	51.6
441	Osteopontin	Osteopontin	3.7e-181	615.2

TABLE 5
	POSITION OF SIGNAL	maxS	meanS
	IN AMINO ACID	(MAXIMUM	(MEAN
SEQ ID NO:	SEQUENCE	SCORE)	SCORE)
2	1-32	0.995	0.681
4	1-37	0.979	0.718
22	1-31	0.947	0.775
25	1-30	0.924	0.720
27	1-28	0.982	0.649
39	1-27	0.983	0.898
40	1-17	0.991	0.955
46	1-22	0.990	0.921
56	1-18	0.925	0.822
58	1-18	0.981	0.951
62	1-28	0.939	0.749
64	1-33	0.979	0.757
72	1-41	0.989	0.690
81	1-26	0.960	0.674
85	1-18	0.979	0.963
86	1-22	0.967	0.792
89	1-25	0.980	0.867
99	1-16	0.973	0.925
100	1-24	0.978	0.760
101	1-17	0.978	0.925
115	1-18	0.887	0.579
117	1-18	0.952	0.670
122	1-42	0.977	0.587
139	1-21	0.966	0.848
142	1-25	0.993	0.954
155	1-28	0.909	0.664
158	1-18	0.954	0.747
176	1-23	0.913	0.597
177	1-20	0.986	0.936
180	1-42	0.978	0.689
182	1-32	0.929	0.583
186	1-21	0.979	0.941
194	1-21	0.930	0.662
202	1-45	0.985	0.714
214	1-37	0.992	0.855
227	1-24	0.971	0.882
230	1-20	0.979	0.911
239	1-17	0.982	0.964
253	1-13	0.918	0.692
254	1-13	0.918	0.692
258	1-20	0.912	0.693
259	1-20	0.912	0.693
262	1-26	0.974	0.824
264	1-18	0.965	0.833
269	1-25	0.956	0.765
290	1-16	0.912	0.705
291	1-18	0.896	0.634
292	1-19	0.966	0.897
296	1-18	0.991	0.973
297	1-20	0.906	0.580
301	1-27	0.957	0.652
309	1-19	0.983	0.871
312	1-22	0.968	0.844
322	1-23	0.952	0.812
326	1-27	0.982	0.911
329	1-18	0.983	0.941
330	1-18	0.932	0.884
334	1-27	0.990	0.923
337	1-45	0.983	0.793
338	1-45	0.983	0.793
348	1-29	0.991	0.729
356	1-22	0.978	0.877
366	1-26	0.939	0.709
367	1-22	0.966	0.843
378	1-29	0.961	0.842
382	1-16	0.951	0.777
404	1-44	0.975	0.876
410	1-33	0.977	0.822
420	1-17	0.989	0.969
421	1-23	0.974	0.799
425	1-18	0.981	0.952
429	1-21	0.982	0.912
431	1-30	0.936	0.679
432	1-43	0.978	0.712
436	1-28	0.993	0.948
437	1-43	0.930	0.624
440	1-24	0.993	0.810
441	1-16	0.978	0.939

Claims

1. An isolated polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO:231.

2. A composition comprising the polypeptide of claim 1 and a carrier.

3. An isolated polypeptide having glucosamine-6-phosphate isomerase activity that is encoded by the polynucleotide comprising a nucleic acid sequence which is 99% identical to the nucleic acid sequence of SEQ ID NO: 231.