EP1190058A2 - Human genes and gene expression products i - Google Patents

Abstract

This invention relates to novel human polynucleotides and variants thereof, their encoded polypeptides and variants thereof, to genes corresponding to these polynucleotides and to proteins expressed by the genes. The invention also relates to diagnostic and therapeutic agents employing such novel human polynucleotides, their corresponding genes or gene products, e.g., these genes and proteins, including probes, antisense constructs, and antibodies.

Description

NOVEL HUMAN GENES AND GENE EXPRESSION PRODUCTS I

Cross-References to Related Applications

This application is a continuation-in-part of U.S. provisional patent application serial no. 60/068,755, filed December 23, 1997, and of U.S. provisional patent application serial no. 60/080,664, filed April 3, 1998, and of U.S. provisional patent application serial no. 60/105,234, filed October 21, 1998, each of which applications are incorporated herein by reference.

Field of the Invention

The present invention relates to novel polynucleotides, particularly to novel polynucleotides of human origin that are expressed in a selected cell type, are differentially expressed in one cell type relative to another cell type (e.g., in cancerous cells, or in cells of a specific tissue origin) and/or share homology to polynucleotides encoding a gene product having an identified functional domain and/or activity.

Background of the Invention

Identification of novel polynucleotides, particularly those that encode an expressed gene product, is important in the advancement of drug discovery, diagnostic technologies, and the understanding of the progression and nature of complex diseases such as cancer. Identification of genes expressed in different cell types isolated from sources that differ in disease state or stage, developmental stage, exposure to various environmental factors, the tissue of origin, the species from which the tissue was isolated, and the like is key to identifying the genetic factors that are responsible for the phenotypes associated with these various differences

This invention provides novel human polynucleotides, the polypeptides encoded by these polynucleotides, and the genes and proteins corresponding to these novel polynucleotides.

Summary of the Invention

This invention relates to novel human polynucleotides and variants thereof, their encoded polypeptides and variants thereof, to genes corresponding to these polynucleotides and to proteins expressed by the genes. The invention also relates to diagnostic and therapeutic agents employing such novel human polynucleotides, their corresponding genes or gene products, e.g., these genes and proteins, including probes, antisense constructs, and antibodies. Accordingly, in one embodiment, the present invention features a library of polynucleotides, the library comprising the sequence information of at least one of SEQ ID NOS: 1-844. In related aspects, the invention features a library provided on a nucleic acid array, or in a computer-readable format.

In one embodiment, the library is comprises a differentially expressed polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOS:9, 39, 42, 52, 62. 74, 119, 172, 317, and 379. In specific related embodiments, the library comprises: 1) a polynucleotide that is differentially expressed in a human breast cancer cell, where the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS: 4, 9, 39, 42, 52, 62, 65, 66, 68, 74, 81, 114, 123, 144, 130, 157, 162, 172, 178, 183, 202, 214, 219, 223, 258, 298, 317, 338, 379, 384, 386, and 388; 2) a polynucleotide differentially expressed in a human colon cancer cell, where the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS: 1, 39, 52, 97, 119, 134, 172, 176, 241, 288, 317, 357, 362, and 374; or 3) a polynucleotide differentially expressed in a human lung cancer cell, where the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS: 9, 34, 42, 62, 74, 106, 1 19, 135, 154, 160, 260, 308, 323, 349, 361, 369, 371, 379, 395, 381, and 400.

In another aspect, the invention features an isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to an identifying sequence of SEQ ID NOS: 1 -844 or a degenerate variant thereof. In related aspects, the invention features recombinant host cells and vectors comprising the polynucleotides of the invention, as well as isolated polypeptides encoded by the polynucleotides of the invention and antibodies that specifically bind such polypeptides.

In one embodiment, the invention features an isolated polynucleotide comprising a sequence encoding a polypeptide of a protein family selected from the group consisting of: 4 transmembrane segments integral membrane proteins, 7 transmembrane receptors,

ATPases associated with various cellular activities (AAA), eukaryotic aspartyl proteases, GATA family of transcription factors, G-protein alpha subunit, phorbol esters/diacylglycerol binding proteins, protein kinase, protein phosphatase 2C, protein tyrosine phosphatase, trypsin, wnt family of developmental signaling proteins, and WW/rsp5/WWP domain containing proteins. In a specific related embodiment, the invention features a polynucleotide comprising a sequence of one of SEQ ID NOS: 24, 41, 101, 157, 291, 305, 315, 341, 63, 116, 134, 136, 151, 384, 404, 308, 213, 367, 188, 251, 202, 315, 367, 397, 256, 382, 169, 23, 291, 324, 330, 341, 353, 188, 379 , and 395.

In another embodiment, the invention features a polynucleotide comprising a sequence encoding a polypeptide having a functional domain selected from the group consisting of: Ank repeat, basic region plus leucine zipper transcription factors, bromodomain, EF-hand, SH3 domain, WD domain/G-beta repeats, zinc finger (C2H2 type), zinc finger (CCHC class), and zinc-binding metalloprotease domain. In a specific related embodiment, the invention features a polynucleotide comprising a sequence of one of SEQ ID NOS: 116, 251, 374, 97, 136, 242, 379, 306, 386, 18, 335, 61, 306, 386, 322, 306, and 395.

In another aspect, the invention features a method of detecting differentially expressed genes correlated with a cancerous state of a mammalian cell, where the method comprises the step of detecting at least one differentially expressed gene product in a test sample derived from a cell suspected of being cancerous, where the gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS:4, 9, 39, 42, 52, 62, 65, 66, 68, 74, 81, 114, 123, 144, 130, 157, 162, 172, 178, 183, 202, 214, 219, 223, 258, 298, 317, 338, 379, 384, 386, 388, 1, 39, 52, 97, 119, 134, 172, 176, 241, 288, 317, 357, 362, 374, 9, 34, 42, 62, 74, 106, 119, 135, 154, 160, 260, 308, 323, 349, 361, 369, 371, 379, 395, 381, and 400. Detection of the differentially expressed gene product is correlated with a cancerous state of the cell from which the test sample was derived. In one embodiment, the detecting is by hybridization of the test sample to a reference array, wherein the reference array comprises an identifying sequence of at least one of SEQ ID NOS: 1-844.

In one embodiment of the method of the invention, the cell is a breast tissue derived cell, and the differentially expressed gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS: 4, 9, 39, 42, 52, 62, 65, 66, 68, 74, 81, 114, 123, 144, 130, 157, 162, 172, 178, 183, 202, 214, 219, 223, 258, 298, 317, 338, 379, 384, 386, and 388.

In another embodiment of the method of the invention, the cell is a colon tissue derived cell, and differentially expressed gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS: 1, 39, 52, 97, 1 19, 134, 172, 176, 241, 288, 317, 357, 362, and 374.

In yet another embodiment of the method of the invention, the cell is a lung tissue derived cell, and differentially expressed gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS: 9, 34, 42, 62, 74, 106, 119, 135, 154, 160, 260, 308, 323, 349, 361, 369, 371, 379, 395, 381, and 400.

Other aspects and embodiments of the invention will be readily apparent to the ordinarily skilled artisan upon reading the description provided herein.

Detailed Description of the Invention The invention relates to polynucleotides comprising the disclosed nucleotide sequences, to full length cDNA, mRNA and genes corresponding to these sequences, and to polypeptides and proteins encoded by these polynucleotides and genes.

Also included are polynucleotides that encode polypeptides and proteins encoded by the polynucleotides of the Sequence Listing. The various polynucleotides that can encode these polypeptides and proteins differ because of the degeneracy of the genetic code, in that most amino acids are encoded by more than one triplet codon. The identity of such codons is well-known in this art, and this information can be used for the construction of the polynucleotides within the scope of the invention.

Polynucleotides encoding polypeptides and proteins that are variants of the polypeptides and proteins encoded by the polynucleotides and related cDNA and genes are also within the scope of the invention. The variants differ from wild type protein in having one or more amino acid substitutions that either enhance, add, or diminish a biological activity of the wild type protein. Once the amino acid change is selected, a polynucleotide encoding that variant is constructed according to the invention. The following detailed description describes the polynucleotide compositions encompassed by the invention, methods for obtaining cDNA or genomic DNA encoding a full-length gene product, expression of these polynucleotides and genes, identification of structural motifs of the polynucleotides and genes, identification of the function of a gene product encoded by a gene corresponding to a polynucleotide of the invention, use of the provided polynucleotides as probes and in mapping and in tissue profiling, use of the corresponding polypeptides and other gene products to raise antibodies, and use of the polynucleotides and their encoded gene products for therapeutic and diagnostic purposes.

I. Polynucleotide Compositions

The scope of the invention with respect to polynucleotide compositions includes, but is not necessarily limited to, polynucleotides having a sequence set forth in any one of SEQ ID NOS: 1 -844; polynucleotides obtained from the biological materials described herein or other biological sources (particularly human sources) by hybridization under stringent conditions (particularly conditions of high stringency); genes corresponding to the provided polynucleotides; variants of the provided polynucleotides and their corresponding genes, particularly those variants that retain a biological activity of the encoded gene product (e.g., a biological activity ascribed to a gene product corresponding to the provided polynucleotides as a result of the assignment of the gene product to a protein family(ies) and/or identification of a functional domain present in the gene product). Other nucleic acid compositions contemplated by and within the scope of the present invention will be readily apparent to one of ordinary skill in the art when provided with the disclosure here. The invention features polynucleotides that are expressed in cells of human tissue, specifically human colon, breast, and/or lung tissue. Novel nucleic acid compositions of the invention of particular interest comprise a sequence set forth in any one of SEQ ID NOS:l- 844 or an identifying sequence thereof. An "identifying sequence" is a contiguous sequence of residues at least about 10 nt to about 20 nt in length, usually at least about 50 nt to about 100 nt in length, that uniquely identifies a polynucleotide sequence, e.g. , exhibits less than 90%, usually less than about 80% to about 85% sequence identity to any contiguous nucleotide sequence of more than about 20 nt. Thus, the subject novel nucleic acid compositions include full length cDNAs or mRNAs that encompass an identifying sequence of contiguous nucleotides from any one of SEQ ID NOS: 1-844. The polynucleotides of the invention also include polynucleotides having sequence similarity or sequence identity. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in lXSSC. Sequence identity can be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM saline/0.9 mM sodium citrate). Hybridization methods and conditions are well known in the art, see, e.g., U.S. Patent No. 5,707,829. Nucleic acids that are substantially identical to the provided polynucleotide sequences, e.g. allelic variants, genetically altered versions of the gene, etc., bind to the provided polynucleotide sequences (SEQ ID NOS: 1-844) under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes can be any species, e.g. primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, nematodes, etc.

Preferably, hybridization is performed using at least 15 contiguous nucleotides of at least one of SEQ ID NOS: 1-844. That is, when at least 15 contiguous nucleotides of one of the disclosed SEQ ID NOs. is used as a probe, the probe will preferentially hybridize with a gene or mRNA (of the biological material) comprising the complementary sequence, allowing the identification and retrieval of the nucleic acids of the biological material that uniquely hybridize to the selected probe. Probes from more than one SEQ ID NO. will hybridize with the same gene or mRNA if the cDNA from which they were derived corresponds to one mRNA. Probes of more than 15 nucleotides can be used, but 15 nucleotides represents enough sequence for unique identification.

The polynucleotides of the invention also include naturally occurring variants of the nucleotide sequences (e.g., degenerate variants, allelic variants, etc.). Variants of the polynucleotides of the invention are identified by hybridization of putative variants with nucleotide sequences disclosed herein, preferably by hybridization under stringent conditions For example, by using appropriate wash conditions, variants of the polynucleotides of the invention can be identified where the allelic variant exhibits at most about 25-30% base pair mismatches relative to the selected polynucleotide probe. In general, allelic variants contain 15-25% base pair mismatches, and can contain as little as even 5-15%, or 2-5%, or 1-2% base pair mismatches, as well as a single base-pair mismatch. The invention also encompasses homologs corresponding to the polynucleotides of

SEQ ID NOS: 1-844, where the source of homologous genes can be any mammalian species, e.g., primate species, particularly human; rodents, such as rats, canines, felines, bovines, ovines, equines, yeast, nematodes, etc. Between mammalian species, e.g., human and mouse, homologs have substantial sequence similarity, e.g., at least 75% sequence identity, usually at least 90%, more usually at least 95% between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 contiguous nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., J. Mol. Biol. (1990) 275:403-10.

In general, variants of the invention have a sequence identity greater than at least about 65%, preferably at least about 75%, more preferably at least about 85%, and can be greater than at least about 90% or more as determined by the Smith- Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular). For the purposes of this invention, a preferred method of calculating percent identity is the Smith- Waterman algorithm, using the following. Global DNA sequence identity must be greater than 65% as determined by the Smith- Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty, 12; and gap extension penalty, 1.

The subject nucleic acids can be cDNAs or genomic DNAs, as well as fragments thereof, particularly fragments that encode a biologically active gene product and/or are useful in the methods disclosed herein (e.g., in diagnosis, as a unique identifier of a differentially expressed gene of interest, etc.). The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3 and 5 non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, being removed by nuclear RNA splicing, to create a continuous open reading frame encoding a polypeptide of the invention. A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It can further include the 3 and 5 untranslated regions found in the mature mRNA. It can further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5 and 3 end of the transcribed region. The genomic DNA can be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3 and 5 , or internal regulatory sequences as sometimes found in introns, contains sequences required for proper tissue, stage-specific, or disease- state specific expression.

The nucleic acid compositions of the subject invention can encode all or a part of the subject differentially expressed polypeptides. Double or single stranded fragments can be obtained from the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. Isolated polynucleotides and polynucleotide fragments of the invention comprise at least about 10, about 15, about 20, about 35, about 50, about 100, about 150 to about 200, about 250 to about 300, or about 350 contiguous nucleotides selected from the polynucleotide sequences as shown in SEQ ID NOS: 1-844. For the most part, fragments will be of at least 15 nt, usually at least 18 nt or 25 nt, and up to at least about 50 contiguous nt in length or more. In a preferred embodiment, the polynucleotide molecules comprise a contiguous sequence of at least twelve nucleotides selected from the group consisting of the polynucleotides shown in SEQ ID NOS: 1-844.

Probes specific to the polynucleotides of the invention can be generated using the polynucleotide sequences disclosed in SEQ ID NOS: 1-844. The probes are preferably at least about 12, 15, 16, 18, 20, 22, 24, or 25 nucleotide fragment of a corresponding contiguous sequence of SEQ ID NOS: 1-844, and can be less than 2, 1, 0.5, 0.1, or 0.05 kb in length. The probes can be synthesized chemically or can be generated from longer polynucleotides using restriction enzymes. The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag. Preferably, probes are designed based upon an identifying sequence of a polynucleotide of one of SEQ ID NOS: 1 -844. More preferably, probes are designed based on a contiguous sequence of one of the subject polynucleotides that remain unmasked following application of a masking program for masking low complexity (e.g., XBLAST) to the sequence., i.e., one would select an unmasked region, as indicated by the polynucleotides outside the poly-n stretches of the masked sequence produced by the masking program.

The polynucleotides of the subject invention are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the polynucleotides, either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", e.g. , flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

The polynucleotides of the invention can be provided as a linear molecule or within a circular molecule. They can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. They can be regulated by their own or by other regulatory sequences, as is known in the art. The polynucleotides of the invention can be introduced into suitable host cells using a variety of techniques which are available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.

The subject nucleic acid compositions can be used to, for example, produce polypeptides, as probes for the detection of mRNA of the invention in biological samples (e.g., extracts of human cells) to generate additional copies of the polynucleotides, to generate ribozymes or antisense oligonucleotides, and as single stranded DNA probes or as triple-strand forming oligonucleotides. The probes described herein can be used to, for example, determine the presence or absence of the polynucleotide sequences as shown in SEQ ID NOS: 1-844 or variants thereof in a sample. These and other uses are described in more detail below. Use of Polynucleotides to Obtain Full-Length cDNA and Full-Length Human Gene and

Promoter Region

Full-length cDNA molecules comprising the disclosed polynucleotides are obtained as follows. A polynucleotide having a sequence of one of SEQ ID NOS: 1-844, or a portion thereof comprising at least 12, 15, 18, or 20 nucleotides, is used as a hybridization probe to detect hybridizing members of a cDNA library using probe design methods, cloning methods, and clone selection techniques such as those described in U.S. Patent No. 5,654,173. Libraries of cDNA are made from selected tissues, such as normal or tumor tissue, or from tissues of a mammal treated with, for example, a pharmaceutical agent. Preferably, the tissue is the same as the tissue from which the polynucleotides of the invention were isolated, as both the polynucleotides described herein and the cDNA represent expressed genes. Most preferably, the cDNA library is made from the biological material described herein in the Examples. Alternatively, many cDNA libraries are available commercially. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY). The choice of cell type for library construction can be made after the identity of the protein encoded by the gene corresponding to the polynucleotide of the invention is known. This will indicate which tissue and cell types are likely to express the related gene, and thus represent a suitable source for the mRNA for generating the cDNA. Where the provided polynucleotides are isolated from cDNA libraries, the libraries are prepared from mRNA of human colon cells, more preferably, human colon cancer cells, even more preferably, from a highly metastatic colon cell, Kml2L4-A.

Techniques for producing and probing nucleic acid sequence libraries are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY. The cDNA can be prepared by using primers based on sequence from SEQ ID NOS: 1-844. In one embodiment, the cDNA library can be made from only poly-adenylated mRNA. Thus, poly-T primers can be used to prepare cDNA from the mRNA.

Members of the library that are larger than the provided polynucleotides, and preferably that encompass the complete coding sequence of the native message, are obtained. In order to confirm that the entire cDNA has been obtained, RNA protection experiments are performed as follows. Hybridization of a full-length cDNA to an mRNA will protect the

RNA from RNase degradation. If the cDNA is not full length, then the portions of the mRNA that are not hybridized will be subject to RNase degradation. This is assayed, as is known in the art, by changes in electrophoretic mobility on polyacrylamide gels, or by detection of released monoribonucleotides. Sambrook et al., Molecular Cloning: A

Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY. In order to obtain additional sequences 5' to the end of a partial cDNA, 5' RACE (PCR Protocols: A Guide to Methods and Applications, (1990) Academic Press, Inc.) is performed. Genomic DNA is isolated using the provided polynucleotides in a manner similar to the isolation of full-length cDNAs. Briefly, the provided polynucleotides, or portions thereof, are used as probes to libraries of genomic DNA. Preferably, the library is obtained from the cell type that was used to generate the polynucleotides of the invention, but this is not essential. Most preferably, the genomic DNA is obtained from the biological material described herein in the Examples. Such libraries can be in vectors suitable for carrying large segments of a genome, such as PI or YAC, as described in detail in Sambrook et al., 9.4- 9.30. In addition, genomic sequences can be isolated from human BAC libraries, which are commercially available from Research Genetics, Inc., Huntville, Alabama, USA, for example. In order to obtain additional 5' or 3' sequences, chromosome walking is performed, as described in Sambrook et al., such that adjacent and overlapping fragments of genomic DNA are isolated. These are mapped and pieced together, as is known in the art, using restriction digestion enzymes and DNA ligase.

Using the polynucleotide sequences of the invention, corresponding full-length genes can be isolated using both classical and PCR methods to construct and probe cDNA libraries. Using either method, Northern blots, preferably, are performed on a number of cell types to determine which cell lines express the gene of interest at the highest level. Classical methods of constructing cDNA libraries are taught in Sambrook et al., supra. With these methods, cDNA can be produced from mRNA and inserted into viral or expression vectors. Typically, libraries of mRNA comprising poly(A) tails can be produced with poly(T) primers. Similarly, cDNA libraries can be produced using the instant sequences as primers. PCR methods are used to amplify the members of a cDNA library that comprise the desired insert. In this case, the desired insert will contain sequence from the full length cDNA that corresponds to the instant polynucleotides. Such PCR methods include gene trapping and RACE methods. Gene trapping entails inserting a member of a cDNA library into a vector. The vector then is denatured to produce single stranded molecules. Next, a substrate-bound probe, such a biotinylated oligo, is used to trap cDNA inserts of interest. Biotinylated probes can be linked to an avidin-bound solid substrate. PCR methods can be used to amplify the trapped cDNA. To trap sequences corresponding to the full length genes, the labeled probe sequence is based on the polynucleotide sequences of the invention.

Random primers or primers specific to the library vector can be used to amplify the trapped cDNA. Such gene trapping techniques are described in Gruber et al., WO 95/04745 and Gruber et al., U.S. Pat. No. 5,500,356. Kits are commercially available to perform gene trapping experiments from, for example, Life Technologies, Gaithersburg, Maryland, USA.

"Rapid amplification of cDN A ends," or RACE, is a PCR method of amplifying cDNAs from a number of different RNAs. The cDNAs are ligated to an oligonucleotide linker, and amplified by PCR using two primers. One primer is based on sequence from the instant polynucleotides, for which full length sequence is desired, and a second primer comprises sequence that hybridizes to the oligonucleotide linker to amplify the cDNA. A description of this methods is reported in WO 97/19110. In preferred embodiments of RACE, a common primer is designed to anneal to an arbitrary adaptor sequence ligated to cDNA ends (Apte and Siebert, Biotechniques (1993) 75:890-893; Edwards et al., Nuc. Acids Res. (1991) 79:5227-5232). When a single gene-specific RACE primer is paired with the common primer, preferential amplification of sequences between the single gene specific primer and the common primer occurs. Commercial cDNA pools modified for use in RACE are available.

Another PCR-based method generates full-length cDNA library with anchored ends without needing specific knowledge of the cDNA sequence. The method uses lock-docking primers (I-VI), where one primer, poly TV (I-III) locks over the polyA tail of eukaryotic mRNA producing first strand synthesis and a second primer, polyGH (IV -VI) locks onto the polyC tail added by terminal deoxynucleotidyl transferase (TdT). This method is described in WO 96/40998. The promoter region of a gene generally is located 5' to the initiation site for RNA polymerase II. Hundreds of promoter regions contain the "TATA" box, a sequence such as TATTA or TATAA, which is sensitive to mutations. The promoter region can be obtained by performing 5' RACE using a primer from the coding region of the gene. Alternatively, the cDNA can be used as a probe for the genomic sequence, and the region 5' to the coding region is identified by "walking up." If the gene is highly expressed or differentially expressed, the promoter from the gene can be of use in a regulatory construct for a heterologous gene.

Once the full-length cDNA or gene is obtained, DNA encoding variants can be prepared by site-directed mutagenesis, described in detail in Sambrook et al., 15.3-15.63. The choice of codon or nucleotide to be replaced can be based on disclosure herein on optional changes in amino acids to achieve altered protein structure and/or function.

As an alternative method to obtaining DNA or RNA from a biological material, nucleic acid comprising nucleotides having the sequence of one or more polynucleotides of the invention can be synthesized. Thus, the invention encompasses nucleic acid molecules ranging in length from 15 nucleotides (corresponding to at least 15 contiguous nucleotides of one of SEQ ID NOS: 1 -844) up to a maximum length suitable for one or more biological manipulations, including replication and expression, of the nucleic acid molecule. The invention includes but is not limited to (a) nucleic acid having the size of a full gene, and comprising at least one of SEQ ID NOS: 1-844; (b) the nucleic acid of (a) also comprising at least one additional gene, operably linked to permit expression of a fusion protein; (c) an expression vector comprising (a) or (b); (d) a plasmid comprising (a) or (b) ; and (e) a recombinant viral particle comprising (a) or (b). Once provided with the polynucleotides disclosed herein, construction or preparation of (a) - (e) are well within the skill in the art. The sequence of a nucleic acid comprising at least 15 contiguous nucleotides of at least any one of SEQ ID NOS: 1-844, preferably the entire sequence of at least any one of SEQ ID NOS : 1 -844, is not limited and can be any sequence of A, T, G, and/or C (for DNA) and A, U, G, and/or C (for RNA) or modified bases thereof, including inosine and pseudouridine. The choice of sequence will depend on the desired function and can be dictated by coding regions desired, the intron-like regions desired, and the regulatory regions desired. Where the entire sequence of any one of SEQ ID NOS: 1-844 is within the nucleic acid, the nucleic acid obtained is referred to herein as a polynucleotide comprising the sequence of any one of SEQ ID NOS: 1-844. II. Expression of Polypeptide Encoded by Full-Length cDNA or Full-Length Gene

The provided polynucleotide (e.g., a polynucleotide having a sequence of one of SEQ ID NOS: 1-844), the corresponding cDNA, or the full-length gene is used to express a partial or complete gene product.

Constructs of polynucleotides having sequences of SEQ ID NOS: 1-844 can be generated synthetically. Alternatively, single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides is described by, e.g., Stemmer et al., Gene (Amsterdam) (1995) 164(l):49-53. In this method, assembly PCR (the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos)) is described. The method is derived from DNA shuffling (Stemmer, Nature (1994) 570:389-391), and does not rely on DΝA ligase, but instead relies on DΝA polymerase to build increasingly longer DΝA fragments during the assembly process. For example, a 1.1 -kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) can be assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene can be PCR amplified and cloned in a vector containing the tetracycline-resistance gene (Tc-R) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the Tc-R colonies were Ap-R, making this approach a general method for the rapid and cost-effective synthesis of any gene. Appropriate polynucleotide constructs are purified using standard recombinant DΝA techniques as described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, ΝY, and under current regulations described in United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research. The gene product encoded by a polynucleotide of the invention is expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Suitable vectors and host cells are described in U.S. Patent No. 5,654,173.

Bacteria. Expression systems in bacteria include those described in Chang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979) 257:544; Goeddel et al., Nucleic Acids Res. (1980) 5:4057; EP 0 036,776; U.S. Patent No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA) (1983) 50:21-25; and Siebenlist et al., Cell (1980) 20:269. Yeast. Expression systems in yeast include those described in Hinnen et al, Proc.

Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al, J. Bacteriol. (1983) 755:163; Kurtz et al, Mol. Cell. Biol. (1986) 6:142; Kunze et al, J. Basic Microbiol. (1985) 25:141 ; Gleeson et al, J. Gen. Microbiol (1986) 752:3459; Roggenkamp et al, Mol. Gen. Genet. (1986) 202:302; Das et al, J. Bacteriol (1984) 755: 1165; De Louvencourt et al, J. Bacteriol (1983) 154:737; Van den Berg et al, Bio/Technology (1990) 5:135; Kunze et al, J. Basic Microbiol (1985) 25:141; Cregg et al, Mol. Cell. Biol. (1985) 5:3376; U.S. Patent Nos. 4,837,148 and 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al, Curr. Genet. (1985) 70:380; Gaillardin et al, Curr. Genet. (1985) 70:49; Ballance et al, Biochem. Biophys. Res. Commun. (1983) 772:284-289; Tilburn et al, Gene (1983) 26:205-221; Yelton et al, Proc. Natl. Acad. Sci. (USA) (1984) 57:1470-1474; Kelly and Hynes, EMBO J. (1985) 4.475479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects is accomplished as described in U.S. Patent No. 4,745,051; Friesen et al, "The Regulation of Baculovirus Gene Expression", in: The Molecular Biology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0 155,476; and Vlak et al, J. Gen. Virol. (1988) 69:165-776; Miller et al, Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al, Gene (1988) 75:409; Maeda et al, Nature (1985) 575:592-594; Lebacq-Verheyden et al, Mol. Cell. Biol. (1988) 5:3129; Smith et al, Proc. Natl. Acad. Sci. (USA) (1985) 52:8844; Miyajima et al, Gene (1987) 55:273; and Martin et al, DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al, Bio/Technology (1988) 6:47-55, Miller et al, Generic Engineering (1986) 5:277-279, and Maeda et al, Nature (1985) 575:592-594.

Mammalian Cells. Mammalian expression is accomplished as described in Dijkema et al, EMBO J. (1985) 4:761, Gorman et al, Proc. Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al, Cell (1985) 41:52 and U.S. Patent No. 4,399,216. Other features of mammalian expression are facilitated as described in Ham and Wallace, Meth. Enz. (1979) 55:44, Barnes and Sato, Λwα/. Biochem. (1980) 102:255, U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985. Polynucleotide molecules comprising a polynucleotide sequence provided herein propagated by placing the molecule in a vector. Viral and non-viral vectors are used, including plasmids. The choice of plasmid will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially. The partial or full-length polynucleotide is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo. Typically this is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.

The polynucleotides set forth in SEQ ID NOS: 1-844 or their corresponding full- length polynucleotides are linked to regulatory sequences as appropriate to obtain the desired expression properties. These can include promoters (attached either at the 5' end of the sense strand or at the 3' end of the antisense strand), enhancers, terminators, operators, repressors, and inducers. The promoters can be regulated or constitutive. In some situations it may be desirable to use conditionally active promoters, such as tissue-specific or developmental stage-specific promoters. These are linked to the desired nucleotide sequence using the techniques described above for linkage to vectors. Any techniques known in the art can be ^■ used.

When any of the above host cells, or other appropriate host cells or organisms, are used to replicate and/or express the polynucleotides or nucleic acids of the invention, the resulting replicated nucleic acid, RNA, expressed protein or polypeptide, is within the scope of the invention as a product of the host cell or organism. The product is recovered by any appropriate means known in the art.

Once the gene corresponding to a selected polynucleotide is identified, its expression can be regulated in the cell to which the gene is native. For example, an endogenous gene of a cell can be regulated by an exogenous regulatory sequence as disclosed in U.S. Patent No. 5,641,670. III. Identification of Functional and Structural Motifs of Novel Genes

A. Screening Polynucleotide Sequences and Amino Acid Sequences Against Publicly Available Databases Translations of the nucleotide sequence of the provided polynucleotides. cDNAs or full genes can be aligned with individual known sequences. Similarity with individual sequences can be used to determine the activity of the polypeptides encoded by the polynucleotides of the invention. For example, sequences that show similarity with a chemokine sequence can exhibit chemokine activities. Also, sequences exhibiting similarity with more than one individual sequence can exhibit activities that are characteristic of either or both individual sequences.

The full length sequences and fragments of the polynucleotide sequences of the nearest neighbors can be used as probes and primers to identify and isolate the full length sequence corresponding to provided polynucleotides. The nearest neighbors can indicate a tissue or cell type to be used to construct a library for the full-length sequences corresponding to the provided polynucleotides..

Typically, a selected polynucleotide is translated in all six frames to determine the best alignment with the individual sequences. The sequences disclosed herein in the Sequence Listing are in a 5' to 3' orientation and translation in three frames can be sufficient (with a few specific exceptions as described in the Examples). These amino acid sequences are referred to, generally, as query sequences, which will be aligned with the individual sequences. Databases with individual sequences are described in "Computer Methods for Macromolecular Sequence Analysis" Methods in Enzymology (1996) 266, Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Databases include Genbank, EMBL, and DNA Database of Japan (DDBJ).

Query and individual sequences can be aligned using the methods and computer programs described above, and include BLAST, available over the world wide web at http://ww.ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is Fasta, available in the Genetics Computing Group (GCG) package, Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Doolittle, supra. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. (1997) 70: 173-187. Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to identify sequences that are distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Amino acid sequences encoded by the provided polynucleotides can be used to search both protein and DNA databases. Results of individual and query sequence alignments can be divided into three categories, high similarity, weak similarity, and no similarity. Individual alignment results ranging from high similarity to weak similarity provide a basis for determining polypeptide activity and/or structure. Parameters for categorizing individual results include: percentage of the alignment region length where the strongest alignment is found, percent sequence identity, and p value.

The percentage of the alignment region length is calculated by counting the number of residues of the individual sequence found in the region of strongest alignment, e.g., contiguous region of the individual sequence that contains the greatest number of residues that are identical to the residues of the corresponding region of the aligned query sequence. This number is divided by the total residue length of the query sequence to calculate a percentage. For example, a query sequence of 20 amino acid residues might be aligned with a 20 amino acid region of an individual sequence. The individual sequence might be identical to amino acid residues 5, 9-15, and 17-19 of the query sequence. The region of strongest alignment is thus the region stretching from residue 9-19, an 11 amino acid stretch. The percentage of the alignment region length is: 11 (length of the region of strongest alignment) divided by (query sequence length) 20 or 55%.

Percent sequence identity is calculated by counting the number of amino acid matches between the query and individual sequence and dividing total number of matches by the number of residues of the individual sequences found in the region of strongest alignment. Thus, the percent identity in the example above would be 10 matches divided by 11 amino acids, or approximately, 90.9% P value is the probability that the alignment was produced by chance. For a single alignment, the p value can be calculated according to Karlin et al, Proc. Natl. Acad. Sci. (1990) 57:2264 and Karlin et al, Proc. Natl. Acad. Sci. (1993) 90. The p value of multiple alignments using the same query sequence can be calculated using an heuristic approach described in Altschul et al., Nat. Genet. (1994) 6:1 19. Alignment programs such as BLAST program can calculate the p value.

Another factor to consider for determining identity or similarity is the location of the similarity or identity. Strong local alignment can indicate similarity even if the length of alignment is short. Sequence identity scattered throughout the length of the query sequence also can indicate a similarity between the query and profile sequences. The boundaries of the region where the sequences align can be determined according to Doolittle. supra; BLAST or FAST programs; or by determining the area where sequence identity is highest. High Similarity. In general, in alignment results considered to be of high similarity, the percent of the alignment region length is typically at least about 55% of total length query sequence; more typically, at least about 58%; even more typically; at least about 60% of the total residue length of the query sequence. Usually, percent length of the alignment region can be as much as about 62%; more usually, as much as about 64%; even more usually, as much as about 66%. Further, for high similarity, the region of alignment, typically, exhibits at least about 75% of sequence identity; more typically, at least about 78%; even more typically; at least about 80% sequence identity. Usually, percent sequence identity can be as much as about 82%; more usually, as much as about 84%; even more usually, as much as about 86%.

The p value is used in conjunction with these methods. If high similarity is found, the query sequence is considered to have high similarity with a profile sequence when the p value is less than or equal to about 10^"2; more usually; less than or equal to about 10^"3; even more usually; less than or equal to about 10^"". More typically, the p value is no more than about 10^"5; more typically; no more than or equal to about 10^"10; even more typically; no more than or equal to about 10^"!5 for the query sequence to be considered high similarity. Weak Similarity. In general, where alignment results considered to be of weak similarity, there is no minimum percent length of the alignment region nor minimum length of alignment. A better showing of weak similarity is considered when the region of alignment is, typically, at least about 15 amino acid residues in length; more typically, at least about 20; even more typically; at least about 25 amino acid residues in length. Usually, length of the alignment region can be as much as about 30 amino acid residues; more usually, as much as about 40; even more usually, as much as about 60 amino acid residues. Further, for weak similarity, the region of alignment, typically, exhibits at least about 35% of sequence identity; more typically, at least about 40%; even more typically; at least about 45% sequence identity. Usually, percent sequence identity can be as much as about 50%; more usually, as much as about 55%; even more usually, as much as about 60%.

If low similarity is found, the query sequence is considered to have weak similarity with a profile sequence when the p value is usually less than or equal to about 10^"2; more usually; less than or equal to about 10^"3; even more usually; less than or equal to about 10^"4. More typically, the p value is no more than about 10^"5; more usually; no more than or equal to about 10^'10; even more usually; no more than or equal to about 10^"15 for the query sequence to be considered weak similarity. Similarity Determined by Sequence Identity Alone. Sequence identity alone can be used to determine similarity of a query sequence to an individual sequence and can indicate the activity of the sequence. Such an alignment, preferably, permits gaps to align sequences. Typically, the query sequence is related to the profile sequence if the sequence identity over the entire query sequence is at least about 15%; more typically, at least about 20%; even more typically, at least about 25%; even more typically, at least about 50%. Sequence identity alone as a measure of similarity is most useful when the query sequence is usually, at least 80 residues in length; more usually, 90 residues; even more usually, at least 95 amino acid residues in length. More typically, similarity can be concluded based on sequence identity alone when the query sequence is preferably 100 residues in length; more preferably, 120 residues in length; even more preferably, 150 amino acid residues in length.

Determining Activity from Alignments with Profile and Multiple Aligned Sequences. Translations of the provided polynucleotides can be aligned with amino acid profiles that define either protein families or common motifs. Also, translations of the provided polynucleotides can be aligned to multiple sequence alignments (MSA) comprising the polypeptide sequences of members of protein families or motifs. Similarity or identity with profile sequences or MS As can be used to determine the activity of the gene products (e.g., polypeptides) encoded by the provided polynucleotides or corresponding cDNA or genes.

For example, sequences that show an identity or similarity with a chemokine profile or MSA can exhibit chemokine activities.

Profiles can designed manually by (1) creating an MSA, which is an alignment of the amino acid sequence of members that belong to the family and (2) constructing a statistical representation of the alignment. Such methods are described, for example, in Birney et al, Nucl. Acid Res. (1996) 24(14): 2730-2739. MSAs of some protein families and motifs are publicly available. For example, http://genome.wustl.edu/Pfam/ includes MSAs of 547 different families and motifs. These MSAs are described also in Sonnhammer et al, Proteins (1997) 25: 405-420. Other sources over the world wide web include the site at http://wvvv..embl-heidelberg.de/argos/ali/ali.htm 1 ; alternatively, a message can be sent to ALI@EMBL-HEIDELBERG.DE for the information. A brief description of these MSAs is reported in Pascarella et al, Prot. Eng. (1996) 9(3):249-25\. Techniques for building profiles from MSAs are described in Sonnhammer et al, supra; Birney et al., supra; and "Computer Methods for Macromolecular Sequence Analysis," Methods in Enzymology

(1996) 266, Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA.

Similarity between a query sequence and a protein family or motif can be determined by (a) comparing the query sequence against the profile and or (b) aligning the query sequence with the members of the family or motif. Typically, a program such as Searchwise is used to compare the query sequence to the statistical representation of the multiple alignment, also known as a profile. The program is described in Birney et al, supra. Other techniques to compare the sequence and profile are described in Sonnhammer et al, supra and Doolittle, supra. Next, methods described by Feng et al, J. Mol. Evol. (1987) 25:351 and Higgins et al, CABIOS (1989) 5:151 can be used align the query sequence with the members of a family or motif, also known as a MSA. Computer programs, such as PILEUP, can be used. See Feng et al, infra. In general, the following factors are used to determine if a similarity between a query sequence and a profile or MSA exists: (1) number of conserved residues found in the query sequence, (2) percentage of conserved residues found in the query sequence, (3) number of frameshifts, and (4) spacing between conserved residues. Some alignment programs that both translate and align sequences can make any number of frameshifts when translating the nucleotide sequence to produce the best alignment. The fewer frameshifts needed to produce an alignment, the stronger the similarity or identity between the query and profile or MSAs. For example, a weak similarity resulting from no frameshifts can be a better indication of activity or structure of a query sequence, than a strong similarity resulting from two frameshifts. Preferably, three or fewer frameshifts are found in an alignment; more preferably two or fewer frameshifts; even more preferably, one or fewer frameshifts; even more preferably, no frameshifts are found in an alignment of query and profile or MSAs. Conserved residues are those amino acids found at a particular position in all or some of the family or motif members. For example, most chemokines contain four conserved cysteines. Alternatively, a position is considered conserved if only a certain class of amino acids is found in a particular position in all or some of the family members. For example, the N-terminal position can contain a positively charged amino acid, such as lysine, arginine, or histidine.

Typically, a residue of a polypeptide is conserved when a class of amino acids or a single amino acid is found at a particular position in at least about 40% of all class members; more typically, at least about 50%; even more typically, at least about 60% of the members. Usually, a residue is conserved when a class or single amino acid is found in at least about 70% of the members of a family or motif; more usually, at least about 80%; even more usually, at least about 90%; even more usually, at least about 95%.

A residue is considered conserved when three unrelated amino acids are found at a particular position in the some or all of the members; more usually, two unrelated amino acids. These residues are conserved when the unrelated amino acids are found at particular positions in at least about 40% of all class member; more typically, at least about 50%; even more typically, at least about 60% of the members. Usually, a residue is conserved when a class or single amino acid is found in at least about 70% of the members of a family or motif; more usually, at least about 80%; even more usually, at least about 90%; even more usually, at least about 95%. A query sequence has similarity to a profile or MSA when the query sequence comprises at least about 25% of the conserved residues of the profile or MSA; more usually, at least about 30%; even more usually; at least about 40%. Typically, the query sequence has a stronger similarity to a profile sequence or MSA when the query sequence comprises at least about 45% of the conserved residues of the profile or MSA; more typically, at least about 50%; even more typically; at least about 55%. B. Screening Polynucleotide and Amino Acid Sequences Against Protein

Profiles The identify and function of the gene that correlates to a polynucleotide described herein can be determined by screening the polynucleotides or their corresponding amino acid sequences against profiles of protein families. Such profiles focus on common structural motifs among proteins of each family. Publicly available profiles are described above in

Section IVA. Additional or alternative profiles are described below.

In comparing a novel polynucleotide with known sequences, several alignment tools are available. Examples include PileUp, which creates a multiple sequence alignment, and is described in Feng et al, J. Mol. Evol. (1987) 25:351. Another method, GAP, uses the alignment method of Needleman et al, J. Mol. Biol. (1970) 48:443. GAP is best suited for global alignment of sequences. A third method, BestFit, functions by inserting gaps to maximize the number of matches using the local homology algorithm of Smith et al., Adv.

Appl. Math. (1981) 2:482. Exemplary protein profiles are provided below and in the examples. Chemokines. Chemokines are a family of proteins that have been implicated in lymphocyte trafficking, inflammatory diseases, angiogenesis, hematopoiesis, and viral infection. See, for example, Rollins, Blood (1997) °0(5 :909-928, and Wells et al, J. Leuk.

Biol. (1997) 67:545-550. U.S. Patent No. 5,605,817 discloses DNA encoding a chemokine expressed in fetal spleen. U.S. Patent No. 5,656,724 discloses chemokine-like proteins and methods of use. U.S. Patent No. 5,602,008 discloses DNA encoding a chemokine expressed by liver.

Chemokine mutants are polypeptides having an amino acid sequence that possesses at least one amino acid substitution, addition, or deletion as compared to native chemokines.

Fragments possess the same amino acid sequence of the native chemokines; mutants can lack the amino and/or carboxyl terminal sequences. Fusions are mutants, fragments, or native chemokines that also include amino and/or carboxyl terminal amino acid extensions. The number or type of the amino acid changes is not critical, nor is the length or number of the amino acid deletions, or amino acid extensions that are incorporated in the chemokines as compared to the native chemokine amino acid sequences. A polynucleotide encoding one of these variant polypeptides will retain at least about 80% amino acid identity with at least one known chemokine. Preferably, these polypeptides will retain at least about 85% amino acid sequence identity, more preferably, at least about 90%; even more preferably, at least about 95%. In addition, the variants exhibit at least 80%; preferably about 90%; more preferably about 95% of at least one activity exhibited by a native chemokine, which includes immunological, biological, receptor binding, and signal transduction functions.

Assays for chemotaxis relating to neutrophils are described in Walz et al, Biochem. Biophys. Res. Commun. (1987) 149:155, Yoshimura et al, Proc. Natl. Acad. Sci. (USA)

(1987) 84:9233, and Schroder et al, J. Immunol. (1987) 759:3474; to lymphocytes, Larsen et al, Science (1989) 245:1464, Carr et al, Proc. Natl. Acad. Sci. (USA) (1994) 97:3652; to tumor-infiltrating lymphocytes, Liao et al, J. Exp. Med (1995). 752:1301; to hematopoietic progenitors, Aiuti et al, J. Exp. Med. (1997) 755:111; to monocytes, Valente et al, Biochem.

(1988) 27:4162; and to natural killer cells, Loetscher et al, J. Immunol. (1996) 756:322, and Allavena et al, Eur. J. Immunol. (1994) 24:3233.

Assays for determining the biological activity of attracting eosinophils are described in Dahinden et al, J. Exp. Med. (1994) 779:751, Weber et al, J. Immunol. (1995) 754:4166, and Noso et al, Biochem. Biophys. Res. Commun. (1994) 200:1470; for attracting dendritic cells, Sozzani et al, J. Immunol. (1995) 755:3292; for attracting basophils, in Dahinden et al, J. Exp. Med. (1994) 779:751, Ala et al, J. Immunol. (1994) 752:1298, Alam et al, J.

Exp. Med. (1992) 776:781; and for activating neutrophils, Maghazaci et al, Eur. J. Immunol. (1996) 26:315, and Taub et al, J. Immunol. (1995) 755:3877. Native chemokines can act as mitogens for fibroblasts, assayed as described in Mullenbach et al, J. Biol. Chem. (1986)

267:719.

Native chemokines exhibit binding activity with a number of receptors. Description of such receptors and assays to detect binding are described in, for example, Murphy et al, Science (1991) 255:1280; Combadiere et al, J. Biol. Chem. (1995) 270:29671 ; Daugherty et al, J. Exp. Med. (1996) 755:2349; Samson et al, Biochem. (1996) 55:3362; Raport et al, J. Biol. Chem. (1996) 277:17161 ; Combadiere et al, J. Leukoc. Biol. (1996) 60:147; Baba et al, J. Biol. Chem. (1997) 25:14893; Yosida et α/., J. Biol. Chem. (1997) 272:13803; Arvannitakis et al, Nature (1997) 555:347, and other assays are known in the art.

Assays for kinase activation of chemokines are described by Yen et al, J. Leukoc. Biol. (1997) 67:529; Dubois et al, J. Immunol. (1996) 756:1356; Turner et al, J. Immunol.

(1995) 755:2437. Assays for inhibition of angiogenesis or cell proliferation are described in Maione et al, Science (1990) 247:11. Glycosaminoglycan production can be induced by native chemokines, assayed as described in Castor et al, Proc. Natl. Acad. Sci. (USA) (1983) 50:765. Chemokine-mediated histamine release from basophils is assayed as described in Dahinden et al, J. Exp. Med. (1989) 770:1787; and White et al, Immunol. Lett. (1989) 22:151. Heparin binding is described in Luster et al, J. Exp. Med. (1995) 752:219.

Chemokines can possess dimerization activity, which can be assayed according to Burrows et al, Biochem. (1994) 55:12741; and Zhang et al, Mol. Cell. Biol. (1995) 75:4851. Native chemokines can play a role in the inflammatory response of viruses. This activity can be assayed as described in Bleul et al, Nature (1996) 552:829; and Oberlin et al, Nature

(1996) 552:833. Exocytosis of monocytes can be promoted by native chemokines. The assay for such activity is described in Uguccioni et al, Eur. J. Immunol. (1995) 25:64. Native chemokines also can inhibit hematopoietic stem cell proliferation. The method for testing for such activity is reported in Graham et al, Nature (1990) 544:442. Death Domain Proteins. Several protein families contain death domain motifs

(Feinstein and Kimchi, TIBS Letters (1995) 20:242). Some death domain containing proteins are implicated in cytotoxic intracellular signaling (Cleveland et al, Cell (1995) 57:479, Pan et al, Science (1997) 276:111; Duan et al, Nature (1997) 555:86-89, and Chinnaiyan et al, Science (1996) 274:990). U.S. Patent No. 5,563,039 describes a protein homologous to TRADD (Tumor Necrosis Factor Receptor- 1 Associated Death Domain containing protein), and modifications of the active domain of TRADD that retain the functional characteristics of the protein, as well as apoptosis assays for testing the function of such death domain containing proteins. U.S. Patent No. 5,658,883 discloses biologically active TGF-B1 peptides. U.S. Patent No. 5,674,734 discloses RIP, which contains a C- terminal death domain and an N-terminal kinase domain. Leukemia Inhibitory Factor (LIF). An LIF profile is constructed from sequences of leukemia inhibitor factor, CT-1 (cardiotrophin-1), CNTF (ciliary neurotrophic factor), OSM (oncostatin M), and IL-6 (interleukin-6). This profile encompasses a family of secreted cytokines that have pleiotropic effects on many cell types including hepatocytes, osteoclasts, neuronal cells and cardiac myocytes, and can be used to detect additional genes encoding such proteins. These molecules are all structurally related and share a common co-receptor gpl30 which mediates intracellular signal transduction by cytoplasmic tyrosine kinases such as src.

Novel proteins related to this family are also likely to be secreted, to activate gpl30 and to function in the development of a variety of cell types. Thus new members of this family would be candidates to be developed as growth or survival factors for the cell types that they stimulate. For more details on this family of cytokines, see Pennica et al, Cytokine and Growth Factor Reviews (1996) 7:81-91. U.S. Patent No. 5,420,247 discloses LIF receptor and fusion proteins. U.S. Patent No. 5,443,825 discloses human LIF. Angiopoietin. Angiopoietin-1 is a secreted ligand of the TIE-2 tyrosine kinase; it functions as an angiogenic factor critical for normal vascular development. Angiopoietin-2 is a natural antagonist of angiopoietin- 1 and thus functions as an anti-angiogenic factor. These two proteins are structurally similar and activate the same receptor (Folkman et al. , Cell (1996) 57:1153, and Davis et al, Cell (1996) 57:1161). The angiopoietin molecules are composed of two domains: a coiled-coil region and a region related to fibrinogen. The fibrinogen domain is found in many molecules including ficolin and tesascin, and is well defined structurally with many members.

Receptor Protein-Tyrosine Kinases. Receptor Protein-Tyrosine Kinases or RPTKs are described in Lindberg, Annu. Rev. Cell Biol. (1994) 70:251-337. Growth Factors: (Epidermal Growth Factor) EGF and (Fibroblast Growth Factor)

FGF. For a discussion of growth factor superfamilies, see Growth Factors: A Practical Approach, (Appendix Al) (1993) McKay and Leigh, Oxford University Press, NY, 237-243. U.S. Patent No. 4,444,760 discloses acidic brain fibroblast growth factor, which is active in the promotion of cell division and wound healing. U.S. Patent No. 5,439,818 discloses DNA encoding human recombinant basic fibroblast growth factor, which is active in wound healing. U.S. Patent No. 5,604,293 discloses recombinant human basic fibroblast growth factor, which is useful for wound healing. U.S. Patent No. 5,410,832 discloses brain-derived and recombinant acidic fibroblast growth factor, which act as mitogens for mesoderm and neuroectoderm-derived cells in culture, and promote wound healing in soft tissue, cartilaginous tissue and musculo-skeletal tissue. U.S. Patent No. 5,387,673 discloses biologically active fragments of FGF.

Proteins of the TNF Family. A profile derived from the TNF family is created by aligning sequences of the following TNF family members: nerve growth factor (NGF), lymphotoxin, Fas ligand, tumor necrosis factor (TNFα), CD40 ligand, TRAIL, ox40 ligand, 4- IBB ligand, CD27 ligand, and CD30 ligand. The profile is designed to identify sequences of proteins that constitute new members or homologues of this family of proteins. U.S. Patent No. 5,606,023 discloses mutant TNF proteins; U.S. Patent No. 5,597,899 and U.S. Patent No. 5,486,463 disclose TNF muteins; and U.S. Patent No. 5,652,353 discloses DNA encoding TNFα muteins.

Members of the TNF family of proteins have been show in vitro to multimerize, as described in Burrows et al, Biochem. (1994) 55:12741 and Zhang et al, Mol. Cell. Biol. (1995) 75:4851 and bind receptors as described in Browning et al, J. Immunol. (1994) 747:1230, Androlewicz et al, J. Biol. Chem.(\992) 267:2542, and Crowe et al, Science (1994) 264:707.

In vivo, TNFs proteolytically cleave a target protein as described in Kriegel et al, Cell (1988) 53 :45 and Mohler et al. , Nature ( 1994) 70:218 and demonstrate cell proliferation and differentiation activity. T-cell or thymocyte proliferation is assayed as described in Armitage et al, Eur. J. Immunol (1992) 22:447; Current Protocols in Immunology, ed. J.E. Coligan et al, 3.1-3.19; Takai et al, J. Immunol. (1986) 757:3494- 3500, Bertagnoli et al, J. Immunol. (1990) 745:1706, Bertagnoli et al, J. Immunol. (1991) 133:321, Bertagnoli et al, J. Immunol. (1992) 749:3778, and Bowman et al, J. Immunol. (1994) 752:1756. B cell proliferation and Ig secretion are assayed as described in Maliszewski, J Immunol. (1990) 744:3028, and Assays for B Cell Function: In Vitro Antibody Production, Mond and Brunswick, Current Protocols in Immunol., Coligan Ed vol 1 pp 3.8.1-3.8.16, John Wiley and Sons, Toronto 1994, Kehrl et al, Science (1987) 255:1144 and Boussiotis et al, PNAS USA (1994) 97:7007. Other in vivo activities include upregulation of cell surface antigens, upregulation of costimulatory molecules, and cellular aggregation/adhesion as described in Barrett et al, J. Immunol. (1991) 746:1722; Bjorck et al, Eur. J. Immunol. (1993) 25:1771; Clark et al, Annu Rev. Immunol. (1991) 9:97;

Ranheim et al., J. Exp. Med. (1994) 777:925; Yellin, J Immunol. (1994) 755:666; and Gruss et al, Blood ( 994) 84:2305. Proliferation and differentiation of hematopoietic and lymphopoietic cells has also been shown in vivo for TNFs, using assays for embryonic differentiation and hematopoiesis as described in Johansson et al, Cellular Biology (1995) 75:141, Keller et al, Mol. Cell.

Biol (1993) 75:473, McClanahan et al, Blood (1993) 57:2903 and using assays to detect stem cell survival and differentiation as described in Culture of Hematopoietic Cells, Freshney et al. eds, pp 1-21, 23-29, 139-162, 163-179, and 265-268, Wiley-Liss, Inc., New

York, NY, 1994, and Hirajama et al, PNAS USA (1992) 59:5907.

In vivo activities of TNFs also include lymphocyte survival and apoptosis, assayed as described in Darzynkewicz et al, Cytometry (1992) 75:795; Gorczca et al, Leukemia (1993)

7:659; Itoh et al, Cell (1991) 66:233; Zacharduk, J. Immunol. (1990) 745:4037; Zamai et al, Cytometry (1993) 74:891 ; and Gorczyca et al, Int'U. Oncol. (1992) 7:639. Some members of the TNF family are cleaved from the cell surface; others remain membrane bound. The three-dimensional structure of TNF is discussed in Sprang and Eck, Tumor

Necrosis Factors; supra.

TNF proteins include a transmembrane domain. The protein is cleaved into a shorter soluble version, as described in Kriegler et al. , Cell (1988) 55:45, Perez et al. , Cell (1990)

65:251, and Shaw et al, Cell (1986) 46:659. The transmembrane domain is between amino acid 46 and 77 and the cytoplasmic domain is between position 1 and 45 on the human form of TNFα. The 3-dimensional motifs of TNF include a sandwich of two pleated β sheets.

Each sheet is composed of anti-parallel β strands, β strands facing each other on opposite sites of the sandwich are connected by short polypeptide loops, as described in Van Ostade et al, Protein Engineering (1994) 7(1):5, and Sprang et al, Tumor Necrosis Factors; supra.

Residues of the TNF family proteins that are involved in the β sheet secondary structure have been identified as described in Van Ostade et al, Protein Eng. (1994) 7(1):5, and

Sprang et al, supra. TNF receptors are disclosed in U.S. Patent No. 5,395,760. A profile derived from the

TNF receptor family is created by aligning sequences of the TNF receptor family, including Apol/Fas, TNFR I and II, death receptor 3 (DR3), CD40, ox40, CD27, and CD30. Thus, the profile is designed to identify from the polynucleotides of the invention sequences of proteins that constitute new members or homologues of this family of proteins.

Tumor necrosis factor receptors exist in two forms in humans: p55 TNFR and p75 TNFR, both of which provide intracellular signals upon binding with a ligand. The extracellular domains of these receptor proteins are cysteine rich. The receptors can remain membrane bound, although some forms of the receptors are cleaved forming soluble receptors. The regulation, diagnostic, prognostic, and therapeutic value of soluble TNF receptors is discussed in Aderka, Cytokine and Growth Factor Reviews, ( 1996) 7(5).231. PDGF Family. U.S. Patent No. 5,326,695 discloses platelet derived growth factor agonists; bioactive portions of PDGF-B are used as agonists. U.S. Patent No. 4,845,075 discloses biologically active B-chain homodimers, and also includes variants and derivatives of the PDGF-B chain. U.S. Patent No. 5,128,321 discloses PDGF analogs and methods of use. Proteins having the same bioactivity as PDGF are disclosed, including A and B chain proteins.

Kinase (Including MKK) Family. U.S. Patent No. 5,650,501 discloses serine/threonine kinase, associated with mitotic and meiotic cell division; the protein has a kinase domain in its N-terminal and 3 PEST regions in the C-terminus. U.S. Patent No. 5,605,825 discloses human PAK65, a serine protein kinase. The foregoing discussion provides a few examples of the protein profiles that can be compared with the polynucleotides of the invention. One skilled in the art can use these and other protein profiles to identify the genes that correlate with the provided polynucleotides. C. Identification of Secreted & Membrane-Bound Polypeptides Both secreted and membrane-bound polypeptides of the present invention are of particular interest. For example, levels of secreted polypeptides can be assayed in body fluids that are convenient, such as blood, urine, prostatic fluid and semen. Membrane-bound polypeptides are useful for constructing vaccine antigens or inducing an immune response. Such antigens would comprise all or part of the extracellular region of the membrane-bound polypeptides. Because both secreted and membrane-bound polypeptides comprise a fragment of contiguous hydrophobic amino acids, hydrophobicity predicting algorithms can be used to identify such polypeptides. A signal sequence is usually encoded by both secreted and membrane-bound polypeptide genes to direct a polypeptide to the surface of the cell. The signal sequence usually comprises a stretch of hydrophobic residues. Such signal sequences can fold into helical structures. Membrane-bound polypeptides typically comprise at least one transmembrane region that possesses a stretch of hydrophobic amino acids that can transverse the membrane. Some transmembrane regions also exhibit a helical structure. Hydrophobic fragments within a polypeptide can be identified by using computer algorithms. Such algorithms include Hopp & Woods_Δ Proc. Natl. Acad. Sci. USA (1981) 75:3824-3828; Kyte & Doolittle, J. Mol. Biol. (1982) 757: 105-132; and RAOAR algorithm, Degli Esposti et al, Eur. J. Biochem. (1990) 790: 207-219.

Another method of identifying secreted and membrane-bound polypeptides is to translate the polynucleotides of the invention in all six frames and determine if at least 8 contiguous hydrophobic amino acids are present. Those translated polypeptides with at least 8; more typically, 10; even more typically, 12 contiguous hydrophobic amino acids are considered to be either a putative secreted or membrane bound polypeptide. Hydrophobic amino acids include alanine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine.

IV. Identification of the Function of an Expression Product of a Full-Length Gene Corresponding to a Polynucleotide

Ribozymes, antisense constructs, and dominant negative mutants can be used to determine function of the expression product of a gene corresponding to a polynucleotide provided herein. These methods and compositions are particularly useful where the provided novel polynucleotide exhibits no significant or substantial homology to a sequence encoding a gene of known function. Antisense molecules and ribozymes can be constructed from synthetic polynucleotides. Typically, the phosphoramidite method of oligonucleotide synthesis is used. See Beaucage et al, Tet. Lett. (1981) 22:1859 and U.S. Patent No. 4,668,777. Automated devices for synthesis are available to create oligonucleotides using this chemistry. Examples of such devices include Biosearch 8600, Models 392 and 394 by Applied Biosystems, a division of Perkin-Elmer Corp., Foster City, California, USA; and Expedite by Perceptive Biosystems, Framingham, Massachusetts, USA. Synthetic RNA, phosphate analog oligonucleotides, and chemically derivatized oligonucleotides can also be produced, and can be covalently attached to other molecules. RNA oligonucleotides can be synthesized, for example, using RNA phosphoramidites. This method can be performed on an automated synthesizer, such as Applied Biosystems, Models 392 and 394, Foster City, California, USA. See Applied Biosystems User Bulletin 53 and Ogilvie et al, Pure & Applied Chem. (1987) 59:325.

Phosphorothioate oligonucleotides can also be synthesized for antisense construction.

A sulfurizing reagent, such as tetraethylthiruam disulfide (TETD) in acetonitrile can be used to convert the internucleotide cyanoethyl phosphite to the phosphorothioate triester within 15 minutes at room temperature. TETD replaces the iodine reagent, while all other reagents used for standard phosphoramidite chemistry remain the same. Such a synthesis method can be automated using Models 392 and 394 by Applied Biosystems, for example.

Oligonucleotides of up to 200 nucleotides can be synthesized, more typically, 100 nucleotides, more typically 50 nucleotides; even more typically 30 to 40 nucleotides. These synthetic fragments can be annealed and ligated together to construct larger fragments. See, for example, Sambrook et al, supra. A. Ribozymes

Trans-cleaving catalytic RNAs (ribozymes) are RNA molecules possessing endoribonuclease activity. Ribozymes are specifically designed for a particular target, and the target message must contain a specific nucleotide sequence. They are engineered to cleave any RNA species site-specifically in the background of cellular RNA. The cleavage event renders the mRNA unstable and prevents protein expression. Importantly, ribozymes can be used to inhibit expression of a gene of unknown function for the purpose of determining its function in an in vitro or in vivo context, by detecting the phenotypic effect. One commonly used ribozyme motif is the hammerhead, for which the substrate sequence requirements are minimal. Design of the hammerhead ribozyme is disclosed in Usman et al, Current Opin. Struct. Biol. (1996) 6:527. Usman also discusses the therapeutic uses of ribozymes. Ribozymes can also be prepared and used as described in Long et al, FASEB J. (1993) 7:25; Symons, Ann. Rev. Biochem. (1992) 67:641 ; Perrotta et al, Biochem. (1992) 57:16; Ojwang et al, Proc. Natl. Acad. Sci. (USA) (1992) 59:10802; and U.S. Patent No. 5,254,678. Ribozyme cleavage of HIV-I RNA is described in U.S. Patent No. 5,144,019; methods of cleaving RNA using ribozymes is described in U.S.

Patent No. 5,116,742; and methods for increasing the specificity of ribozymes are described in U.S. Patent No. 5,225,337 and Koizumi et al, Nucleic Acid Res. (1989) 77:7059. Preparation and use of ribozyme fragments in a hammerhead structure are also described by Koizumi et al, Nucleic Acids Res. (1989) 7:7059. Preparation and use of ribozyme fragments in a hairpin structure are described by Chowrira and Burke, Nucleic Acids Res. (1992) 20:2835. Ribozymes can also be made by rolling transcription as described in Daubendiek and Kool, Nat. Biotechnol. (1997) 15(3):213.

The hybridizing region of the ribozyme can be modified or can be prepared as a branched structure as described in Horn and Urdea, Nucleic Acids Res. (1989) 77:6959. The basic structure of the ribozymes can also be chemically altered in ways familiar to those skilled in the art, and chemically synthesized ribozymes can be administered as synthetic oligonucleotide derivatives modified by monomeric units. In a therapeutic context, liposome mediated delivery of ribozymes improves cellular uptake, as described in Birikh et al, Eur. J. Biochem. (1997) 245:1.

Using the polynucleotide sequences of the invention and methods known in the art, ribozymes are designed to specifically bind and cut the corresponding mRNA species. Ribozymes thus provide a means to inhibit the expression of any of the proteins encoded by the disclosed polynucleotides or their full-length genes. The full-length gene need not be known in order to design and use specific inhibitory ribozymes. In the case of a polynucleotide or full-length cDNA of unknown function, ribozymes corresponding to that nucleotide sequence can be tested in vitro for efficacy in cleaving the target transcript. Those ribozymes that effect cleavage in vitro are further tested in vivo. The ribozyme can also be used to generate an animal model for a disease, as described in Birikh et al, supra. An effective ribozyme is used to determine the function of the gene of interest by blocking its transcription and detecting a change in the cell. Where the gene is found to be a mediator in a disease, an effective ribozyme is designed and delivered in a gene therapy for blocking transcription and expression of the gene.

Therapeutic and functional genomic applications of ribozymes proceed beginning with knowledge of a portion of the coding sequence of the gene to be inhibited. Thus, for many genes, a partial polynucleotide sequence provides adequate sequence for constructing an effective ribozyme. A target cleavage site is selected in the target sequence, and a ribozyme is constructed based on the 5' and 3' nucleotide sequences that flank the cleavage site. Retroviral vectors are engineered to express monomeric and multimeric hammerhead ribozymes targeting the mRNA of the target coding sequence. These monomeric and multimeric ribozymes are tested in vitro for an ability to cleave the target mRNA. A cell line is stably transduced with the retroviral vectors expressing the ribozymes, and the transduction is confirmed by Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR). The cells are screened for inactivation of the target mRNA by such indicators as reduction of expression of disease markers or reduction of the gene product of the target mRNA. B. Antisense

Antisense nucleic acids are designed to specifically bind to RNA, resulting in the formation of RNA-DNA or RNA-RNA hybrids, with an arrest of DNA replication, reverse transcription or messenger RNA translation. Antisense polynucleotides based on a selected polynucleotide sequence can interfere with expression of the corresponding gene. Antisense polynucleotides are typically generated within the cell by expression from antisense constructs that contain the antisense strand as the transcribed strand. Antisense polynucleotides based on the disclosed polynucleotides will bind and/or interfere with the translation of mRNA comprising a sequence complementary to the antisense polynucleotide. The expression products of control cells and cells treated with the antisense construct are compared to detect the protein product of the gene corresponding to the polynucleotide upon which the antisense construct is based. The protein is isolated and identified using routine biochemical methods.

One rationale for using antisense methods to determine the function of the gene corresponding to a disclosed polynucleotide is the biological activity of antisense therapeutics. Antisense therapy for a variety of cancers is in clinical phase and has been discussed extensively in the literature. Reed reviewed antisense therapy directed at the Bcl-2 gene in tumors; gene transfer-mediated overexpression of Bcl-2 in tumor cell lines conferred resistance to many types of cancer drugs. (Reed, 3. C, N.C.I. (1997) 59:988). The potential for clinical development of antisense inhibitors of ras is discussed by Cowsert, L.M., Anti- Cancer Drug Design (1997) 72:359. Additional important antisense targets include leukemia (Geurtz, A.M., Anti-Cancer Drug Design (1997) 72:341); human C-ref kinase

(Monia, B.P., Anti-Cancer Drug Design (1997) 72:327); and protein kinase C (McGraw et al, Anti-Cancer Drug Design (1997) 72:315.

Given the extensive background literature and clinical experience in antisense therapy, one skilled in the art can use selected polynucleotides of the invention as additional potential therapeutics. The choice of polynucleotide can be narrowed by first testing them for binding to "hot spot" regions of the genome of cancerous cells. If a polynucleotide is identified as binding to a "hot spot", testing the polynucleotide as an antisense compound in the corresponding cancer cells clearly is warranted. Ogunbiyi et al, Gastroenterology (1997) 113(3):16\ describe prognostic use of allelic loss in colon cancer; Barks et al, Genes, Chromosomes, and Cancer (1997) 19(4):21 describe increased chromosome copy number detected by FISH in malignant melanoma; Nishizake et al, Genes, Chromosomes, and Cancer (1997) 19(4):261 describe genetic alterations in primary breast cancer and their metastases and direct comparison using modified comparative genome hybridization; and Elo et al, Cancer Research (1997)

57(16):3356 disclose that loss of heterozygosity at 16z24.1-q24.2 is significantly associated with metastatic and aggressive behavior of prostate cancer. C. Dominant Negative Mutations As an alternative method for identifying function of the gene corresponding to a polynucleotide disclosed herein, dominant negative mutations are readily generated for corresponding proteins that are active as homomul timers. A mutant polypeptide will interact with wild-type polypeptides (made from the other allele) and form a non-functional multimer. Thus, a mutation is in a substrate-binding domain, a catalytic domain, or a cellular localization domain. Preferably, the mutant polypeptide will be overproduced. Point mutations are made that have such an effect. In addition, fusion of different polypeptides of various lengths to the terminus of a protein can yield dominant negative mutants. General strategies are available for making dominant negative mutants (see, e.g., Herskowitz, Nature (1987) 529:219). Such techniques can be used to create loss of function mutations, which are useful for determining protein function. V. Construction of Polypeptides of the Invention and Variants Thereof

The polypeptides of the invention include those encoded by the disclosed polynucleotides. These polypeptides can also be encoded by nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed polynucleotides. Thus, the invention includes within its scope a polypeptide encoded by a polynucleotide having the sequence of any one of SEQ ID NOS: 1-844 or a variant thereof.

In general, the term "polypeptide" as used herein refers to both the full length polypeptide encoded by the recited polynucleotide, the polypeptide encoded by the gene represented by the recited polynucleotide, as well as portions or fragments thereof. "Polypeptides" also includes variants of the naturally occurring proteins, where such variants are homologous or substantially similar to the naturally occurring protein, and can be of an origin of the same or different species as the naturally occurring protein (e.g., human, murine, or some other species that naturally expresses the recited polypeptide, usually a mammalian species). In general, variant polypeptides have a sequence that has at least about 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a differentially expressed polypeptide of the invention, as measured by BLAST using the parameters described above. The variant polypeptides can be naturally or non- naturally glycosylated, i. e. , the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring protein. The invention also encompasses homologs of the disclosed polypeptides (or fragments thereof) where the homologs are isolated from other species, i.e. other animal or plant species, where such homologs, usually mammalian species, e.g. rodents, such as mice, rats; domestic animals, e.g., horse, cow, dog, cat; and humans. By homolog is meant a polypeptide having at least about 35%, usually at least about 40% and more usually at least about 60% amino acid sequence identity a particular differentially expressed protein as identified above, where sequence identity is determined using the BLAST algorithm, with the parameters described supra.

In general, the polypeptides of the subject invention are provided in a non-naturally occurring environment, e.g. are separated from their naturally occurring environment. In certain embodiments, the subject protein is present in a composition that is enriched for the protein as compared to a control. As such, purified polypeptide is provided, where by purified is meant that the protein is present in a composition that is substantially free of non- differentially expressed polypeptides, where by substantially free is meant that less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of non-differentially expressed polypeptides. Also within the scope of the invention are variants; variants of polypeptides include mutants, fragments, and fusions. Mutants can include amino acid substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid substituted. For example, substitutions between the following groups are conservative: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/ Arg, Asn/Gln, Ser/Cys, Thr, and Phe/Trp/Tyr. Variants can be designed so as to retain biological activity of a particular region of the protein (e.g., a functional domain and/or, where the polypeptide is a member of a protein family, a region associated with a consensus sequence). In a non-limiting example, Osawa et al, Biochem. Mol. Int. (1994) 54:1003, discusses the actin binding region of a protein from several different species. The actin binding regions of the these species are considered homologous based on the fact that they have amino acids that fall within "homologous residue groups." Homologous residues are judged according to the following groups (using single letter amino acid designations): STAG; ILVMF; HRK; DEQN; and FYW. For example, and S, a T, an A or a G can be in a position and the function (in this case actin binding) is retained. Additional guidance on amino acid substitution is available from studies of protein evolution. Go et al, Int. J. Peptide Protein Res. (1980) 75:211, classified amino acid residue sites as interior or exterior depending on their accessibility. More frequent substitution on exterior sites was confirmed to be general in eight sets of homologous protein families regardless of their biological functions and the presence or absence of a prosthetic group. Virtually all types of amino acid residues had higher mutabilities on the exterior than in the interior. No correlation between mutability and polarity was observed of amino acid residues in the interior and exterior, respectively. Amino acid residues were classified into one of three groups depending on their polarity: polar (Arg, Lys, His, Gin, Asn, Asp. and Glu); weak polar (Ala, Pro, Gly, Thr, and Ser), and nonpolar (Cys, Val, Met, He, Leu. Phe, Tyr, and Trp). Amino acid replacements during protein evolution were very conservative: 88% and 76% of them in the interior or exterior, respectively, were within the same group of the three. Inter-group replacements are such that weak polar residues are replaced more often by nonpolar residues in the interior and more often by polar residues on the exterior. Additional guidance for production of polypeptide variants is provided in Querol et al, Prot. Eng. (1996) 9:265, which provides general rules for amino acid substitutions to enhance protein thermostability. New glycosylation sites can be introduced as discussed in Olsen and Thomsen, J. Gen. Microbiol. (1991) 757:579. An additional disulfide bridge can be introduced, as discussed by Perry and Wetzel, Science (1984) 226:555; Pantoliano et al, Biochemistry (1987) 26:2077; Matsumura et al, Nature (1989) 542:291; Nishikawa et al, Protein Eng. (1990) 5:443; Takagi et al, J. Biol. Chem. (1990) 265:6874; Clarke et al, Biochemistry (1993) 52:4322; and Wakarchuk et al, Protein Eng. (1994) 7:1379. Metal binding sites can be introduced, according to Toma et al, Biochemistry (1991) 30:91, and Haezerbrouck et al, Protein Eng. (1993) 6:643. Substitutions with prolines in loops can be made according to Masul et al, Appl. Env. Microbiol. (1994) 60:3579; and Hardy et al, FEBSLett. 317:S9. Cysteine-depleted muteins are considered variants within the scope of the invention.

These variants can be constructed according to methods disclosed in U.S. Patent No. 4,959,314, which discloses substitution of cysteines with other amino acids, and methods for assaying biological activity and effect of the substitution. Such methods are suitable for proteins according to this invention that have cysteine residues suitable for such substitutions, for example to eliminate disulfide bond formation.

Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. Fragments of interest will typically be at least about 10 aa to at least about 15 aa in length, usually at least about 50 aa in length, and can be as long as 300 aa in length or longer, but will usually not exceed about 1000 aa in length, where the fragment will have a stretch of amino acids that is identical to a polypeptide encoded by a polynucleotide having a sequence of any SEQ ID NOS: 1-844, or a homolog thereof.

The protein variants described herein are encoded by polynucleotides that are within the scope of the invention. The genetic code can be used to select the appropriate codons to construct the corresponding variants.

VI. Computer-Related Embodiments

In general, a library of polynucleotides is a collection of sequence information, which information is provided in either biochemical form (e.g., as a collection of polynucleotide molecules), or in electronic form (e.g., as a collection of polynucleotide sequences stored in a computer-readable form, as in a computer system and/or as part of a computer program). The sequence information of the polynucleotides can be used in a variety of ways, e.g., as a resource for gene discovery, as a representation of sequences expressed in a selected cell type (e.g., cell type markers), and/or as markers of a given disease or disease state. In general, a disease marker is a representation of a gene product that is present in all affected by disease either at an increased or decreased level relative to a normal cell (e.g., a cell of the same or similar type that is not substantially affected by disease). For example, a polynucleotide sequence in a library can be a polynucleotide that represents an mRNA, polypeptide, or other gene product encoded by the polynucleotide, that is either overexpressed or underexpressed in a breast ductal cell affected by cancer relative to a normal (i.e., substantially disease-free) breast cell.

The nucleotide sequence information of the library can be embodied in any suitable form, e.g., electronic or biochemical forms. For example, a library of sequence information embodied in electronic form includes an accessible computer data file (or, in biochemical form, a collection of nucleic acid molecules) that contains the representative nucleotide sequences of genes that are differentially expressed (e.g., overexpressed or underexpressed) as between, for example, i) a cancerous cell and a normal cell; ii) a cancerous cell and a dysplastic cell; iii) a cancerous cell and a cell affected by a disease or condition other than cancer; iv) a metastatic cancerous cell and a normal cell and/or non-metastatic cancerous cell; v) a malignant cancerous cell and a non-malignant cancerous cell (or a normal cell) and/or vi) a dysplastic cell relative to a normal cell. Other combinations and comparisons of cells affected by various diseases or stages of disease will be readily apparent to the ordinarily skilled artisan. Biochemical embodiments of the library include a collection of nucleic acids that have the sequences of the genes in the library, where the nucleic acids can correspond to the entire gene in the library or to a fragment thereof, as described in greater detail below.

The polynucleotide libraries of the subject invention include sequence information of a plurality of polynucleotide sequences, where at least one of the polynucleotides has a sequence of any of SEQ ID NOS: 1-844. By plurality is meant at least 2, usually at least 3 and can include up to all of SEQ ID NOS: 1-844. The length and number of polynucleotides in the library will vary with the nature of the library, e.g., if the library is an oligonucleotide array, a cDNA array, a computer database of the sequence information, etc.

Where the library is an electronic library, the nucleic acid sequence information can be present in a variety of media. "Media" refers to a manufacture, other than an isolated nucleic acid molecule, that contains the sequence information of the present invention. Such a manufacture provides the genome sequence or a subset thereof in a form that can be examined by means not directly applicable to the sequence as it exists in a nucleic acid. For example, the nucleotide sequence of the present invention, e.g. the nucleic acid sequences of any of the polynucleotides of SEQ ID NOS: 1-844, can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as a floppy disc, a hard disc storage medium, and a 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. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present sequence information. "Recorded" refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc. In addition to the sequence information, electronic versions of the libraries of the invention can be provided in conjunction or connection with other computer-readable information and/or other types of computer-readable files (e.g. , searchable files, executable files, etc, including, but not limited to, for example, search program software, etc.).

By providing the nucleotide sequence in computer readable form, the information can be accessed for a variety of purposes. Computer software to access sequence information is publicly available. For example, the BLAST (Altschul et al, supra.) and BLAZE (Brutlag et al. Comp. Chem. (1993) 17:203) search algorithms on a Sybase system can be used identify open reading frames (ORFs) within the genome that contain homology to ORFs from other organisms.

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 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 system are suitable for use in the present invention. The data storage means can comprise any manufacture comprising a recording of the present sequence information as described above, or a memory access means that can access such a manufacture.

"Search means" refers to one or more programs implemented on the computer-based system, to compare a target sequence or target structural motif with the stored sequence information. Search means are used to identify fragments or regions of the genome that match a particular target sequence or target motif. A variety of known algorithms are publicly known and commercially available, e.g. MacPattern (EMBL), BLASTN and BLASTX (NCBI). A "target sequence" can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids, preferably from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues.

A "target structural motif," or "target motif," refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration that is formed upon the folding of the target motif, or on consensus sequences of regulatory or active sites. There are a variety of target motifs known in the art. Protein target motifs include, but arc not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, hairpin structures, promoter sequences and other expression elements such as binding sites for transcription factors.

A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. One format for an output means ranks fragments of the genome possessing varying degrees of homology to a target sequence or target motif. Such presentation provides a skilled artisan with a ranking of sequences and identifies the degree of sequence similarity contained in the identified fragment.

A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify sequence fragments of the genome. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer based systems of the present invention.

As discussed above, the "library" of the invention also encompasses biochemical libraries of the polynucleotides of SEQ ID NOS:l-844, e.g., collections of nucleic acids representing the provided polynucleotides. The biochemical libraries can take a variety of forms, e.g., a solution of cDNAs, a pattern of probe nucleic acids stably associated with a surface of a solid support (i.e., an array) and the like. Of particular interest are nucleic acid arrays in which one or more of SEQ ID NOS: 1-844 is represented on the array. By array is meant a an article of manufacture that has at least a substrate with at least two distinct nucleic acid targets on one of its surfaces, where the number of distinct nucleic acids can be considerably higher, typically being at least 10 nt, usually at least 20 nt and often at least 25 nt. A variety of different array formats have been developed and are known to those of skill in the art, including those described in 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,472,672; 5,527,681; 5,529,756; 5,545,531 ; 5,554,501 ; 5,556,752; 5,561,071; 5,599,895; 5,624,711; 5,639,603; 5,658,734; WO 93/17126; WO 95/11995; WO 95/35505; EP 742287; and EP 799897. The arrays of the subject invention find use in a variety of applications, including gene expression analysis, drug screening, mutation analysis and the like, as disclosed in the above-listed exemplary patent documents . In addition to the above nucleic acid libraries, analogous libraries of polypeptides are also provided, where the where the polypeptides of the library will represent at least a portion of the polypeptides encoded by SEQ ID NOS: 1-844.

VII. Utilities

A. Use of Polynucleotide Probes in Mapping, and in Tissue Profiling Polynucleotide probes, generally comprising at least 12 contiguous nucleotides of a polynucleotide as shown in the Sequence Listing, are used for a variety of purposes, such as chromosome mapping of the polynucleotide and detection of transcription levels. Additional disclosure about preferred regions of the disclosed polynucleotide sequences is found in the Examples. A probe that hybridizes specifically to a polynucleotide disclosed herein should provide a detection signal at least 5-, 10-, or 20-fold higher than the background hybridization provided with other unrelated sequences.

Probes in Detection of Expression Levels. Nucleotide probes are used to detect expression of a gene corresponding to the provided polynucleotide. The references describe an example of a sandwich nucleotide hybridization assay. For example, in Northern blots, mRNA is separated electrophoretically and contacted with a probe. A probe is detected as hybridizing to an mRNA species of a particular size. The amount of hybridization is quantitated to determine relative amounts of expression, for example under a particular condition. Probes are also used to detect products of amplification by polymerase chain reaction. The products of the reaction are hybridized to the probe and hybrids are detected. Probes are used for in situ hybridization to cells to detect expression. Probes can also be used in vivo for diagnostic detection of hybridizing sequences. Probes are typically labeled with a radioactive isotope. Other types of detectable labels can be used such as chromophores, fluors, and enzymes. Other examples of nucleotide hybridization assays are described in WO92/02526 and U.S. Patent No. 5,124,246.

Alternatively, the Polymerase Chain Reaction (PCR) is another means for detecting small amounts of target nucleic acids (see, e.g., Mullis et al, Meth. Enzymol. (1987) 755:335; U.S. Patent No. 4,683,195; and U.S. Patent No. 4,683,202). Two primer polynucleotides nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can be composed of sequence within or 3' and 5' to the polynucleotides of the Sequence Listing. Alternatively, if the primers are 3' and 5' to these polynucleotides, they need not hybridize to them or the complements. A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a large amount of target nucleic acids is generated by the polymerase, it is detected by methods such as Southern blots. When using the Southern blot method, the labeled probe will hybridize to a polynucleotide of the Sequence Listing or complement.

Furthermore, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al, "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory, 1989). mRNA or cDNA generated from mRNA using a polymerase enzyme can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labeled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labeled with radioactivity. Mapping. Polynucleotides of the present invention are used to identify a chromosome on which the corresponding gene resides. Such mapping can be useful in identifying the function of the polynucleotide-related gene by its proximity to other genes with known function. Function can also be assigned to the polynucleotide-related gene when particular syndromes or diseases map to the same chromosome. For example, use of polynucleotide probes in identification and quantification of nucleic acid sequence aberrations is described in U.S. Patent No. 5,783,387.

For example, fluorescence in situ hybridization (FISH) on normal metaphase spreads facilitates comparative genomic hybridization to allow total genome assessment of changes in relative copy number of DNA sequences. See Schwartz and Samad, Curr. Opin. Biotechnol. (1994) 5:70; Kallioniemi et al, Sem. Cancer Biol. (1993) 4:41; Valdes et al, Methods in Molecular Biology (1997) 65:1, Boultwood, ed., Human Press, Totowa, NJ. Preparations of human metaphase chromosomes are prepared using standard cytogenetic techniques from human primary tissues or cell lines. Nucleotide probes comprising at least 12 contiguous nucleotides selected from the nucleotide sequence shown in the Sequence Listing are used to identify the corresponding chromosome. The nucleotide probes are labeled, for example, with a radioactive, fluorescent, biotinylated, or chemiluminescent label, and detected by well known methods appropriate for the particular label selected. Protocols for hybridizing nucleotide probes to preparations of metaphase chromosomes are also well known in the art. A nucleotide probe will hybridize specifically to nucleotide sequences in the chromosome preparations that are complementary to the nucleotide sequence of the probe.

Polynucleotides are mapped to particular chromosomes using, for example, radiation hybrids or chromosome-specific hybrid panels. See Leach et al, Advances in Genetics, (1995) 55:63-99; Walter et al, Nature Genetics (1994) 7:22; Walter and Goodfellow, Trends in Genetics (1992) 9:352. Panels for radiation hybrid mapping are available from Research Genetics, Inc., Huntsville, Alabama, USA. Databases for markers using various panels are available via the world wide web at http:/F/shgc-www.stanford.edu; and http://www- genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl. The statistical program RHMAP can be used to construct a map based on the data from radiation hybridization with a measure of the relative likelihood of one order versus another. RHMAP is available via the world wide web at http://www.sph.umich.edu group/statgen software.

In addition, commercial programs are available for identifying regions of chromosomes commonly associated with disease, such as cancer. Polynucleotides based on the polynucleotides of the invention can be used to probe these regions. For example, if through profile searching a provided polynucleotide is identified as corresponding to a gene encoding a kinase, its ability to bind to a cancer-related chromosomal region will suggest its role as a kinase in one or more stages of tumor cell development/growth. Although some experimentation would be required to elucidate the role, the polynucleotide constitutes a new material for isolating a specific protein that has potential for developing a cancer diagnostic or therapeutic. Tissue Typing or Profiling. Expression of specific mRNA corresponding to the provided polynucleotides can vary in different cell types and can be tissue-specific. This variation of mRNA levels in different cell types can be exploited with nucleic acid probe assays to determine tissue types. For example, PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes substantially identical or complementary to polynucleotides listed in the Sequence Listing can determine the presence or absence of the corresponding cDNA or mRNA. For example, a metastatic lesion is identified by its developmental organ or tissue source by identifying the expression of a particular marker of that organ or tissue. If a polynucleotide is expressed only in a specific tissue type, and a metastatic lesion is found to express that polynucleotide, then the developmental source of the lesion has been identified. Expression of a particular polynucleotide is assayed by detection of either the corresponding mRNA or the protein product. Immunological methods, such as antibody staining, are used to detect a particular protein product. Hybridization methods can be used to detect particular mRNA species, including but not limited to in situ hybridization and Northern blotting.

Use of Polymorphisms. A polynucleotide of the invention will be useful in forensics, genetic analysis, mapping, and diagnostic applications if the corresponding region of a gene is polymorphic in the human population. Particular polymorphic forms of the provided polynucleotides can be used to either identify a sample as deriving from a suspect or rule out the possibility that the sample derives from the suspect. Any means for detecting a polymorphism in a gene are used, including but not limited to electrophoresis of protein polymorphic variants, differential sensitivity to restriction enzyme cleavage, and hybridization to allele-specific probes. B. Antibody Production

Expression products of a polynucleotide of the invention, the corresponding mRNA or cDNA, or the corresponding complete gene are prepared and used for raising antibodies for experimental, diagnostic, and therapeutic purposes. For polynucleotides to which a corresponding gene has not been assigned, this provides an additional method of identifying the corresponding gene. The polynucleotide or related cDNA is expressed as described above, and antibodies are prepared. These antibodies are specific to an epitope on the polypeptide encoded by the polynucleotide, and can precipitate or bind to the corresponding native protein in a cell or tissue preparation or in a cell-free extract of an in vitro expression system.

Immunogens for raising antibodies are prepared by mixing the polypeptides encoded by the polynucleotides of the present invention with adjuvants. Alternatively, polypeptides are made as fusion proteins to larger immunogenic proteins. Polypeptides are also covalently linked to other larger immunogenic proteins, such as keyhole limpet hemocyanin. Immunogens are typically administered intradermally, subcutaneously, or intramuscularly. Immunogens are administered to experimental animals such as rabbits, sheep, and mice, to generate antibodies. Optionally, the animal spleen cells are isolated and fused with myeloma cells to form hybridomas which secrete monoclonal antibodies. Such methods are well known in the art. According to another method known in the art, the selected polynucleotide is administered directly, such as by intramuscular injection, and expressed in vivo. The expressed protein generates a variety of protein-specific immune responses, including production of antibodies, comparable to administration of the protein.

Preparations of polyclonal and monoclonal antibodies specific for polypeptides encoded by a selected polynucleotide are made using standard methods known in the art. The antibodies specifically bind to epitopes present in the polypeptides encoded by polynucleotides disclosed in the Sequence Listing. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, for example at least 15, 25, or 50 amino acids. A short sequence of a polynucleotide may then be unsuitable for use as an epitope to raise antibodies for identifying the corresponding novel protein, because of the potential for cross-reactivity with a known protein. However, the antibodies can be useful for other purposes, particularly if they identify common structural features of a known protein and a novel polypeptide encoded by a polynucleotide of the invention.

Antibodies that specifically bind to human polypeptides encoded by the provided polypeptides should provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in Western blots or other immunochemical assays. Preferably, antibodies that specifically polypeptides of the invention do not bind to other proteins in immunochemical assays at detectable levels and can immunoprecipitate the specific polypeptide from solution. To test for the presence of serum antibodies to the polypeptide of the invention in a human population, human antibodies are purified by methods well known in the art. Preferably, the antibodies are affinity purified by passing antiserum over a column to which the corresponding selected polypeptide or fusion protein is bound. The bound antibodies can then be eluted from the column, for example using a buffer with a high salt concentration. In addition to the antibodies discussed above, genetically engineered antibody derivatives are made, such as single chain antibodies, according to methods well known in the art.

C. Use of Polynucleotides to Construct Arrays for Diagnostics Polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotide sequences in a sample. This technology can be used as a diagnostic and as a tool to test for differential expression to determine function of an encoded protein. Arrays can be created by spotting polynucleotide probes onto a substrate (e.g., glass, nitrocelllose, etc.) in a two-dimensional matrix or array having bound probes. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. Samples of polynucleotides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled. sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Techniques for constructing arrays and methods of using these arrays are described in EP No. 0 799 897; PCT No. WO 97/29212; PCT No. WO 97/27317; EP No. 0 785 280; PCT No. WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP No. 0 728 520; U.S. Pat. No. 5,599,695; EP No. 0 721 016; U.S. Pat. No. 5,556,752; PCT No. WO 95/22058; and U.S. Pat. No. 5,631,734. As discussed in some detail above, arrays can be used to examine differential expression of genes and can be used to determine gene function. For example, arrays of the instant polynucleotide sequences can be used to determine if any of the provided polynucleotides are differentially expressed between a test cell and control cell (e.g., cancer cells and normal cells). For example, high expression of a particular message in a cancer cell, which is not observed in a corresponding normal cell, can indicate a cancer specific protein. Exemplary uses of arrays are further described in, for example, Pappalarado et al, Sem. Radiation Oncol. (1998) 5:217; and Ramsay Nature Biotechnol. (1998) 76:40.

D. Differential Expression

The polynucleotides of the invention can also be used to detect differences in expression levels between two cells, e.g. , as a method to identify abnormal or diseased tissue in a human. For polynucleotides corresponding to profiles of protein families as described above, the choice of tissue can be selected according to the putative biological function. In general, the expression of a gene corresponding to a specific polynucleotide is compared between a first tissue that is suspected of being diseased and a second, normal tissue of the human. The tissue suspected of being abnormal or diseased can be derived from a different tissue type of the human, but preferably it is derived from the same tissue type; for example an intestinal polyp or other abnormal growth should be compared with normal intestinal tissue. The normal tissue can be the same tissue as that of the test sample, or any normal tissue of the patient, especially those that express the polynucleotide-related gene of interest (e.g., brain, thymus, testis, heart, prostate, placenta, spleen, small intestine, skeletal muscle, pancreas, and the mucosal lining of the colon). A difference between the polynucleotide- related gene, mRNA, or protein in the two tissues which are compared, for example in molecular weight, amino acid or nucleotide sequence, or relative abundance, indicates a change in the gene, or a gene which regulates it, in the tissue of the human that was suspected of being diseased. Examples of detection of differential expression and its use in diagnosis of cancer are described in U.S. Patent Nos. 5,688,641 and 5,677,125.

The polynucleotide-related genes in the two tissues are compared by any means known in the art. For example, the two genes can be sequenced, and the sequence of the gene in the tissue suspected of being diseased compared with the gene sequence in the normal tissue. The genes corresponding to a provided polynucleotide, or portions thereof, in the two tissues are amplified, for example using nucleotide primers based on the nucleotide sequence shown in the Sequence Listing, using the polymerase chain reaction. The amplified genes or portions of genes are hybridized to detectably labeled nucleotide probes selected from a nucleotide sequence shown in the Sequence Listing. A difference in the nucleotide sequence of the isolated gene in the tissue suspected of being diseased compared with the normal nucleotide sequence suggests a role of the gene product encoded by the subject polynucleotide in the disease, and provides guidance for preparing a therapeutic agent.

Alternatively, mRNA corresponding to a provided polynucleotide in the two tissues is compared. PolyA⁺ RNA is isolated from the two tissues as is known in the art. For example, one of skill in the art can readily determine differences in the size or amount of mRNA transcripts between the two tissues using Northern blots and detectably labeled nucleotide probes selected from the nucleotide sequence shown in the Sequence Listing.

Increased or decreased expression of a given mRNA in a tissue sample suspected of being diseased, compared with the expression of the same mRNA in a normal tissue, suggests that the expressed protein has a role in the disease, and also provides a lead for preparing a therapeutic agent.

The comparison can also be accomplished by analyzing polypeptides between the matched samples. The sizes of the proteins in the two tissues are compared, for example, using antibodies of the present invention to detect polypeptides in Western blots of protein extracts from the two tissues. Other changes, such as expression levels and subcellular localization, can also be detected immunologically, using antibodies to the corresponding protein. A higher or lower level of expression of a given polypeptide in a tissue suspected of being diseased, compared with the same protein expression level in a normal tissue, is indicative that the expressed protein has a role in the disease, and provides guidance for preparing a therapeutic agent. Similarly, comparison of polynucleotide sequences or of gene expression products, e.g., mRNA and protein, between a human tissue that is suspected of being diseased and a normal tissue of a human, are used to follow disease progression or remission in the human. Such comparisons are made as described above. For example, increased or decreased expression of a gene corresponding to an inventive polynucleotide in the tissue suspected of being neoplastic can indicate the presence of neoplastic cells in the tissue. The degree of increased expression of a given gene in the neoplastic tissue relative to expression of the same gene in normal tissue, or differences in the amount of increased expression of a given gene in the neoplastic tissue over time, is used to assess the progression of the neoplasia in that tissue or to monitor the response of the neoplastic tissue to a therapeutic protocol over time.

The expression pattern of any two cell types can be compared, such as low and high metastatic tumor cell lines, malignant or non-malignant cells, or cells from tissue which have and have not been exposed to a therapeutic agent. A genetic predisposition to disease in a human is detected by comparing expression levels of an mRNA or protein corresponding to a polynucleotide of the invention in a fetal tissue with levels associated in normal fetal tissue. Fetal tissues that are used for this purpose include, but are not limited to, amniotic fluid, chorionic villi, blood, and the blastomere of an in vitro-fertilized embryo. The comparable normal polynucleotide-related gene is obtained from any tissue. The mRNA or protein is obtained from a normal tissue of a human in which the polynucleotide-related gene is expressed. Differences such as alterations in the nucleotide sequence or size of the same product of the fetal polynucleotide-related gene or mRNA, or alterations in the molecular weight, amino acid sequence, or relative abundance of fetal protein, can indicate a germline mutation in the polynucleotide-related gene of the fetus, which indicates a genetic predisposition to disease. Particular diagnostic and prognostic uses of the disclosed polynucleotides are described in more detail below. E. Diagnostic. Prognostic, and Other Uses Based On Differential Expression

In general, diagnostic methods of the invention for involve detection of a level or amount of a gene product, particularly a differentially expressed gene product, in a test sample obtained from a patient suspected of having or being susceptible to a disease (e.g., breast cancer, lung cancer, colon cancer and/or metastatic forms thereof), and comparing the detected levels to those levels found in normal cells (e.g., cells substantially unaffected by cancer) and/or other control cells (e.g., to differentiate a cancerous cell from a cell affected by dysplasia). Furthermore, the severity of the disease can be assessed by comparing the detected levels of a differentially expressed gene product with those levels detected in samples representing the levels of differentially gene product associated with varying degrees of severity of disease.

The term "differentially expressed gene" is intended to encompass a polynucleotide that can, for example, include an open reading frame encoding a gene product (e.g., a polypeptide), and/or introns of such genes and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 20 kb beyond the coding region, but possibly further in either direction. The gene can be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome. In general, a difference in expression level associated with a decrease in expression level of at least about 25%, usually at least about 50% to 75%, more usually at least about 90% or more is indicative of a differentially expressed gene of interest, /. e. , a gene that is underexpressed or down-regulated in the test sample relative to a control sample.

Furthermore, a difference in expression level associated with an increase in expression of at least about 25%, usually at least about 50% to 75%, more usually at least about 90% and can be at least about 1 '/--fold, usually at least about 2-fold to about 10-fold, and can be about 100-fold to about 1, 000-fold increase relative to a control sample is indicative of a differentially expressed gene of interest, i.e., an overexpressed or up-regulated gene. "Differentially expressed polynucleotide" as used herein means a nucleic acid molecule (RNA or DNA) having a sequence that represents a differentially expressed gene, e.g., the differentially expressed polynucleotide comprises a sequence (e.g., an open reading frame encoding a gene product) that uniquely identifies a differentially expressed gene so that detection of the differentially expressed polynucleotide in a sample is correlated with the presence of a differentially expressed gene in a sample. "Differentially expressed polynucleotides" is also meant to encompass fragments of the disclosed polynucleotides, e.g., fragments retaining biological activity, as well as nucleic acids homologous, substantially similar, or substantially identical (e.g., having about 90% sequence identity) to the disclosed polynucleotides. Methods of the subject invention useful in diagnosis or prognosis typically involve comparison of the abundance of a selected differentially expressed gene product in a sample of interest with that of a control to determine any relative differences in the expression of the gene product, where the difference can be measured qualitatively and/or quantitatively. Quantitation can be accomplished, for example, by comparing the level of expression product detected in the sample with the amounts of product present in a standard curve. A comparison can be made visually; by using a technique such as densitometry, with or without computerized assistance; by preparing a representative library of cDNA clones of mRNA isolated from a test sample, sequencing the clones in the library to determine that number of cDNA clones corresponding to the same gene product, and analyzing the number of clones corresponding to that same gene product relative to the number of clones of the same gene product in a control sample; or by using an array to detect relative levels of hybridization to a selected sequence or set of sequences, and comparing the hybridization pattern to that of a control. The differences in expression are then correlated with the presence or absence of an abnormal expression pattern. A variety of different methods for determining the nucleic acid abundance in a sample are known to those of skill in the art, where particular methods of interest include those described in: Pietu et al. Genome Res. (1996) 6:492; Zhao et al, Gene (1995) 756:207; Soares , Curr. Opin. Biotechnol. (1977) 5:

542; Raval, J. Pharmacol Toxicol Methods (1994) 52:125; Chalifour et al, Anal. Biochem (1994) 276:299; Stolz et al, Mol. Biotechnol. (1996) 6:225; Hong et al, Biosci. Reports (1982) 2:907; and McGraw, Anal. Biochem. (1984) 745:298. Also of interest are the methods disclosed in WO 97/27317, the disclosure of which is herein incorporated by reference.

In general, diagnostic assays of the invention involve detection of a gene product of a the polynucleotide sequence (e.g., mRNA or polypeptide) that corresponds to a sequence of SEQ ID NOS: 1-844. The patient from whom the sample is obtained can be apparently healthy, susceptible to disease (e.g., as determined by family history or exposure to certain environmental factors), or can already be identified as having a condition in which altered expression of a gene product of the invention is implicated.

In the assays of the invention, the diagnosis can be determined based on detected gene product expression levels of a gene product encoded by at least one, preferably at least two or more, at least 3 or more, or at least 4 or more of the polynucleotides having a sequence set forth in SEQ ID NOS: 1-844, and can involve detection of expression of genes corresponding to all of SEQ ID NOS: 1-844 and/or additional sequences that can serve as additional diagnostic markers and/or reference sequences. Where the diagnostic method is designed to detect the presence or susceptibility of a patient to cancer, the assay preferably involves detection of a gene product encoded by a gene corresponding to a polynucleotide that is differentially expressed in cancer. For example, a higher level of expression of a polynucleotide corresponding to SEQ ID NO:52 relative to a level associated with a normal sample can indicate the presence of cancer in the patient from whom the sample is derived. In another example, detection of a lower level of a polynucleotide corresponding to SEQ ID NO:39 relative to a normal level is indicative of the presence of cancer in the patient. Further examples of such differentially expressed polynucleotides are described in the Examples below. Given the provided polynucleotides and information regarding their relative expression levels provided herein, assays using such polynucleotides and detection of their expression levels in diagnosis and prognosis will be readily apparent to the ordinarily skilled artisan. Any of a variety of detectable labels can be used in connection with the various embodiments of the diagnostic methods of the invention. Suitable detectable labels include fluorochromes,(e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'- dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-

2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'- tetramethyl-6-carboxyrhodamine (TAMRA)), radioactive labels, (e.g. ³²P, ³³S, ³H, etc.), and the like. The detectable label can involve a two stage systems (e.g., biotin-avidin, hapten- anti-hapten antibody, etc.) Reagents specific for the polynucleotides and polypeptides of the invention, such as antibodies and nucleotide probes, can be supplied in a kit for detecting the presence of an expression product in a biological sample. The kit can also contain buffers or labeling components, as well as instructions for using the reagents to detect and quantify expression products in the biological sample. Exemplary embodiments of the diagnostic methods of the invention are described below in more detail.

Polypeptide detection in diagnosis. In one embodiment, the test sample is assayed for the level of a differentially expressed polypeptide. Diagnosis can be accomplished using any of a number of methods to determine the absence or presence or altered amounts of the differentially expressed polypeptide in the test sample. For example, detection can utilize staining of cells or histological sections with labeled antibodies, performed in accordance with conventional methods. Cells can be permeabilized to stain cytoplasmic molecules. In general, antibodies that specifically bind a differentially expressed polypeptide of the invention are added to a sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody can be detectably labeled for direct detection (e.g., using radioisotopes, enzymes, fluorescers, chemiluminescers, and the like), or can be used in conjunction with a second stage antibody or reagent to detect binding (e.g., biotin with horseradish peroxidase-conjugated avidin, a secondary antibody conjugated to a fluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc.). The absence or presence of antibody binding can be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc. Any suitable alternative methods can of qualitative or quantitative detection of levels or amounts of differentially expressed polypeptide can be used, for example ELISA, western blot, immunoprecipitation, radioimmunoassay, etc.

In general, the detected level of differentially expressed polypeptide in the test sample is compared to a level of the differentially expressed gene product in a reference or control sample, e.g., in a normal cell (negative control) or in a cell having a known disease state (positive control). For example, a higher level of expression of a polypeptide encoded by SEQ ID NO:52 relative to a level associated with a normal sample can indicate the presence of cancer in the patient from whom the sample is derived. In another example, detection of a lower level of the polypeptide encoded by SEQ ID NO:39 relative to a normal level is indicative of the presence of cancer in the patient. mRNA detection. The diagnostic methods of the invention can also or alternatively involve detection of mRNA encoded by a gene corresponding to a differentially expressed polynucleotides of the invention. Any suitable qualitative or quantitative methods known in the art for detecting specific mRNAs can be used. mRNA can be detected by, for example, in situ hybridization in tissue sections, by reverse transcriptase-PCR, or in Northern blots containing poly A+ mRNA. One of skill in the art can readily use these methods to determine differences in the size or amount of mRNA transcripts between two samples. For example, the level of mRNA of the invention in a tissue sample suspected of being cancerous or dysplastic is compared with the expression of the mRNA in a reference sample, e.g., a positive or negative control sample (e.g., normal tissue, cancerous tissue, etc.). In a specific non-limiting example, a higher level of mRNA corresponding to SEQ ID NO:52 relative to a level associated with a normal sample can indicate the presence of cancer in the patient from whom the sample is derived. In another example, detection of a lower level of mRNA corresponding to SEQ ID NO:39 relative to a normal level is indicative of the presence of cancer in the patient.

Any suitable method for detecting and comparing mRNA expression levels in a sample can be used in connection with the diagnostic methods of the invention (see, e.g., U.S. 5,804,382). For example, mRNA expression levels in a sample can be determined by generation of a library of expressed sequence tags (ESTs) from the sample, where the EST library is representative of sequences present in the sample (Adams, et al., (1991) Science

252:1651). Enumeration of the relative representation of ESTs within the library can be used to approximate the relative representation of the gene transcript within the starting sample.

The results of EST analysis of a test sample can then be compared to EST analysis of a reference sample to determine the relative expression levels of a selected polynucleotide, particularly a polynucleotide corresponding to one or more of the differentially expressed genes described herein.

Alternatively, gene expression in a test sample can be performed using serial analysis of gene expression (SAGE) methodology (Velculescu et al., Science (1995) 270:484). In short, SAGE involves the isolation of short unique sequence tags from a specific location within each transcript (e.g., a sequence of any one of SEQ ID NOS:l-6). The sequence tags are concatenated, cloned, and sequenced. The frequency of particular transcripts within the starting sample is reflected by the number of times the associated sequence tag is encountered with the sequence population.

Gene expression in a test sample can also be analyzed using differential display (DD) methodology. In DD, fragments defined by specific sequence delimiters (e.g., restriction enzyme sites) are used as unique identifiers of genes, coupled with information about fragment length or fragment location within the expressed gene. The relative representation of an expressed gene with a sample can then be estimated based on the relative representation of the fragment associated with that gene within the pool of all possible fragments. Methods and compositions for carrying out DD are well known in the art, see, e.g., U.S. 5,776,683; and U.S. 5,807,680.

Alternatively, gene expression in a sample using hybridization analysis, which is based on the specificity of nucleotide interactions. Oligonucleotides or cDNA can be used to selectively identify or capture DNA or RNA of specific sequence composition, and the amount of RNA or cDNA hybridized to a known capture sequence determined qualitatively or quantitatively, to provide information about the relative representation of a particular message within the pool of cellular messages in a sample. Hybridization analysis can be designed to allow for concurrent screening of the relative expression of hundreds to thousands of genes by using, for example, array-based technologies having high density formats, including filters, microscope slides, or microchips, or solution-based technologies that use spectroscopic analysis (e.g., mass spectrometry). One exemplary use of arrays in the diagnostic methods of the invention is described below in more detail. Use of a single gene in diagnostic applications. The diagnostic methods of the invention can focus on the expression of a single differentially expressed gene. For example, the diagnostic method can involve detecting a differentially expressed gene, or a polymorphism of such a gene (e.g., a polymorphism in an coding region or control region), that is associated with disease. Disease-associated polymorphisms can include deletion or truncation of the gene, mutations that alter expression level and/or affect activity of the encoded protein, etc.

Changes in the promoter or enhancer sequence that affect expression levels of an differentially gene can be compared to expression levels of the normal allele by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as β-galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that provides for convenient quantitation; and the like.

A number of methods are available for analyzing nucleic acids for the presence of a specific sequence, e.g. a disease associated polymorphism. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. Cells that express a differentially expressed gene can be used as a source of mRNA, which can be assayed directly or reverse transcribed into cDNA for analysis. The nucleic acid can be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis, and a detectable label can be included in the amplification reaction (e.g., using a detectably labeled primer or detectably labeled oligonucleotides) to facilitate detection. The use of the polymerase chain reaction is described in Saiki, et αl, Science (1985) 259:487, and a review of techniques can be found in Sambrook, et αl., Molecular Cloning: A Laboratory Manual, (1989) pp. 14.2. Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al, Nucl. Acids Res. (1990) 75:2887; and Delahunty et al, Am. J. Hum. Genet. (1996) 55:1239.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by one of a number of methods known in the art. The nucleic acid can be sequenced by dideoxy or other methods, and the sequence of bases compared to a selected sequence, e.g., to a wild-type sequence. Hybridization with the polymorphic or variant sequence can also be used to determine its presence in a sample (e.g. , by Southern blot, dot blot, etc.). The hybridization pattern of a polymorphic or variant sequence and a control sequence to an array of oligonucleotide probes immobilized on a solid support, as described in US 5,445,934, or in WO 95/35505, can also be used as a means of identifying polymorphic or variant sequences associated with disease. Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease, the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.

Screening for mutations in an differentially expressed gene can be based on the functional or antigenic characteristics of the protein. Protein truncation assays are useful in detecting deletions that can affect the biological activity of the protein. Various immunoassays designed to detect polymorphisms in proteins can be used in screening. Where many diverse genetic mutations lead to a particular disease phenotype, functional protein assays have proven to be effective screening tools. The activity of the encoded protein can be determined by comparison with the wild-type protein.

Pattern matching in diagnosis using arrays. In another embodiment, the diagnostic and/or prognostic methods of the invention involve detection of expression of a selected set of genes in a test sample to produce a test expression pattern (TEP). The TEP is compared to a reference expression pattern (REP), which is generated by detection of expression of the selected set of genes in a reference sample (e.g., a positive or negative control sample). The selected set of genes includes at least one of the genes of the invention, which genes correspond to the polynucleotide sequences of SEQ ID NOS: 1-844. Of particular interest is a selected set of genes that includes gene differentially expressed in the disease for which the test sample is to be screened. "Reference sequences" or "reference polynucleotides" as used herein in the context of differential gene expression analysis and diagnosis/prognosis refers to a selected set of polynucleotides, which selected set includes at least one or more of the differentially expressed polynucleotides described herein. A plurality of reference sequences, preferably comprising positive and negative control sequences, can be included as reference sequences. Additional suitable reference sequences are found in Genbank, Unigene, and other nucleotide sequence databases (including, e.g., expressed sequence tag (EST), partial, and full-length sequences).

"Reference array" means an array having reference sequences for use in hybridization with a sample, where the reference sequences include all, at least one of, or any subset of the differentially expressed polynucleotides described herein. Usually such an array will include at least 3 different reference sequences, and can include any one or all of the provided differentially expressed sequences. Arrays of interest can further comprise sequences, including polymorphisms, of other genetic sequences, particularly other sequences of interest for screening for a disease or disorder (e.g., cancer, dysplasia, or other related or unrelated diseases, disorders, or conditions). The oligonucleotide sequence on the array will usually be at least about 12 nt in length, and can be of about the length of the provided sequences, or can extend into the flanking regions to generate fragments of 100 nt to 200 nt in length or more.

A "reference expression pattern" or "REP" as used herein refers to the relative levels of expression of a selected set of genes, particularly of differentially expressed genes, that is associated with a selected cell type, e.g., a normal cell, a cancerous cell, a cell exposed to an environmental stimulus, and the like. A "test expression pattern" or "TEP" refers to relative levels of expression of a selected set of genes, particularly of differentially expressed genes, in a test sample (e.g., a cell of unknown or suspected disease state, from which mRNA is isolated). "Diagnosis" as used herein generally includes determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, as well as to the prognosis of a subject affected by a disease or disorder (e.g., identification of pre-metastatic or metastatic cancerous states, stages of cancer, or responsiveness of cancer to therapy). The present invention particularly encompasses diagnosis of subjects in the context of breast cancer (e.g. , carcinoma in situ (e.g. , ductal carcinoma in situ), estrogen receptor (ER)-positive breast cancer, ER-negative breast cancer, or other forms and/or stages of breast cancer), lung cancer (e.g., small cell carcinoma, non-small cell carcinoma, mesothelioma, and other forms and/or stages of lung cancer), and colon cancer (e.g., adenomatous polyp, colorectal carcinoma, and other forms and/or stages of colon cancer). "Sample" or "biological sample" as used throughout here are generally meant to refer to samples of biological fluids or tissues, particularly samples obtained from tissues, especially from cells of the type associated with the disease for which the diagnostic application is designed (e.g., ductal adenocarcinoma), and the like. "Samples" is also meant to encompass derivatives and fractions of such samples (e.g., cell lysates). Where the sample is solid tissue, the cells of the tissue can be dissociated or tissue sections can be analyzed. REPs can be generated in a variety of ways according to methods well known in the art. For example, REPs can be generated by hybridizing a control sample to an array having a selected set of polynucleotides (particularly a selected set of differentially expressed polynucleotides), acquiring the hybridization data from the array, and storing the data in a format that allows for ready comparison of the REP with a TEP. Alternatively, all expressed sequences in a control sample can be isolated and sequenced, e.g., by isolating mRNA from a control sample, converting the mRNA into cDNA, and sequencing the cDNA. The resulting sequence information roughly or precisely reflects the identity and relative number of expressed sequences in the sample. The sequence information can then be stored in a format (e.g., a computer-readable format) that allows for ready comparison of the REP with a TEP. The REP can be normalized prior to or after data storage, and/or can be processed to selectively remove sequences of expressed genes that are of less interest or that might complicate analysis (e.g., some or all of the sequences associated with housekeeping genes can be eliminated from REP data). TEPs can be generated in a manner similar to REPs, e.g. , by hybridizing a test sample to an array having a selected set of polynucleotides, particularly a selected set of differentially expressed polynucleotides, acquiring the hybridization data from the array, and storing the data in a format that allows for ready comparison of the TEP with a REP. The REP and TEP to be used in a comparison can be generated simultaneously, or the TEP can be compared to previously generated and stored REPs. In one embodiment of the invention, comparison of a TEP with a REP involves hybridizing a test sample with a reference array, where the reference array has one or more reference sequences for use in hybridization with a sample. The reference sequences include all, at least one of, or any subset of the differentially expressed polynucleotides described herein. Hybridization data for the test sample is acquired, the data normalized, and the produced TEP compared with a REP generated using an array having the same or similar selected set of differentially expressed polynucleotides. Probes that correspond to sequences differentially expressed between the two samples will show decreased or increased hybridization efficiency for one of the samples relative to the other. Reference arrays can be produced according to any suitable methods known in the art. For example, methods of producing large arrays of oligonucleotides are described in U.S. 5,134,854, and U.S. 5,445,934 using light-directed synthesis techniques. Using a computer controlled system, a heterogeneous array of monomers is converted, through simultaneous coupling at a number of reaction sites, into a heterogeneous array of polymers. Alternatively, microarrays are generated by deposition of pre-synthesized oligonucleotides onto a solid substrate, for example as described in PCT published application no. WO 95/35505.

Methods for collection of data from hybridization of samples with a reference arrays are also well known in the art. For example, the polynucleotides of the reference and test samples can be generated using a detectable fluorescent label, and hybridization of the polynucleotides in the samples detected by scanning the microarrays for the presence of the detectable label. Methods and devices for detecting fluorescently marked targets on devices are known in the art. Generally, such detection devices include a microscope and light source for directing light at a substrate. A photon counter detects fluorescence from the substrate, while an x-y translation stage varies the location of the substrate. A confocal detection device that can be used in the subject methods is described in U.S. Patent no. 5,631,734. A scanning laser microscope is described in Shalon et al., Genome Res. (1996) 6:639. A scan, using the appropriate excitation line, is performed for each fluorophore used. The digital images generated from the scan are then combined for subsequent analysis. For any particular array element, the ratio of the fluorescent signal from one sample (e.g., a test sample) is compared to the fluorescent signal from another sample (e.g., a reference sample), and the relative signal intensity determined.

Methods for analyzing the data collected from hybridization to arrays are well known in the art. For example, where detection of hybridization involves a fluorescent label, data analysis can include the steps of determining fluorescent intensity as a function of substrate position from the data collected, removing outliers, i.e. data deviating from a predetermined statistical distribution, and calculating the relative binding affinity of the targets from the remaining data. The resulting data can be displayed as an image with the intensity in each region varying according to the binding affinity between targets and probes. In general, the test sample is classified as having a gene expression profile corresponding to that associated with a disease or non-disease state by comparing the TEP generated from the test sample to one or more REPs generated from reference samples (e.g., from samples associated with cancer or specific stages of cancer, dysplasia, samples affected by a disease other than cancer, normal samples, etc.). The criteria for a match or a substantial match between a TEP and a REP include expression of the same or substantially the same set of reference genes, as well as expression of these reference genes at substantially the same levels (e.g., no significant difference between the samples for a signal associated with a selected reference sequence after normalization of the samples, or at least no greater than about 25% to about 40% difference in signal strength for a given reference sequence. In general, a pattern match between a TEP and a REP includes a match in expression, preferably a match in qualitative or quantitative expression level, of at least one of, all or any subset of the differentially expressed genes of the invention.

Pattern matching can be performed manually, or can be performed using a computer program. Methods for preparation of substrate matrices (e.g., arrays), design of oligonucleotides for use with such matrices, labeling of probes, hybridization conditions, scanning of hybridized matrices, and analysis of patterns generated, including comparison analysis, are described in, for example, U.S. 5,800,992.

F. Use of the Polynucleotides of the Invention in Cancer Oncogenesis involves the unbridled growth, dedifferentiation and abnormal migration of cells. Cancerous cells can have the ability to compress, invade, and destroy normal tissue. Cancerous cells may also metastasize to other parts of the body via the bloodstream or the lymph system and colonize in these other areas. Different cancers are classified by the cell from which the cancerous cell is derived and from its cellular morphology and/or state of differentiation.

Somatic genetic abnormalities cause cancer initiation and progression. Cancer generally is clonally formed, i.e. gain of function of oncogenes and loss of function of tumor suppressor genes within a single cell transform the cell to be cancerous, and that single cell grows and divides to form a cancerous lesion. The genes known to be involved in cancer initiation and progression are involved in numerous cellular functions, including developmental differentiation, cell cycle regulation, cell signaling, immunological response, DNA replication, and DNA repair.

The identification and characterization of genetic or biochemical markers in blood or tissues that will detect the earliest changes along the carcinogenesis pathway and monitor the efficacy of various therapies and preventive interventions is a major goal of cancer research. Scientists have identified genetic changes in stool specimens that indicate the stages of colon cancer, and other biomarkers such as gene mutations, hormone receptors, proteins that inhibit metastasis, and enzymes that metabolize drugs are all being used to determine the severity and predict the course of breast, prostate, lung, and other cancers.

Recent advances in the pathogenesis of certain cancers has been helpful in determining patient treatment. The level of expression of certain polynucleotides can be indicative of a poorer prognosis, and therefore warrant more aggressive chemo- or radiotherapy for a patient. The correlation of novel surrogate tumor specific features with response to treatment and outcome in patients has defined certain prognostic indicators that allow the design of tailored therapy based on the molecular profile of the tumor. These therapies include antibody targeting and gene therapy. Moreover, a promising level of one or more marker polynucleotides can provide impetus for not aggressively treating a particular patient, thus sparing the patient the deleterious side effects of aggressive therapy. Determining expression of certain polynucleotides and comparison of a patients profile with known expression in normal tissue and variants of the disease allows a determination of the best possible treatment for a patient, both in terms of specificity of treatment and in terms of comfort level of the patient. Surrogate tumor markers, such as polynucleotide expression, can also be used to better classify, and thus diagnose and treat, different forms and disease states of cancer.

Two classifications widely used in oncology that can benefit from identification of the expression levels of the polynucleotides of the invention are staging of the cancerous disorder, and grading the nature of the cancerous tissue.

Staging. Staging is a process used by physicians to describe how advanced the cancerous state is in a patient. Staging assists the physician in determining a prognosis, planning treatment and evaluating the results of such treatment. Different staging systems are used for different types of cancer, but each generally involves the following determinations: the type of tumor, indicated by T; whether the cancer has metastasized to nearby lymph nodes, indicated by N; and whether the cancer has metastasized to more distant parts of the body, indicated by M. This system of staging is called the TNM system. Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II. In Stage III, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or another site, are called Stage IV, the most advanced stage.

Currently, the determination of staging is done using pathological techniques and is based more on the presence or absence of malignant tissue rather than the characteristics of the tumor type. Presence or absence of malignant tissue is based primarily on the gross morphology of the cells in the areas biopsied. The polynucleotides of the invention can facilitate fine-tuning of the staging process by identifying markers for the aggresivity of a cancer, e.g. the metastatic potential, as well as the presence in different areas of the body. Thus, a Stage II cancer with a polynucleotide signifying a high metastatic potential cancer can be used to change a borderline Stage II tumor to a Stage III tumor, justifying more aggressive therapy. Conversely, the presence of a polynucleotide signifying a lower metastatic potential allows more conservative staging of a tumor.

Grading of cancers. Grade is a term used to describe how closely a tumor resembles normal tissue of its same type. Based on the microscopic appearance of a tumor, pathologists will identify the grade of a tumor based on parameters such as cell morphology, cellular organization, and other markers of differentiation. As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness. < That is, undifferentiated or high- grade tumors grow more quickly than well differentiated or low-grade tumors. Information about tumor grade is useful in planning treatment and predicting prognosis. The American Joint Commission on Cancer has recommended the following guidelines for grading tumors: 1) GX Grade cannot be assessed; 2) GI Well differentiated; G2 Moderately well differentiated; 3) G3 Poorly differentiated; 4) G4 Undifferentiated. Although grading is used by pathologists to describe most cancers, it plays a more important role in treatment planning for certain types than for others. An example is the Gleason system that is specific for prostate cancer, which uses grade numbers to describe the degree of differentiation. Lower Gleason scores indicate well-differentiated cells. Intermediate scores denote tumors with moderately differentiated cells. Higher scores describe poorly differentiated cells. Grade is also important in some types of brain tumors and soft tissue sarcomas. The polynucleotides of the invention can be especially valuable in determining the grade of the tumor, as they not only can aid in determining the differentiation status of the cells of a tumor, they can also identify factors other than differentiation that are valuable in determining the aggressivity of a tumor, such as metastatic potential.

Familial Cancer Genes. A number of cancer syndromes are linked to Mendelian inheritance of a predisposition to develop particular cancers. The following table contains a list of cancer types that can be inherited, and for which the gene or genes responsible have been identified. Most of the cancer types listed can occur as part of several different genetic conditions, each caused by alterations in a different gene.

Cancer Type Genetic Condition Gene

Brain Li-Fraumeni syndrome TP53

Neurofibromatosis 1 NFI

Neurofibromatosis 2 NF2 von Hippel-Lindau syndrome VHL

Tuberous sclerosis 2 TSC2

Breast Hereditary breast/ovarian cancer 1 BRCA1

Hereditary breast/ovarian cancer 2 BRCA2

Li-Fraumeni syndrome TP53

Ataxia telangiectasia ATM

Colon Familial adenomatous polyposis (FAP) APC

Hereditary non-polyposis colon cancer (HNPCC) 1 HMSH2

Hereditary non-polyposis colon cancer (HNPCC) 2 hMLHl Cancer Type Genetic Condition Gene

Hereditary non-polyposis colon cancer (HNPCC) 3 hPMSl Hereditary non-polyposis colon cancer (HNPCC) 4 hPMS2

Endocrine Multiple endocrine neoplasia 1 (MEN1) MEN1

(parathyroid, pituitary, GI endocrine)

Endocrine Multiple endocrine neoplasia 2 (MEN2) RET

(pheochromacytoma, medullary thyroid)

Endometrial Hereditary non-polyposis colon cancer (HNPCC) 1 hMSH2 Hereditary non-polyposis colon cancer (HNPCC) 2 hMLHl Hereditary non-polyposis colon cancer (HNPCC) 3 hPMSl Hereditary non-polyposis colon cancer (HNPCC) 4 hPMS2

Eye Hereditary retinoblastoma RBI

Hematologic Li-Fraumeni syndrome TP53 (lymphomas and leukemia) Ataxia telangiectasia ATM

Kidney Hereditary Wilms' tumor WT1 von Hippel-Lindau syndrome VHL Tuberous sclerosis 2 TSC2

Ovary Hereditary breast/ovarian cancer 1 BRCA1 Hereditary breast/ovarian cancer 2 BRCA2

Sarcoma Hereditary retinoblastoma RBI Li-Fraumeni syndrome TP53 Neurofibromatosis 1 NFI

Skin Hereditary melanoma 1 CDKN2 Hereditary melanoma 2 CDK4 Basal cell naevus (Gorlin) syndrome PTCH

Stomach Hereditary non-polyposis colon cancer (HNPCC) 1 hMSH2 Hereditary non-polyposis colon cancer (HNPCC) 2 hMLHl Hereditary non-polyposis colon cancer (HNPCC) 3 hPMSl Hereditary non-polyposis colon cancer (HNPCC) 4 hPMS2

The polynucleotides of the invention can be especially useful to monitor patients having any of the above syndromes to detect potentially malignant events at a molecular level before they are detectable at a gross morphological level. As can be seen from the table, a number of genes are involved in multiple forms of cancer. Thus, a polynucleotide of the invention identified as important for metastatic colon cancer can also have clinical implications for a patient diagnosed with stomach cancer or endometrial cancer.

Lung Cancer. Lung cancer is one of the most common cancers in the United States, accounting for about 15 percent of all cancer cases, or 170,000 new cases each year. At this time, over half of the lung cancer cases in the United States are in men, but the number found in women is increasing and will soon equal that in men. Today more women die of lung cancer than of breast cancer. Lung cancer is especially difficult to diagnose and treat because of the large size of the lungs, which allows cancer to develop for years undetected. In fact, lung cancer can spread outside the lungs without causing any symptoms. Adding to the confusion, the most common symptom of lung cancer, a persistent cough, can often be mistaken for a cold or bronchitis.

Although there are more than a dozen different kinds of lung cancer, the two main types of lung cancer are small cell and nonsmall cell, which encompass about 90% of all lung cancer cases. Small cell carcinoma (also called oat cell carcinoma), which usually starts in one of the larger bronchial tubes, grows fairly rapidly, and is likely to be large by the time of diagnosis. Nonsmall cell lung cancer (NSCLC) is made up of three general subtypes of lung cancer. Epidermoid carcinoma (also called squamous cell carcinoma) usually starts in one of the larger bronchial tubes and grows relatively slowly. The size of these tumors can range from very small to quite large. Adenocarcinoma starts growing near the outside surface of the lung and can vary in both size and growth rate. Some slowly growing adenocarcinomas are described as alveolar cell cancer. Large cell carcinoma starts near the surface of the lung, grows rapidly, and the growth is usually fairly large when diagnosed. Other less common forms of lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignant mesothelioma.

Currently, CT scans, MRIs, X-rays, sputum cytology, and biopsies are used to diagnose nonsmall cell lung cancer. The form and cellular origin of the lung cancer is diagnosed primarily through biopsy from either a surgical biopsy or a needle aspiration of lung tissue, and usually the biopsy is prompted from an abnormality identified on an X-ray. In some cases, sputum cytology can reveal lung cancers in patients with normal X-rays or can determine the type of lung cancer, but because it cannot pinpoint the tumor's location, a positive sputum cytology test is usually followed by further tests. Since these tests are based in large part on gross morphology of the tissue, the diagnosis of a particular kind of tumor is largely subjective, and the diagnosis can vary significantly between clinicians.

The polynucleotides of the invention can be used to distinguish types of lung cancer as well as identifying traits specific to a certain patient's cancer. For example, if the patient's biopsy expresses a polynucleotide that is associated with a low metastatic potential, it may justify leaving a larger portion of the patient's lung in surgery to remove the lesion. Alternatively, a smaller lesion with expression of a polynucleotide that is associated with high metastatic potential may justify a more radical removal of lung tissue and/or the surrounding lymph nodes, even if no metastasis can be identified through pathological examination.

Similarly, the expression of polynucleotides of the invention can be used in the diagnosis, prognosis and management of colorectal cancer. The differential expression of a polynucleotide in hyperplasia can be used as a diagnostic marker for metastatic lung cancer. The polynucleotides of the invention that would be especially useful for this purpose are those that exhibit differential expression between high metastatic versus low metastatic lung cancer , i.e. SEQ ID NOS: 9, 34, 42, 62, 74, 106, 119, 135, 154, 160, 260, 308, 323, 349, 361, 369, 371, 381, 395, and 400. Detection of malignant lung cancer with a higher metastatic potential can be determined using expression levels of any of these sequences alone or in combination with the levels of expression of other known genes.

Breast Cancer. The National Cancer Institute (NCI) estimates that about 1 in 8 women in the United States will develop breast cancer during her lifetime. Clinical breast examination and mammography are recommended as combined modalities for breast cancer screening, and the nature of the cancer will often depend upon the location of the tumor and the cell type from which the tumor is derived. The majority of breast cancers are adenocarcinomas subtypes, which can be summarized as follows:

Ductal carcinoma in situ (DCIS): Ductal carcinoma in situ is the most common type of noninvasive breast cancer. In DCIS, the malignant cells have not metastasized through the walls of the ducts into the fatty tissue of the breast. Comedocarcinoma is a type of DCIS that is more likely than other types of DCIS to come back in the same area after lumpectomy. It is more closely linked to eventual development of invasive ductal carcinoma than other forms of DCIS.

Infiltrating (or invasive) ductal carcinoma (IDC): this type of cancer has metastasized through the wall of the duct and invaded the fatty tissue of the breast. At this point, it has the potential to use the lymphatic system and bloodstream for metastasis to more distant parts of the body. Infiltrating ductal carcinoma accounts for about 80% of breast cancers.

Lobular carcinoma in situ (LCIS): While not a true cancer, LCIS (also called lobular neoplasia) is sometimes classified as a type of noninvasive breast cancer. It does not penetrate through the wall of the lobules. Although it does not itself usually become an invasive cancer, women with this condition have a higher risk of developing an invasive breast cancer in the same breast, or in the opposite breast.

Infiltrating (or invasive) lobular carcinoma (ILC): ILC is similar to IDC, in that it has the potential metastasize elsewhere in the body. About 10% to 15% of invasive breast cancers are invasive lobular carcinomas. ILC can be more difficult to detect by mammogram than IDC.

Inflammatory breast cancer: This rare type of invasive breast cancer accounts for about 1% of all breast cancers and is extremely aggressive. Multiple skin symptoms associated with this cancer are caused by cancer cells blocking lymph vessels or channels in the skin over the breast.

Medullary carcinoma: This special type of infiltrating breast cancer has a relatively well defined, distinct boundary between tumor tissue and normal tissue. It accounts for about 5% of breast cancers. The prognosis for this kind of breast cancer is better than for other types of invasive breast cancer. Mucinous carcinoma: This rare type of invasive breast cancer originates from mucus- producing cells. The prognosis for mucinous carcinoma is better than for the more common types of invasive breast cancer.

Paget's disease of the nipple: This type of breast cancer starts in the ducts and spreads to the skin of the nipple and the areola. It is a rare type of breast cancer, occurring in only 1% of all cases. Paget's disease can be associated with in situ carcinoma, or with infiltrating breast carcinoma. If no lump can be felt in the breast tissue, and the biopsy shows DCIS but no invasive cancer, the prognosis is excellent.

Phyllodes tumor: This very rare type of breast tumor forms from the stroma of the breast, in contrast to carcinomas which develop in the ducts or lobules. Phyllodes (also spelled phylloides) tumors are usually benign, but are malignant on rare occasions.

Nevertheless, malignant phyllodes tumors are very rare and less than 10 women per year in the US die of this disease. Benign phyllodes tumors are successfully treated by removing the mass and a narrow margin of normal breast tissue.

Tubular carcinoma: Accounting for about 2% of all breast cancers, tubular carcinomas are a special type of infiltrating breast carcinoma. They have a better prognosis than usual infiltrating ductal or lobularcarcinomas. High-quality mammography combined with clinical breast exam remains the only screening method clearly tied to reduction in breast cancer mortality. Lower dose x-rays, digitized computer rather than film images, and the use of computer programs to assist diagnosis, are almost ready for widespread dissemination. Other technologies also are being developed, including magnetic resonance imaging and ultrasound. In addition, a very low radiation exposure technique, positron emission tomography has the potential for detecting early breast cancer.

It is also possible to differentiate between non-cancerous breast tissue and malignant breast tissue by analyzing differential gene expression between tissues. In addition, there may be several possible alterations that lead to the various possible types of breast cancer. The different types of breast tumors (e.g., invasive vs. non-invasive, ductal vs. axillary lymph node) can be differentiable from one another by the identification of the differences in genes expressed by different types of breast tumor tissues (Porter- Jordan et al, Hematol Oncol Clin North Am (1994) 5:73). Breast cancer can thus be generally diagnosed by detection of expression of a gene or genes associated with breast tumors. Where enough information is available about the differential gene expression between various types of breast tumor tissues, the specific type of breast tumor can also be diagnosed.

For example, increased estrogen receptor (ER) expression in normal breast epithileum, while not itself indicative of malignant tissue, is a known risk marker for development of breast cancer. Khan S A et al, Cancer Res (1994) 54:993. Malignant breast cancer is often divided into two groups, ER-positive and ER-negative, based on the estrogen receptor status of the tissue. The ER status represents different survival length and response to hormone therapy, and is thought to represent either: 1 ) an indicator of different stages of the disease, or 2) an indicator that allows differentiation between two similar but distinct diseases. K. Zhu et al, Med. Hypoth. (1997) 49:69. A number of other genes are known to vary expression between either different stages of cancer or different types of similar breast cancer.

Similarly, the expression of polynucleotides of the invention can be used in the diagnosis and management of breast cancer. The differential expression of a polynucleotide in human breast tumor tissue can be used as a diagnostic marker for human breast cancer. The polynucleotides of the invention that would be especially useful for this purpose are those that exhibit differential expression between breast cancer tissue with a high metastatic potential and a low metastatic potential, i.e. SEQ ID NOS: 9, 42, 52, 62, 65, 66, 68, 114, 123, 144, 172, 178, 214, 219, 223, 258, 317, and 379. Detection of breast cancer can be determined using expression levels of any of these sequences alone or in combination. Determination of the aggressive nature and/or the metastatic potential of a breast cancer can also be determined by comparing levels of one or more polynucleotides of the invention and comparing levels of another sequence known to vary in cancerous tissue, e.g. ER expression. In addition, development of breast cancer can be detected by examining the ratio of SEQ ID NO: to the levels of steroid hormones (e.g. , testosterone or estrogen) or to other hormones (e.g. , growth hormone, insulin). Thus expression of specific marker polynucleotides can be used to discriminate between normal and cancerous breast tissue, to discriminate between breast cancers with different cells of origin, to discriminate between breast cancers with different potential metastatic rates, etc.

Diagnosis of breast cancer can also involve comparing the expression of a polynucleotide of the invention with the expression of other sequences in non-malignant breast tissue samples in comparison to one or more forms of the diseased tissue. A comparison of expression of one or more polynucleotides of the invention between the samples provides information on relative levels of these polynucleotides as well as the ratio of these polynucleotides to the expression of other sequences in the tissue of interest compared to normal.

This risk of breast cancer is elevated significantly by the presence of an inherited risk for breast cancer, such as a mutation in BRCA-1 or BRCA-2. New diagnostic tools are being developed to address the needs of higher risk patients to complement mammography and physical examinations for early detection of breast cancer, particularly among younger women. The presence of antigen or expression markers in nipple aspirate fluid (NAF) samples collected from one or both breasts can be useful for useful for risk assessment or early cancer detection. Breast cytology and biomarkers obtained by random fine needle aspiration have been used to identify hyperplasia with atypia and overexpression of p53 and EGFR. The polynucleotides of the invention can be used in multivariate analysis with expression studies with genes such as p53 and EGFR as risk predictors and as surrogate endpoint biomarkers for breast cancer. As well as being used for diagnosis and risk assessment, the expression of certain genes can also correlated to prognosis of a disease state. The expression of particular gene have been used as prognostic indicators for breast cancer including increased expression of c-erbB-2, pS2, ER, progesterone receptor, epidermal growth factor receptor (EGFR), neu, myc, bcl-2, int2, cytosolic tyrosine kinase, cyclin E,prad-1, hst, uPA, PAI-1, PAI-2, cathepsin D, as well as the presence of a number of cancer-specific antigens, e.g. CEA, CA M26, CA M29 and CA 15.3. Davis, Br. J. BiomedSci. (1996) 55:157. Poor prognosis has also been linked to a decrease in expression of certain genes, such as p53, Rb, nm23. The expression of the polynucleotides of the invention can be of prognostic value for determining the metastatic potential of a malignant breast cancer, as this molecules are differentially expressed between high and low metastatic potential tissues tumors. The levels of these polynucleotides in patients with malignant breast cancer can compared to normal tissue, malignant tissue with a known high potential metastatic level, and malignant tissue with a known lower level of metastatic potential to provide a prognosis for a particular patient. Such a prognosis is predictive of the extent and nature of the cancer. The determined prognosis is useful in determining the prognosis of a patient with breast cancer, both for initial treatment of the disease and for longer-term monitoring of the same patient. If samples are taken from the same individual over a period of time, differences in polynucleotide expression that are specific to that patient can be identified and closely watched.

Colon Cancer. Colorectal cancer is one of the most common neoplasms in humans and perhaps the most frequent form of hereditary neoplasia. Prevention and early detection are key factors in controlling and curing colorectal cancer. Indeed, colorectal cancer is the second most preventable cancer, after lung cancer. Colorectal cancer begins as polyps, which are small, benign growths of cells that form on the inner lining of the colon. Over a period of several years, some of these polyps accumulate additional mutations and become cancerous. About 20 percent of all cases of colon cancer are thought to be related to heredity. Currently, multiple familial colorectal cancer disorders have been identified, which are summarized as follows: Familial adenomatous polyposis (FAP): This condition results in a person having hundreds or even thousands of polyps in the colon and rectum that usually first appear during the teenage years. Cancer nearly always develops in one or more of these polyps between the ages of 30 and 50.

Gardner's syndrome: Like FAP, Gardner's syndrome results in polyps and colorectal cancers that develop at a young age. It can also cause benign tumors of the skin, soft connective tissue and bones.

Hereditary nonpolyposis colon cancer (HNPCC): People with this condition tend to develop colorectal cancer at a young age, without first having many polyps. HNPCC has an autosomal dominant pattern of inheritance with variable but high penetrance estimated to be about 90%. HNPCC underlies 0.5%- 10% of all cases of colorectal cancer. An understanding of the mechanisms behind the development of HNPCC is emerging, and genetic presymptomatic testing, now being conducted in research settings, soon will be available on a widespread basis for individuals identified at risk for this disease.

Familial colorectal cancer in Ashkenazi Jews: Recent research has found an inherited tendency to developing colorectal cancer among some Jews of Eastern European descent. Like people with FAP, Gardner's syndrome, and HNPCC, their increased risk is due to an inherited mutation present in about 6% of American Jews.

Several tests are currently used to screen for colorectal cancer, including digital rectal examination, fecal occult blood test, sigmoidoscopy, colonoscopy, virtual colonoscopy and

MRI. Each of these tests identifies potential colorectal cancer lesions, or a risk of development of these lesions, at a fairly gross morphological level.

The sequential alteration of a number of genes is associated with malignant adenocarcinoma, including the genes DCC, p53, ras, and FAP. For a review, see e.g. Fearon

ER, et al, Cell (1990) 61(5):159; Hamilton SR et al, Cancer (1993) 72:957; Bodmer W, et al, Nat Genet. (1994) 4(3) :217; Fearon ER, Ann N Y Acad Sci. (1995) 765:101. Molecular genetic alterations are thus promising as potential diagnostic and prognostic indicators in colorectal carcinoma and molecular genetics of colorectal carcinoma since it is possible to differentiate between different types of colorectal neoplasias using molecular markers.

Colorectal cancer can thus be generally diagnosed by detection of expression of a gene or genes associated with colorectal tumors. Similarly, the expression of polynucleotides of the invention can be used in the diagnosis, prognosis and management of colorectal cancer. The differential expression of a polynucleotide in hyperplasia can be used as a diagnostic marker for colon cancer. The polynucleotides of the invention that would be especially useful for this purpose are those that exhibit differential expression between malignant metastatic colon cancer and normal patient tissue , i.e. SEQ ID NOS: 52, 119, 172, 288. Detection of malignant colon cancer can be determined using expression levels of any of these sequences alone or in combination with the levels of expression.

Determination of the aggressive nature and/or the metastatic potential of a colon cancer can also be determined by comparing levels of one or more polynucleotides of the invention and comparing total levels of another sequence known to vary in cancerous tissue, e.g. p53 expression. In addition, development of colon cancer can be detected by examining the ratio of any of the polynucleotides of the invention to the levels of oncogenes (e.g. ras) or tumor suppressor genes (e.g. FAP or p53). Thus expression of specific marker polynucleotides can be used to discriminate between normal and cancerous breast tissue, to discriminate between breast cancers with different cells of origin, to discriminate between breast cancers with different potential metastatic rates, etc.

G. Use of Polynucleotides to Screen for Peptide Analogs and Antagonists Polypeptides encoded by the instant polynucleotides and corresponding full length genes can be used to screen peptide libraries to identify binding partners, such as receptors, from among the encoded polypeptides. A library of peptides can be synthesized following the methods disclosed in U.S. Pat.

No. 5,010,175 (' 175), and in WO 91/17823. As described below in brief, one prepares a mixture of peptides, which is then screened to identify the peptides exhibiting the desired signal transduction and receptor binding activity. In the '175 method, a suitable peptide synthesis support (e.g., a resin) is coupled to a mixture of appropriately protected, activated amino acids. The concentration of each amino acid in the reaction mixture is balanced or adjusted in inverse proportion to its coupling reaction rate so that the product is an equimolar mixture of amino acids coupled to the starting resin. The bound amino acids are then deprotected, and reacted with another balanced amino acid mixture to form an equimolar mixture of all possible dipeptides. This process is repeated until a mixture of peptides of the desired length (e.g., hexamers) is formed. Note that one need not include all amino acids in each step: one can include only one or two amino acids in some steps (e.g., where it is known that a particular amino acid is essential in a given position), thus reducing the complexity of the mixture. After the synthesis of the peptide library is completed, the mixture of peptides is screened for binding to the selected polypeptide. The peptides are then tested for their ability to inhibit or enhance activity. Peptides exhibiting the desired activity are then isolated and sequenced.

The method described in WO 91/17823 is similar. However, instead of reacting the synthesis resin with a mixture of activated amino acids, the resin is divided into twenty equal portions (or into a number of portions corresponding to the number of different amino acids to be added in that step), and each amino acid is coupled individually to its portion of resin. The resin portions are then combined, mixed, and again divided into a number of equal portions for reaction with the second amino acid. In this manner, each reaction can be easily driven to completion. Additionally, one can maintain separate "subpools" by treating portions in parallel, rather than combining all resins at each step. This simplifies the process of determining which peptides are responsible for any observed receptor binding or signal transduction activity.

In such cases, the subpools containing, e.g., 1-2,000 candidates each are exposed to one or more polypeptides of the invention. Each subpool that produces a positive result is then resynthesized as a group of smaller subpools (sub-subpools) containing, e.g., 20-100 candidates, and reassayed. Positive sub-subpools can be resynthesized as individual compounds, and assayed finally to determine the peptides that exhibit a high binding constant. These peptides can be tested for their ability to inhibit or enhance the native activity. The methods described in WO 91/7823 and U.S. Patent No. 5,194,392 (herein incorporated by reference) enable the preparation of such pools and subpools by automated techniques in parallel, such that all synthesis and resynthesis can be performed in a matter of days.

Peptide agonists or antagonists are screened using any available method, such as signal transduction, antibody binding, receptor binding, mitogenic assays, chemotaxis assays, etc. The methods described herein are presently preferred. The assay conditions ideally should resemble the conditions under which the native activity is exhibited in vivo, that is, under physiologic pH, temperature, and ionic strength. Suitable agonists or antagonists will exhibit strong inhibition or enhancement of the native activity at concentrations that do not cause toxic side effects in the subject. Agonists or antagonists that compete for binding to the native polypeptide can require concentrations equal to or greater than the native concentration, while inhibitors capable of binding irreversibly to the polypeptide can be added in concentrations on the order of the native concentration. The end results of such screening and experimentation will be at least one novel polypeptide binding partner, such as a receptor, encoded by a gene or a cDNA corresponding to a polynucleotide of the invention, and at least one peptide agonist or antagonist of the novel binding partner. Such agonists and antagonists can be used to modulate, enhance, or inhibit receptor function in cells to which the receptor is native, or in cells that possess the receptor as a result of genetic engineering. Further, if the novel receptor shares biologically important characteristics with a known receptor, information about agonist/antagonist binding can facilitate development of improved agonists/antagonists of the known receptor. H. Pharmaceutical Compositions and Therapeutic Uses Pharmaceutical compositions can comprise polypeptides, antibodies, or polynucleotides of the claimed invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, an effective dose will generally be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered. A pharmaceutical composition can also contain a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.

The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington 's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Delivery Methods. Once formulated, the compositions of the invention can be

(1) administered directly to the subject (e.g., as polynucleotide or polypeptides); (2) delivered ex vivo, to cells derived from the subject (e.g., as in ex vivo gene therapy); or (3) delivered in vitro for expression of recombinant proteins (e.g., polynucleotides). Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a tumor or lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment can be a single dose schedule or a multiple dose schedule. Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells. Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Once a gene corresponding to a polynucleotide of the invention has been found to correlate with a proliferative disorder, such as neoplasia, dysplasia, and hyperplasia, the disorder can be amenable to treatment by administration of a therapeutic agent based on the provided polynucleotide or corresponding polypeptide.

Preparation of antisense polynucleotides is discussed above. Neoplasias that are treated with the antisense composition include, but are not limited to, cervical cancers, melanomas, colorectal adenocarcinomas, Wilms' tumor, retinoblastoma, sarcomas, myosarcomas, lung carcinomas, leukemias, such as chronic myelogenous leukemia, promyelocytic leukemia, monocytic leukemia, and myeloid leukemia, and lymphomas, such as histiocytic lymphoma. Proliferative disorders that are treated with the therapeutic composition include disorders such as anhydric hereditary ectodermal dysplasia, congenital alveolar dysplasia, epithelial dysplasia of the cervix, fibrous dysplasia of bone, and mammary dysplasia. Hyperplasias, for example, endometrial, adrenal, breast, prostate, or thyroid hyperplasias or pseudoepitheliomatous hyperplasia of the skin, are treated with antisense therapeutic compositions based upon a polynucleotide of the invention. Even in disorders in which mutations in the corresponding gene are not implicated, downregulation or inhibition of expression of a gene corresponding to a polynucleotide of the invention can have therapeutic application. For example, decreasing gene expression can help to suppress tumors in which enhanced expression of the gene is implicated.

Both the dose of the antisense composition and the means of administration are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. Administration of the therapeutic antisense agents of the invention includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. Preferably, the therapeutic antisense composition contains an expression construct comprising a promoter and a polynucleotide segment of at least 12, 22, 25, 30, or 35 contiguous nucleotides of the antisense strand of a polynucleotide disclosed herein. Within the expression construct, the polynucleotide segment is located downstream from the promoter, and transcription of the polynucleotide segment initiates at the promoter.

Various methods are used to administer the therapeutic composition directly to a specific site in the body. For example, a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of tumor. Alternatively, arteries which serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor. A tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor. The antisense composition is directly administered to the surface of the tumor, for example, by topical application of the composition. X-ray imaging is used to assist in certain of the above delivery methods.

Receptor-mediated targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues is also used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al, Trends Biotechnol. (1993) 77:202; Chiou et al, Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al, J. Biol. Chem. (1988) 265:621; Wu et al, J. Biol. Chem. (1994) 269:542; Zenke et al, Proc. Natl. Acad. Sci. (USA) (1990) 57:3655; Wu et al, J. Biol. Chem. (1991) 266:338. Preferably, receptor- mediated targeted delivery of therapeutic compositions containing antibodies of the invention is used to deliver the antibodies to specific tissue. Therapeutic compositions containing antisense subgenomic polynucleotides are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of antisense subgenomic polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. A more complete description of gene therapy vectors, especially retroviral vectors, is contained in U.S. Serial No. 08/869,309, which is expressly incorporated herein, and in section G below.

For polynucleotide-related genes encoding polypeptides or proteins with anti- inflammatory activity, suitable use, doses, and administration are described in U.S. Patent No. 5,654,173. Therapeutic agents also include antibodies to proteins and polypeptides encoded by the polynucleotides of the invention and related genes, as described in U.S. Patent No. 5,654,173.

I. Gene Therapy The therapeutic polynucleotides and polypeptides of the present invention can be utilized in gene delivery vehicles. The gene delivery vehicle can be of viral or non- viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 7:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 7:185; and Kaplitt, Nature Genetics (1994) 6:148). Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches. Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

The present invention can employ recombinant retroviruses which are constructed to carry or express a selected nucleic acid molecule of interest. Retrovirus vectors that can be employed include those described in EP 0 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5, 219,740; WO 93/11230; WO 93/10218; Vile and Hart, Cancer Res. (1993) 55:3860; Vile et al, Cancer Res. (1993) 55:962; Ram et al., Cancer Res. (1993) 55:83; Takamiya et al, J. Neurosci. Res. (1992) 55:493; Baba et al, J. Neurosurg. (1993) 79:729; U.S. Patent No. 4,777,127; GB Patent No. 2,200,651; and EP 0 345 242. Preferred recombinant retroviruses include those described in WO 91/02805. Packaging cell lines suitable for use with the above-described retroviral vector constructs can be readily prepared (see, e.g., WO 95/30763 and WO 92/05266), and used to create producer cell lines (also termed vector cell lines) for the production of recombinant vector particles. Within particularly preferred embodiments of the invention, packaging cell lines are made from human (such as HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviruses that can survive inactivation in human serum.

The present invention also employs alphavirus-based vectors that can function as gene delivery vehicles. Such vectors can be constructed from a wide variety of alphaviruses, including, for example, Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532). Representative examples of such vector systems include those described in U.S. Patent Nos. 5,091,309; 5,217,879; and 5,185,440; WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO 95/07994. Gene delivery vehicles of the present invention can also employ parvovirus such as adeno-associated virus (AAV) vectors. Representative examples include the AAV vectors disclosed by Srivastava in WO 93/09239, Samulski et al., J. Virol. (1989) 65:3822; Mendelson et al, Virol. (1988) 766:154; and Flotte et al, PNAS (1993) 90:10613. Representative examples of adeno viral vectors include those described by Berkner,

Biotechniques (1988) 6:616; Rosenfeld et al, Science (1991) 252:431; WO 93/19191 ; Kolls et al, PNAS (1994) 97:215; Kass-Eisler et al, PNAS (1993) 90:11498; Guzman et al, Circulation (1993) 55:2838; Guzman et al., Cir. Res. (1993) 75:1202; Zabner et al, Cell (1993) 75:207; Li et al, Hum. Gene Ther. (1993) 4:403; Cailaud et al, Eur. J. Neurosci. (1993) 5:1287; Vincent et al, Nat. Genet. (1993) 5:130; Jaffe et al, Nat. Genet. (1992)

7:372; and Levrero et al, Gene (1991) 707:195. Exemplary adenoviral gene therapy vectors employable in this invention also include those described in WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 5:147 can be employed. Other gene delivery vehicles and methods can be employed, including polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example Curiel. Hum. Gene Ther. (1992) 5:147; ligand linked DNA, for example see Wu, J. Biol. Chem. (1989) 264:16985; eukaryotic cell delivery vehicles cells, for example see U.S. Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338; deposition of photopolymerized hydrogel materials; hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; ionizing radiation as described in U.S. Patent No. 5,206,152 and in WO92/11033; nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 74:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 97 : 1581.

Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Patent No. 5,580,859. Uptake efficiency can be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method can be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968.

Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al, Proc. Natl. Acad. Sci. USA (1994)

97 (24): 11581. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Patent No. 5,206,152 and WO 92/11033.

The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way. EXAMPLES

The present invention is now illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, these embodiments are illustrative and are not meant to be construed as restricting the invention in any way.

Example 1 : Source of Biological Materials and Overview of Novel Polynucleotides

Expressed by the Biological Materials

Human colon cancer cell line Kml2L4-A (Morika, W. A. K. et al., Cancer Research

(1988) 45:6863) was used to construct a cDNA library from mRNA isolated from the cells. As described in the above overview, a total of 4,693 sequences expressed by the Kml2L4-A cell line were isolated and analyzed; most sequences were about 275-300 nucleotides in length. The KM12L4-A cell line is derived from the KM12C cell line. The KM12C cell line, which is poorly metastatic (low metastatic) was established in culture from a Dukes' stage B₂ surgical specimen (Morikawa et al. Cancer Res. (1988) 45:6863). The KML4-A is a highly metastatic subline derived from KM12C (Yeatman et al. Nucl. Acids. Res. (1995) 25:4007; Bao-Ling et al. Proc. Annu. Meet. Am. Assoc. Cancer. Res. (1995) 27:3269). The KM12C and KM12C-derived cell lines (e.g., KM12L4, KM12L4-A, etc.) are well- recognized in the art as a model cell line for the study of colon cancer (see, e.g., Moriakawa et al, supra; Radinsky et al. Clin. Cancer Res. (1995) 7:19; Yeatman et al, (1995) supra; Yeatman et al. Clin. Exp. Metastasis (1996) 74:246).

The sequences were first masked to eliminate low complexity sequences using the XBLAST masking program (Claverie "Effective Large-Scale Sequence Similarity Searches," In: Computer Methods for Macromolecular Sequence Analysis. Doolittle, ed., Meth. Enzymol. 266:212-227 Academic Press, NY, NY (1996); see particularly Claverie, in "Automated DNA Sequencing and Analysis Techniques" Adams et al, eds., Chap. 36, p. 267 Academic Press, San Diego, 1994 and Claverie et al. Comput. Chem. (1993) 17:191 ). Generally, masking does not influence the final search results, except to eliminate of relative little interest due to their lox complexity, and to eliminate multiple "hits" based on similarity to repetitive regions common to multiple sequences, e.g., Alu repeats. Masking resulted in the elimination of 43 sequences. The remaining sequences were then used in a BLASTN vs.

Genbank search with search parameters of greater than 70% overlap, 99% identity, and a p value of less than 1 x 10^"40, which search resulted in the discarding of 1,432 sequences.

Sequences from this search also were discarded if the inclusive parameters were met, but the sequence was ribosomal or vector-derived. The resulting sequences from the previous search were classified into three groups

(1, 2 and 3 below) and searched in a BLASTX vs. NRP (non-redundant proteins) database search: (1) unknown (no hits in the Genbank search), (2) weak similarity (greater than 45% identity and p value of less than 1 x 10^"5), and (3) high similarity (greater than 60% overlap, greater than 80% identity, and p value less than 1 x 10^"5). This search resulted in discard of 98 sequences as having greater than 70% overlap, greater than 99% identity, and p value of less than l x lO^"40.

The remaining sequences were classified as unknown (no hits), weak similarity, and high similarity (parameters as above). Two searches were performed on these sequences. First, a BLAST vs. EST database search resulted in discard of 1771 sequences (sequences with greater than 99% overlap, greater than 99% similarity and a p value of less than 1 x 10^{" 40} ; sequences with a p value of less than 1 x 10^"65 when compared to a database sequence of human origin were also excluded). Second, a BLASTN vs. Patent GeneSeq database resulted in discard of 15 sequences (greater than 99% identity; p value less than 1 x 10^"40; greater than 99% overlap).

The remaining sequences were subjected to screening using other rules and redundancies in the dataset. Sequences with a p value of less than 1 x 10 ^{_ι n} in relation to a database sequence of human origin were specifically excluded. The final result provided the 404 sequences listed in the accompanying Sequence Listing. The Sequence Listing is arranged beginning with sequences with no similarity to any sequence in a database searched, and ending with sequences with the greatest similarity. Each identified polynucleotide represents sequence from at least a partial mRNA transcript. Polynucleotides that were determined to be novel were assigned a sequence identification number.

The novel polynucleotides and were assigned sequence identification numbers SEQ ID NOS: 1-404. The DNA sequences corresponding to the novel polynucleotides are provided in the Sequence Listing. The majority of the sequences are presented in the Sequence Listing in the 5' to 3' direction. A small number, 25, are listed in the Sequence Listing in the 5' to 3' direction but the sequence as written is actually 3' to 5'. These sequences are readily identified with the designation "AR" in the Sequence Name in Table 1 (inserted before the claims). The sequences correctly listed in the 5' to 3' direction in the

Sequence Listing are designated "AF." The Sequence Listing filed herewith therefore contains 25 sequences listed in the reverse order, namely SEQ ID NOS:47, 97, 137, 171,

173, 179, 182, 194, 200, 202, 213, 227, 258, 264, 275, 302, 313, 324, 329, 330, 331, 338,

358, 379, and 404. Because the provided polynucleotides represent partial mRNA transcripts, two or more polynucleotides of the invention may represent different regions of the same mRNA transcript and the same gene. Thus, if two or more SEQ ID NOS: are identified as belonging to the same clone, then either sequence can be used to obtain the full-length mRNA or gene. In order to confirm the sequences of SEQ ID NOS : 1 -404, inserts of the clones corresponding to these polynucleotides were re-sequenced. These "validation" sequences are provided in SEQ ID NOS:405-800. These validation sequences were often longer than the original polynucleotide sequences. They validate, and thus often provide additional sequence information. Validation sequences can be correlated with the original sequences they validate by identifying those sequences of SEQ ID NOS: 1 -404 and the validation sequences of SEQ ID NOS:405-800 that share the same clone name in Table 1.

Example 2: Results of Public Database Search to Identify Function of Gene Products

SEQ ID NOS: 1-404, as well as the validation sequences SEQ ID NOS:405-800, were translated in all three reading frames to determine the best alignment with the individual sequences. These amino acid sequences and nucleotide sequences are referred, generally, as query sequences, which are aligned with the individual sequences. Query and individual sequences were aligned using the BLAST programs, available over the world wide web at http://ww.ncbi.nlm.nih.gov/BLAST/. Again the sequences were masked to various extents to prevent searching of repetitive sequences or poly-A sequences, using the XBLAST program for masking low complexity as described above in Example 1.

Table 2 (inserted before the claims) shows the results of the alignments. Table 2 refers to each sequence by its SEQ ID NO:, the accession numbers and descriptions of nearest neighbors from the Genbank and Non-Redundant Protein searches, and the p values of the search results. Table 1 identifies each SEQ ID NO: by SEQ name, clone ID, and cluster. As discussed above, a single cluster includes polynucleotides representing the same gene or gene family, and generally represents sequences encoding the same gene product.

For each of SEQ ID NOS: 1-800, the best alignment to a protein or DNA sequence is included in Table 2. The activity of the polypeptide encoded by SEQ ID NOS: 1-800 is the same or similar to the nearest neighbor reported in Table 2. The accession number of the nearest neighbor is reported, providing a reference to the activities exhibited by the nearest neighbor. The search program and database used for the alignment also are indicated as well as a calculation of the p value. Full length sequences or fragments of the polynucleotide sequences of the nearest neighbors can be used as probes and primers to identify and isolate the full length sequence of SEQ ID NOS: 1-800. The nearest neighbors can indicate a tissue or cell type to be used to construct a library for the full-length sequences of SEQ ID NOS: 1-800.

SEQ ID NOS: 1-800 and the translations thereof may be human homologs of known genes of other species or novel allelic variants of known human genes. In such cases, these new human sequences are suitable as diagnostics or therapeutics. As diagnostics, the human sequences SEQ ID NOS: 1-800 exhibit greater specificity in detecting and differentiating human cell lines and types than homologs of other species. The human polypeptides encoded by SEQ ID NOS: 1-800 are likely to be less immunogenic when administered to humans than homologs from other species. Further, on administration to humans, the polypeptides encoded by SEQ ID NOS: 1-800 can show greater specificity or can be better regulated by other human proteins than are homologs from other species.

Example 3 : Members of Protein Families

After conducting a profile search as described in the specification above, several of the polynucleotides of the invention were found to encode polypeptides having characteristics of a polypeptide belonging to a known protein families (and thus represent new members of these protein families) and/or comprising a known functional domain (Table 3). Thus the invention encompasses fragments, fusions, and variants of such polynucleotides that retain biological activity associated with the protein family and/or functional domain identified herein.

Table 3 Polynucleotides encoding gene products of a protein family or having a known functional domain(s).

Table 3 Polynucleotides encoding gene products of a protein family or having a known functional domain(s).

Start and stop indicate the position within the individual sequenes that align with the query sequence having the indicated SEQ ID NO. The direction (Dir) indicates the orientation of the query sequence with respect to the individual sequence, where forward (for) indicates that the alignment is in the same direction (left to right) as the sequence provided in the Sequence Listing and reverse (rev) indicates that the alignment is with a sequence complementary to the sequence provided in the Sequence Listing.

Some polynucleotides exhibited multiple profile hits because, for example, the particular sequence contains overlapping profile regions, and/or the sequence contains two different functional domains. These profile hits are described in more detail below. a) Four Transmembrane Integral Membrane Proteins. SEQ ID NOS: 24, 41, 101, 157, 341, and 395 correspond to a sequence encoding a polypeptide that is a member of the 4 transmembrane segments integral membrane protein family (transmembrane 4 family). The transmembrane 4 family of proteins includes a number of evolutionarily-related eukaryotic cell surface antigens (Levy et al, J. Biol. Chem., (1991) 266:14597; Tomlinson et al, Eur. J. Immunol. (1993) 25:136; Barclay et al. The leucocyte antigen factbooks. (1993) Academic Press, London/San Diego). The proteins belonging to this family include: 1) Mammalian antigen CD9 (MIC3), which is involved in platelet activation and aggregation; 2) Mammalian leukocyte antigen CD37, expressed on B lymphocytes; 3) Mammalian leukocyte antigen CD53 (OX-44), which is implicated in growth regulation in hematopoietic cells; 4) Mammalian lysosomal membrane protein CD63 (melanoma-associated antigen ME491 ; antigen AD1); 5) Mammalian antigen CD81 (cell surface protein TAPA-1), which is implicated in regulation of lymphoma cell growth; 6) Mammalian antigen CD82 (protein R2; antigen C33; Kangai 1 (KAI1)), which associates with CD4 or CD8 and delivers costimulatory signals for the TCR/CD3 pathway; 7) Mammalian antigen CDl 51 (SFA-1 ; platelet-endothelial tetraspan antigen 3 (PETA-3)); 8) Mammalian cell surface glycoprotein A15 (TALLA-1 ; MXS1); 9) Mammalian novel antigen 2 (NAG-2); 10) Human tumor- associated antigen CO-029; 11) Schistosoma mansoni and japonicum 23 Kd surface antigen (SM23 / SJ23).

The members of the 4 transmembrane family share several characteristics. First, they all are apparently type III membrane proteins, which are integral membrane proteins containing an N-terminal membrane-anchoring domain which is not cleaved during biosynthesis and which functions both as a translocation signal and as a membrane anchor. The family members also contain three additional transmembrane regions, at least seven conserved cysteines residues, and are of approximately the same size (218 to 284 residues). These proteins are collectively know as the "transmembrane 4 superfamily" (TM4) because they span plasma membrane four times. A schematic diagram of the domain structure of these proteins is as follows:

1 1 TMa I Extra | TM2| Cyt | TM3 | Extracellular | TM4 | Cyt| +_+. — + + — c C- — + CC— C- — C— +- — C — +

where Cyt is the cytoplasmic domain, TMa is the transmembrane anchor; TM2 to TM4 represents transmembrane regions 2 to 4, 'C are conserved cysteines, and '* 'indicates the position of the consensus pattern. The consensus pattern spans a conserved region including two cysteines located in a short cytoplasmic loop between two transmembrane domains: Consensus pattern: G-x(3)-[LIVMF]-x(2)-[GSA]-[LIVMF](2)-G-C-x-[GA]-[STA]- x(2)- [EG]-x(2)-[CWN]-[LIVM](2). b) Seven Transmembrane Integral Membrane Proteins. SEQ ID NOS: 24, 41, 101, 157, 291, 305, 315, and 341 correspond to a sequence encoding a polypeptide that is a member of the seven transmembrane receptor family. G-protein coupled receptors (Strosberg, Eur. J. Biochem. (1991) 796:1; Kerlavage, Curr. Opin. Struct. Biol. (1991) 7 :394; and Probst et al. , DNA Cell Biol. (1992) 77 : 1 ; and Savarese et al. , Biochem. J. ( 1992)

293: ) (also called R7G) are an extensive group of hormones, neurotransmitters, odorants and light receptors which transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins. The tertiary structure of these receptors is thought to be highly similar. They have seven hydrophobic regions, each of which most probably spans the membrane. The N-terminus is located on the extracellular side of the membrane and is often glycosylated, while the C-terminus is cytoplasmic and generally phosphorylated. Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. Most, but not all of these receptors, lack a signal peptide. The most conserved parts of these proteins are the transmembrane regions and the first two cytoplasmic loops. A conserved acidic-Arg-aromatic triplet is present in the N-terminal extremity of the second cytoplasmic loop (Attwood et al, Gene (1991) 95:153) and could be implicated in the interaction with G proteins.

To detect this widespread family of proteins a pattern is used that contains the conserved triplet and that also spans the major part of the third transmembrane helix.

Additional information about the seven transmembrane receptor family, and methods for their identification and use, is found in U.S. Patent No. 5,759,804. Due in part to their expression on the cell surface and other attractive characteristics, seven transmembrane protein family members are of particular interest as drug targets, as surface antigen markers, and as drug delivery targets (e.g. , using antibody-drug complexes and/or use of anti-seven transmembrane protein antibodies as therapeutics in their own right). c) Ank Repeats. SEQ ID NOS: 116 and 251 represent polynucleotides encoding Ank repeat-containing proteins. The ankyrin motif is a 33 amino acid sequence named after the protein ankyrin which has 24 tandem 33-amino-acid motifs. Ank repeats were originally identified in the cell-cycle-control protein cdclO (Breeden et al, Nature (1987) 529:651).

Proteins containing ankyrin repeats include ankyrin, myotropin, I-kappaB proteins, cell cycle protein cdclO, the Notch receptor (Matsuno et al, Development (1997) 124(21):4265); G9a (or BAT8) of the class III region of the major histocompatibility complex (Biochem J. 290:811-818, 1993), FABP, GABP, 53BP2, Linl2, glp-1, SW14, and SW16. The functions of the ankyrin repeats are compatible with a role in protein-protein interactions (Bork,

Proteins (1993) 17(4):363; Lambert and Bennet, Eur. J. Biochem. (1993) 277:1; Kerr et al, Current Op. Cell Biol. (1992) 4:496; Bennet et al, J. Biol. Chem. (1980) 255:6424).

The 90 kD N-terminal domain of ankyrin contains a series of 24 33-amino-acid ank repeats. (Lux et al, Nature (1990) 544:36-42, Lambert et al, PNAS USA (1990) 57:1730.) The 24 ank repeats form four folded subdomains of 6 repeats each. These four repeat subdomains mediate interactions with at least 7 different families of membrane proteins.

Ankyrin contains two separate binding sites for anion exchanger dimers. One site utilizes repeat subdomain two (repeats 7-12) and the other requires both repeat subdomains 3 and 4

(repeats 13-24). Since the anion exchangers exist in dimers, ankyrin binds 4 anion exchangers at the same time. (Michaely and Bennett, J. Biol. Chem. (1995) 270(37). -22050)

The repeat motifs are involved in ankyrin interaction with tubulin, spectrin, and other membrane proteins. (Lux et al, Nature (1990) 544:36.)

The Rel/NF-kappaB/Dorsal family of transcription factors have activity that is controlled by sequestration in the cytoplasm in association with inhibitory proteins referred to as I-kappaB. (Gilmore, Cell (1990) 62:841; Nolan and Baltimore, Curr Opin Genet Dev. (1992) 2:21 1; Baeuerle, Biochim Biophys Acta (1991) 1072:63; Schmitz et al, Trends Cell Biol. (1991) 7:130.) I-kappaB proteins contain 5 to 8 copies of 33 amino acid ankyrin repeats and certain NF-kappaB/rel proteins are also regulated by cis-acting ankyrin repeat containing domains including pi 05NF-kappaB which contains a series of ankyrin repeats (Diehl and Hannink, J. Virol. (1993) 67(12)1 6 ). The I-kappaBs and Cactus (also containing ankyrin repeats) inhibit activators through differential interactions with the Rel- homology domain. The gene family includes proto-oncogenes, thus broadly implicating I- kappaB in the control of both normal gene expression and the aberrant gene expression that makes cells cancerous. (Nolan and Baltimore, Curr Opin Genet Dev. (1992) 2(2):2\ 1-220). In the case of rel/NF-kappaB and pp40/I-kappaBβ, both the ankyrin repeats and the carboxy- terminal domain are required for inhibiting DNA-binding activity and direct association of pp40/ I-kappaBβ with rel/NF-kappaB protein. The ankyrin repeats and the carboxy-terminal of pp40/I-kappaBβ ( form a structure that associates with the rel homology domain to inhibit DNA binding activity (Inoue et al, PNAS USA (1992) 59:4333).

The 4 ankyrin repeats in the amino terminus of the transcription factor subunit GABPβ are required for its interaction with the GABPα subunit to form a functional high affinity DNA-binding protein. These repeats can be crosslinked to DNA when GABP is bound to its target sequence. (Thompson et al, Science (1991) 253:162; LaMarco et al, Science (1991) 255:789).

Myotrophin, a 12.5 kDa protein having a key role in the initiation of cardiac hypertrophy, comprises ankyrin repeats. The ankyrin repeats are characteristic of a hairpinlike protruding tip followed by a helix-turn-helix motif. The V-shaped helix-turn-helix of the repeats stack sequentially in bundles and are stabilized by compact hydrophobic cores, whereas the protruding tips are less ordered. d) ATPases Associated with Various Cellular Activities ( AAA). SEQ ID NOS: 63, 116, 134, 136, 151, 384, and 404 polynucleotides encoding novel members of the "ATPases Associated with diverse cellular Activities" (AAA) protein family The AAA protein family is composed of a large number of ATPases that share a conserved region of about 220 amino acids that contains an ATP-binding site (Froehlich et al, J. Cell Biol. (1991) 774:443; Erdmann et Ω/. Cell (1991) 64:499; Peters et al, EMBO J. (1990) 9:1757; Kunau et al, Biochimie (1993) 75:209-224; Confalonieri et al, BioEssays (1995) 77:639; http://yeamob.pci.chemie.uni-tuebingen.de/AAA Description.html). The proteins that belong to this family either contain one or two AAA domains.

Proteins containing two AAA domains include: 1) Mammalian and drosophila NSF (N-ethylmaleimide-sensitive fusion protein) and the fungal homolog, SEC 18, which are involved in intracellular transport between the endoplasmic reticulum and Golgi, as well as between different Golgi cisternae; 2) Mammalian transitional endoplasmic reticulum

ATPase (previously known as p97 or VCP), which is involved in the transfer of membranes from the endoplasmic reticulum to the golgi apparatus. This ATPase forms a ring-shaped homooligomer composed of six subunits. The yeast homolog, CDC48, plays a role in spindle pole proliferation; 3) Yeast protein PAS1 essential for peroxisome assembly and the related protein PAS1 from Pichia pastoris; 4) Yeast protein AFG2; 5) Sulfolobus acidocaldarius protein SAV and Halobacterium salinarium cdcH, which may be part of a transduction pathway connecting light to cell division.

Proteins containing a single AAA domain include: 1) Escherichia coli and other bacteria ftsH (or hflB) protein. FtsH is an ATP-dependent zinc metallopeptidase that degrades the heat-shock sigma-32 factor, and is an integral membrane protein with a large cytoplasmic C-terminal domain that contain both the AAA and the protease domains; 2) Yeast protein YME1, a protein important for maintaining the integrity of the mitochondrial compartment. YME1 is also a zinc-dependent protease; 3) Yeast protein AFG3 (or YTA10). This protein also contains an AAA domain followed by a zinc-dependent protease domain; 4) Subunits from regulatory complex of the 26S proteasome (Hilt et al, Trends Biochem. Sci. (1996) 27:96), which is involved in the ATP-dependent degradation of ubiquitinated proteins, which subunits include: a) Mammalian 4 and homologs in other higher eukaryotes, in yeast (gene YTA5) and fission yeast (gene mts2); b) Mammalian 6 (TBP7) and homologs in other higher eukaryotes and in yeast (gene YTA2); c) Mammalian subunit 7 (MSS1) and homologs in other higher eukaryotes and in yeast (gene CIM5 or YTA3); d) Mammalian subunit 8 (P45) and homologs in other higher eukaryotes and in yeast (SUG1 or CIM3 or

TBY1) and fission yeast (gene letl); e) Other probable subunits include human TBP1, which influences HIV gene expression by interacting with the virus tat transactivator protein, and yeast YTA1 and YTA6; 5) Yeast protein BCS1, a mitochondrial protein essential for the expression of the Rieske iron-sulfur protein; 6) Yeast protein MSP1, a protein involved in intramitochondrial sorting of proteins; 7) Yeast protein PAS8, and the corresponding proteins PAS5 from Pichia pastoris and PAY4 from Yarrowia lipolytica; 8) Mouse protein SKD1 and its fission yeast homolog (SpAC2Gl 1.06); 9) Caenorhabditis elegans meiotic spindle formation protein mei-1; 10) Yeast protein SAPl ' 11) Yeast protein YTA7; and 12) Mycobacterium leprae hypothetical protein A2126A.

In general, the AAA domains in these proteins act as ATP-dependent protein clamps(Confalonieri et al. (1995) BioEssays 77:639). In addition to the ATP-binding 'A' and 'B' motifs, which are located in the N-terminal half of this domain, there is a highly conserved region located in the central part of the domain which was used in the development of the signature pattern. The consensus pattern is: [LIVMT]-x-[LIVMT]- [LIVMF]-x-[GATMC]-[ST]-[NS]-x(4)-[LIVM]- D-x-A-[LIFA]-x-R. e) Basic Region Plus Leucine Zipper Transcription Factors. SEQ ID NO:374 correspond to a polynucleotide encoding a novel member of the family of basic region plus leucine zipper transcription factors. The bZIP superfamily (Hurst, Protein Prof. (1995) 2:105; and Ellenberger, Curr. Opin. Struct. Biol. (1994) 4:12) of eukaryotic DNA-binding transcription factors encompasses proteins that contain a basic region mediating sequence- specific DNA-binding followed by a leucine zipper required for dimerization. Members of the family include transcription factor AP-1, which binds selectively to enhancer elements in the cis control regions of SV40 and metallothionein IIA. AP- 1 , also known as c-jun, is the cellular homolog of the avian sarcoma virus 17 (ASV17) oncogene v-jun.

Other members of this protein family include jun-B and jun-D, probable transcription factors that are highly similar to jun AP-1 ; the fos protein, a proto-oncogene that forms a non-covalent dimer with c-jun; the fos-related proteins fra-1, and fos B; and mammalian cAMP response element (CRE) binding proteins CREB, CREM, ATF-1, ATF- 3, ATF-4, ATF-5, ATF-6 and LRF-1. The consensus pattern for this protein family is: [KR]-x( 1 ,3)-[RKSAQ]-N-x(2)-[SAQ](2)-x-[RKTAENQ]-x-R-x-[RK]. f) Bromodomain. SEQ ID NO: 97 corresponds to a polynucleotide encoding a polypeptide having a bromodomain region (Haynes et al., 1992, Nucleic Acids Res. 20:2693-2603, Tamkun et al, 1992, Cell 68:561-572, and Tamkun, 1995, Curr. Opin. Genet.

Dev. 5:473-477), which is a conserved region of about 70 amino acids found in the following proteins: 1) Higher eukaryotes transcription initiation factor TFIID 250 Kd subunit (TBP-associated factor p250) (gene CCG1); P250 is associated with the TFIID

TATA-box binding protein and seems essential for progression of the GI phase of the cell cycle. 2) Human RING3, a protein of unknown function encoded in the MHC class II locus;

3) Mammalian CREB-binding protein (CBP), which mediates cAMP-gene regulation by binding specifically to phosphorylated CREB protein; 4) Mammalian homologs of brahma, including three brahma-like human: SNF2a(hBRM), SNF2b, and BRG1 ; 5) Human BS69, a protein that binds to adeno virus El A and inhibits El A transactivation; 6) Human peregrin

(or Brl40).

The bromodomain is thought to be involved in protein-protein interactions and may be important for the assembly or activity of multicomponent complexes involved in transcriptional activation. The consensus pattern, which spans a major part of the bromodomain, is: [STANVF]-x(2)-F-x(4)-[DNS]-x(5,7)-[DENQTF]-Y-[HFY]-x(2)-

[LIVMFY]-x(3)-[LIVM]-x(4)-[LIVM]-x(6,8)-Y-x(12,13)-[LIVM]-x(2)-N-[SACF]-x(2)- [FY]. g) EF-Hand. SEQ ID NOS: 136, 242, and 379 correspond to polynucleotides encoding a novel protein in the family of EF-hand proteins. Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand (Kawasaki et al, Protein. Prof. (1995) 2:305-490). This type of domain consists of a twelve residue loop flanked on both sides by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, -Y, -X and -Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand).

Proteins known to contain EF-hand regions include: Calmodulin (Ca=4, except in yeast where Ca=3) ("Ca=" indicates approximate number of EF-hand regions); diacylglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2); 2) FAD-dependent glycerol-3- phosphate dehydrogenase (EC 1.1.99.5) from mammals (Ca=l); guanylate cyclase activating protein (GCAP) (Ca=3); MIF related proteins 8 (MRP-8 or CFAG) and 14 (MRP- 14) (Ca=2); myosin regulatory light chains (Ca=l); oncomodulin (Ca=2); osteonectin (basement membrane protein BM-40) (SPARC); and proteins that contain an "osteonectin" domain (QR1, matrix glycoprotein SCI). The consensus pattern includes the complete EF-hand loop as well as the first residue which follows the loop and which seem to always be hydrophobic.

Consensus pattern: D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}- [LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW] h) Eukaryotic Aspartyl Proteases. SEQ ID NO: 308 corresponds to a gene encoding a novel eukaryotic aspartyl protease. Aspartyl proteases, known as acid proteases, (EC 3.4.23.-) are a widely distributed family of proteolytic enzymes (Foltmann B., Essays Biochem. (1981) 77:52; Davies O.R., Annu. Rev. Biophys. Chem. (1990) 79:189; Rao J.K.M., et al, Biochemistry (1991) 50:4663) known to exist in vertebrates, fungi, plants, retroviruses and some plant viruses. Aspartate proteases of eukaryotes are monomeric enzymes which consist of two domains. Each domain contains an active site centered on a catalytic aspartyl residue. The two domains most probably evolved from the duplication of an ancestral gene encoding a primordial domain. Currently known eukaryotic aspartyl proteases include: 1) Vertebrate gastric pepsins A and C (also known as gastricsin); 2) Vertebrate chymosin (rennin), involved in digestion and used for making cheese; 3) Vertebrate lysosomal cathepsins D (EC 3.4.23.5) and E (EC 3.4.23.34); 4) Mammalian renin (EC 3.4.23.15) whose function is to generate angiotensin I from angiotensinogen in the plasma; 5) Fungal proteases such as aspergillopepsin A (EC 3.4.23.18), candidapepsin (EC 3.4.23.24), mucoropepsin (EC 3.4.23.23) (mucor rennin), endothiapepsin (EC 3.4.23.22), polyporopepsin (EC 3.4.23.29), and rhizopuspepsin (EC 3.4.23.21); and 6) Yeast saccharopepsin (EC 3.4.23.25) (proteinase A) (gene PEP4). PEP4 is implicated in posttranslational regulation of vacuolar hydrolases; 7) Yeast barrierpepsin (EC 3.4.23.35) (gene BAR1); a protease that cleaves alpha-factor and thus acts as an antagonist of the mating pheromone; and 8) Fission yeast sxal which is involved in degrading or processing the mating pheromones.

Most retroviruses and some plant viruses, such as badnaviruses, encode for an aspartyl protease which is an homodimer of a chain of about 95 to 125 amino acids. In most retroviruses, the protease is encoded as a segment of a polyprotein which is cleaved during the maturation process of the virus. It is generally part of the pol polyprotein and, more rarely, of the gag polyprotein. Because the sequence around the two aspartates of eukaryotic aspartyl proteases and around the single active site of the viral proteases is conserved, a single signature pattern can be used to identify members of both groups of proteases. The consensus pattern is: [LIVMFGAC]-[LIVMTADN]-[LIVFSA]-D-[ST]-G-[STAV]- [STAPDENQ]- x-[LIVMFSTNC]-x-[LIVMFGTA], where D is the active site residue. i) GATA Family of Transcription Factors. SEQ ID NO:213 corresponds to a novel member of the GATA family of transcription factors. The GATA family of transcription factors are proteins that bind to DNA sites with the consensus sequence (A T)GATA(A G), found within the regulatory region of a number of genes. Proteins currently known to belong to this family are: 1) GATA-1 (Trainor, CD., et al, Nature (1990) 545:92) (also known as

Eryfl, GF-1 or NF-E1), which binds to the GATA region of globin genes and other genes expressed in erythroid cells. It is a transcriptional activator which probably serves as a general 'switch' factor for erythroid development; 2) GATA-2 (Lee, M.E., et al, J. Biol. Chem. (1991) 266:16188), a transcriptional activator which regulates endothelin- 1 gene expression in endothelial cells; 3) GATA-3 (Ho, I.-C, et al, EMBO J. (1991) 70:1 187), a transcriptional activator which binds to the enhancer of the T-cell receptor alpha and delta genes; 4) GATA-4 (Spieth, J., et al, Mol. Cell. Biol. (1991) 77:4651), a transcriptional activator expressed in endodermally derived tissues and heart; 5) Drosophila protein pannier (or DGATAa) (gene pnr) which acts as a repressor of the achaete-scute complex (as-c); 6) Bombyx mori BCFI (Drevet, J.R., et al, J. Biol. Chem. (1994) 269:10660), which regulates the expression of chorion genes; 7) Caenorhabditis elegans elt-1 and elt-2, transcriptional activators of genes containing the GATA region, including vitellogenin genes (Hawkins, M.G., et al, J. Biol. Chem. (1995) 270:14666); 8) Ustilago maydis urbsl (Voisard, C.P.O., et al, Mol. Cell. Biol. (1993) 75:7091), a protein involved in the repression of the biosynthesis of siderophores; 9) Fission yeast protein GAF2.

All these transcription factors contain a pair of highly similar 'zinc finger' type domains with the consensus sequence C-x2-C-xl7-C-x2-C. Some other proteins contain a single zinc finger motif highly related to those of the GATA transcription factors. These proteins are: 1) Drosophila box A-binding factor (ABF) (also known as protein serpent

(gene srp)) which may function as a transcriptional activator protein and may play a key role in the organogenesis of the fat body; 2) Emericella nidulans are (Arst, H.N., Jr., et al, Trends Genet. (1989) 5:291) a transcriptional activator which mediates nitrogen metabolite repression; 3) Neurospora crassa nit-2 (Fu, Y.-H., et al, Mol. Cell. Biol. (1990) 70:1056), a transcriptional activator which turns on the expression of genes coding for enzymes required for the use of a variety of secondary nitrogen sources, during conditions of nitrogen limitation; 4) Neurospora crassa white collar proteins 1 and 2 (WC-1 and WC-2), which control expression of light-regulated genes; 5) Saccharomyces cerevisiae DAL81 (or UGA43), a negative nitrogen regulatory protein; 6) Saccharomyces cerevisiae GLN3, a positive nitrogen regulatory protein; 7) Saccharomyces cerevisiae GATl; 8) Saccharomyces cerevisiae GZF3.

The consensus pattern for the GATA family is: C-x-[DN]-C-x(4,5)-[ST]-x(2)-W- [HR]-[RK]-x(3)-[GN]-x(3,4)-C-N-[AS]-C, where the four C's are zinc ligands. j) G-Protein Alpha Subunit. SEQ ID NO: 367 corresponds to a gene encoding a novel polypeptide of the G-protein alpha subunit family. Guanine nucleotide binding proteins (G- proteins) are a family of membrane-associated proteins that couple extracellularly-activated integral-membrane receptors to intracellular effectors, such as ion channels and enzymes that vary the concentration of second messenger molecules. G-proteins are composed of 3 subunits (alpha, beta and gamma) which, in the resting state, associate as a trimer at the inner face of the plasma membrane. The alpha subunit has a molecule of guanosine diphosphate (GDP) bound to it. Stimulation of the G-protein by an activated receptor leads to its exchange for GTP (guanosine triphosphate). This results in the separation of the alpha from the beta and gamma subunits, which always remain tightly associated as a dimer. Both the alpha and beta-gamma subunits are then able to interact with effectors, either individually or in a cooperative manner. The intrinsic GTPase activity of the alpha subunit hydrolyses the bound GTP to GDP. This returns the alpha subunit to its inactive conformation and allows it to reassociate with the beta-gamma subunit, thus restoring the system to its resting state. G-protein alpha subunits are 350-400 amino acids in length and have molecular weights in the range 40-45 kDa. Seventeen distinct types of alpha subunit have been identified in mammals. These fall into 4 main groups on the basis of both sequence similarity and function: alpha-s, alpha-q, alpha-i and alpha- 12 (Simon et al, Science (1993) 252:802). Many alpha subunits are substrates for ADP-ribosylation by cholera or pertussis toxins. They are often N-terminally acylated, usually with myristate and/or palmitoylate, and these fatty acid modifications are probably important for membrane association and high- affinity interactions with other proteins. The atomic structure of the alpha subunit of the G-protein involved in mammalian vision, transducin, has been elucidated in both GTP- and GDB-bound forms, and shows considerable similarity in both primary and tertiary structure in the nucleotide-binding regions to other guanine nucleotide binding proteins, such as p21-ras and EF-Tu. k) Phorbol Esters/Diacylglvcerol Binding. SEQ ID NO: 188 and 251 represent polynucleotides encoding a protein belonging to the family including phorbol esters/diacylglycerol binding proteins. Diacylglycerol (DAG) is an important second messenger. Phorbol esters (PE) are analogues of DAG and potent tumor promoters that cause a variety of physiological changes when administered to both cells and tissues. DAG activates a family of serine/threonine protein kinases, collectively known as protein kinase C

(PKC) (Azzi et al, Eur. J. Biochem. (1992) 205:547). Phorbol esters can directly stimulate

PKC. The N-terminal region of PKC, known as Cl, has been shown (Ono et al, Proc. Natl. Acad. Sci. USA (1989) 56:4868) to bind PE and DAG in a phospholipid and zinc-dependent fashion. The C 1 region contains one or two copies (depending on the isozyme of PKC) of a cysteine-rich domain about 50 amino-acid residues long and essential for DAG/PE-binding. Such a domain has also been found in, for example, the following proteins. (1) Diacylglycerol kinase (EC 2.7.1.107) (DGK) (Sakane et al, Nature (1990)

344:345), the enzyme that converts DAG into phosphatidate. It contains two copies of the DAG/PE-binding domain in its N-terminal section. At least five different forms of DGK are known in mammals; and

(2) N-chimaerin, a brain specific protein which shows sequence similarities with the BCR protein at its C-terminal part and contains a single copy of the DAG/PE-binding domain at its N-terminal part. It has been shown (Ahmed et al, Biochem. J. (1990) 272:161, and Ahmed et al, Biochem. J. (1991) 250:233) to be able to bind phorbol esters.

The DAG/PE-binding domain binds two zinc ions; the ligands of these metal ions are probably the six cysteines and two histidines that are conserved in this domain. The signature pattern completely spans the DAG/PE domain. The consensus pattern is: H-x-

[LIVMFYW]-x(8,l l)-C-x(2)-C-x(3)-[LIVMFC]-x(5,10)-C-x(2)-C-x(4)-[HD]-x(2)-C-x(5,9)- C. All the C and H are probably involved in binding zinc.

1) Protein Kinase. SEQ ID NOS:202, 315, 367, and 397 represent polynucleotides encoding protein kinases. Protein kinases catalyze phosphorylation of proteins in a variety of pathways, and are implicated in cancer. Eukaryotic protein kinases (Hanks S.K., et al, FASEB J. (1995) 9:576; Hunter T., Meth. Enzymol. (1991) 200:3; Hanks S.K., et al, Meth. Enzymol. (1991) 200:38; Hanks S.K., Curr. Opin. Struct. Biol (1991) 7:369; Hanks S.K., et al, Science (1988) 247:42) are enzymes that belong to a very extensive family of proteins which share a conserved catalytic core common to both serine/threonine and tyrosine protein kinases. There are a number of conserved regions in the catalytic domain of protein kinases. Two of the conserved regions are the basis for the signature pattern in the protein kinase profile. The first region, which is located in the N-terminal extremity of the catalytic domain, is a glycine-rich stretch of residues in the vicinity of a lysine residue, which has been shown to be involved in ATP binding. The second region, which is located in the central part of the catalytic domain, contains a conserved aspartic acid residue which is important for the catalytic activity of the enzyme (Knighton D.R., et al, Science (1991) 255:407). The protein kinase profile includes two signature patterns for this second region: one specific for serine/threonine kinases and the other for tyrosine kinases. A third profile is based on the alignment in (Hanks S.K., et al, FASEB J. (1995) 9:576) and covers the entire catalytic domain. The consensus patterns are as follows:

1) Consensus pattern: [LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}- [LIVCAT]-{PD}-x-[GSTACLIVMFY]-x(5,18)-[LIVMFYWCSTAR]-[AIVP]- [LIVMFAGCKRj-K, where K binds ATP. The majority of known protein kinases are detected by this pattern. Proteins kinases that are not detected by this consensus include viral kinases, which are quite divergent in this region and are completely missed by this pattern.

2) Consensus pattern: [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x(2)-N- [LIVMFYCT](3), where D is an active site residue. This consensus sequence identifies most serine/threonine-specific protein kinases with only 10 exceptions. Half of the exceptions are viral kinases, while the other exceptions include Epstein-Barr virus BGLF4 and Drosophila ninaC, which have Ser and Arg, respectively, instead of the conserved Lys. These latter two protein kinases are detected by the tyrosine kinase specific pattern described below. 3) Consensus pattern: [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-[RSTAC]-x(2)-N-

[LIVMFYC], where D is an active site residue. All tyrosine-specific protein kinases are detected by this consensus pattern, with the exception of human ERBB3 and mouse blk. This pattern also detects most bacterial aminoglycoside phosphotransferases (Benner S., Nature (1987) 529:21; Kirby R., J. Mol. Evol. (1992) 50:489) and herpesviruses ganciclovir kinases (Littler E., et al, Nature (1992) 555:160), which are structurally and evolutionary related to protein kinases.

The protein kinase profile also detects receptor guanylate cyclases and 2-5A- dependent ribonucleases. Sequence similarities between these two families and the eukaryotic protein kinase family have been noticed previously. The profile also detects Arabidopsis thaliana kinase-like protein TMKL1 which seems to have lost its catalytic activity.

If a protein analyzed includes the two of the above protein kinase signatures, the probability of it being a protein kinase is close to 100%. Eukaryotic-type protein kinases have also been found in prokaryotes such as Myxococcus xanthus (Munoz-Dorado J., et al. , Ce/7 (1991) 67:995) and Yersinia pseudotuberculosis. The patterns shown above has been updated since their publication in (Bairoch A., et al, Nature (1988) 331:22). m) Protein Phosphatase 2C. SEQ ID NO:256 corresponds to a polynucleotide encoding a novel protein phosphatase 2C (PP2C), which is one of the four major classes of mammalian serine/threonine specific protein phosphatases. PP2C (Wenk et al, FEBS Lett. (1992) 297:135) is a monomeric enzyme of about 42 Kd which shows broad substrate specificity and is dependent on divalent cations (mainly manganese and magnesium) for its activity. Three isozymes are currently known in mammals: PP2C-alpha, -beta and -gamma, n) Protein Tyrosine Phosphatase. SEQ ID NO:382 represents a polynucleotide encoding a protein tyrosine kinase. Tyrosine specific protein phosphatases (EC 3.1.3.48) (PTPase) (Fischer et al, Science (1991) 255:401; Charbonneau et al, Annu. Rev. Cell Biol. (1992) 5:463; Trowbridge, J. Biol. Chem. (1991) 266:23517; Tonks et al, Trends Biochem. Sci. (1989) 74:497; and Hunter, Cell (1989) 55:1013) catalyze the removal of a phosphate group attached to a tyrosine residue. These enzymes are very important in the control of cell growth, proliferation, differentiation and transformation. Multiple forms of PTPase have been characterized and can be classified into two categories: soluble PTPases and transmembrane receptor proteins that contain PTPase domain(s).

Soluble PTPases include PTPN3 (HI) and PTPN4 (MEG), enzymes that contain an N-terminal band 4.1 -like domain and could act at junctions between the membrane and cytoskeleton; PTPN6 (PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp), enzymes that contain two copies of the SH2 domain at its N-terminal extremity.

Dual specificity PTPases include DUSP1 (PTPN10; MAP kinase phosphatase- 1; MKP-1) which dephosphorylates MAP kinase on both Thr- 183 and Tyr- 185; and DUSP2 (PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 and ERK2 on both Thr and Tyr residues.

Structurally, all known receptor PTPases are made up of a variable length extracellular domain, followed by a transmembrane region and a C-terminal catalytic cytoplasmic domain. Some of the receptor PTPases contain fibronectin type III (FN-III) repeats, immunoglobulin-like domains, MAM domains or carbonic anhydrase-like domains in their extracellular region. The cytoplasmic region generally contains two copies of the

PTPAse domain. The first seems to have enzymatic activity, while the second is inactive but seems to affect substrate specificity of the first. In these domains, the catalytic cysteine is generally conserved but some other, presumably important, residues are not.

PTPase domains consist of about 300 amino acids. There are two conserved cysteines and the second one has been shown to be absolutely required for activity.

Furthermore, a number of conserved residues in its immediate vicinity have also been shown to be important. The consensus pattern for PTPases is: [LIVMF]-H-C-x(2)-G-x(3)-[STC]- [STAGP]-x-[LIVMFY]; C is the active site residue. o) SH3 Domain. SEQ ID NO:306 and 386 represent polynucleotides encoding SH3 domain proteins. The Src homology 3 (SH3) domain is a small protein domain of about 60 amino acid residues first identified as a conserved sequence in the non-catalytic part of several cytoplasmic protein tyrosine kinases (e.g. Src, Abl, Lck) (Mayer et al, Nature (1988) 552:272). The domain has also been found in a variety of intracellular or membrane- associated proteins (Musacchio et al, FEBS Lett. (1992) 507:55; Pawson et al, Curr. Biol. (1993) 5:434; Mayer et al, Trends Cell Biol. (1993) 5:8; and Pawson et al, Nature (1995) 575:573).

The SH3 domain has a characteristic fold that consists of five or six beta-strands arranged as two tightly packed anti-parallel beta sheets. The linker regions may contain short helices (Kuriyan et al, Curr. Opin. Struct. Biol. (1993) 5:828). It is believed that SH3 domain-containing proteins mediate assembly of specific protein complexes via binding to proline-rich peptides (Morton et al, Curr. Biol. (1994) 4:615). In general, SH3 domains are found as single copies in a given protein, but there is a significant number of proteins with two SH3 domains and a few with 3 or 4 copies.

SH3 domains have been identified in, for example, protein tyrosine kinases. such as the Src, Abl, Bkt, Csk and ZAP70 families of kinases; mammalian phosphatidylinositol- specific phospholipase C-gamma-1 and -2; mammalian phosphatidyl inositol 3-kinase regulatory p85 subunit; mammalian Ras GTPase-activating protein (GAP); mammalian Vav oncoprotein, a guanine nucleotide exchange factor of the CDC24 family; Drosophila lethal(l)discs large-1 tumor suppressor protein (gene Dlgl); mammalian tight junction protein ZO-1 ; vertebrate erythrocyte membrane protein p55; Caenorhabditis elegans protein lin-2; rat protein CASK; and mammalian synaptic proteins SAP90/PSD-95, CHAPS YN- 110/PSD-93, SAP97/DLG1 and SAP102. Novel SH3-domain containing polypeptides will facilitate elucidation of the role of such proteins in important biological pathways, such as ras activation. p) Trypsin. SEQ ID NO: 169 corresponds to a novel serine protease of the trypsin family. The catalytic activity of the serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue hydrogen-bonded to a histidine, which itself is hydrogen-bonded to a serine. The sequences in the vicinity of the active site serine and histidine residues are well conserved in this family of proteases (Brenner S., Nature

(1988) 554:528). Proteases known to belong to the trypsin family include: 1) Acrosin; 2)

Blood coagulation factors VII, IX, X, XI and XII, thrombin, plasminogen, and protein C; 3)

Cathepsin G; 4) Chymotrypsins; 5) Complement components Clr, Cis, C2, and complement factors B, D and I; 6) Complement-activating component of RA-reactive factor; 7) Cytotoxic cell proteases (granzymes A to H); 8) Duodenase I; 9) Elastases 1, 2, 3 A, 3B (protease E), leukocyte (medullasin).; 10) Enterokinase (EC 3.4.21.9) (enteropeptidase); 11) Hepatocyte growth factor activator; 12) Hepsin; 13) Glandular (tissue) kallikreins (including EGF- binding protein types A, B, and C, NGF-gamma chain, gamma-renin, prostate specific antigen (PSA) and tonin); 14) Plasma kallikrein; 15) Mast cell proteases (MCP) 1 (chymase) to 8; 16) Myeloblastin (proteinase 3) (Wegener's autoantigen); 17) Plasminogen activators (urokinase-type, and tissue-type); 18) Trypsins I, II, III, and IV; 19) Tryptases; 20) Snake venom proteases such as ancrod, batroxobin, cerastobin, flavoxobin, and protein C activator; 21) Collagenase from common cattle grub and collagenolytic protease from Atlantic sand fiddler crab; 22) Apolipoprotein(a); 23) Blood fluke cercarial protease; 24) Drosophila trypsin like proteases: alpha, easter, snake-locus; 25) Drosophila protease stubble (gene sb); and 26) Major mite fecal allergen Der p III. All the above proteins belong to family SI in the classification of peptidases (Rawlings N.D., et al, Meth. Enzymol. (1994) 244:19; http://www.expasy.ch/cgi-bin/lists7peptidas.txt) and originate from eukaryotic species. It should be noted that bacterial proteases that belong to family S2A are similar enough in the regions of the active site residues that they can be picked up by the same patterns.

The consensus patterns for this trypsin protein family are: 1) [LIVM]-[ST]-A- [STAG]-H-C, where H is the active site residue. All sequences known to belong to this class detected by the pattern, except for complement components Clr and Cis, pig plasminogen, bovine protein C, rodent urokinase, ancrod, gyroxin and two insect trypsins; 2) [DNSTAGC]-[GSTAPIMVQH]-x(2)-G-[DE]-S-G-[GS]-[SAPHV]- [LIVMFYWFΪ]- [LIVMFYSTANQH], where S is the active site residue. All sequences known to belong to this family are detected by the above consensus sequences, except for 18 different proteases which have lost the first conserved glycine. If a protein includes both the serine and the histidine active site signatures, the probability of it being a trypsin family serine protease is 100%. q) WD Domain. G-Beta Repeats. SEQ ID NOS: 188 and 335 represent novel members of the WD domain G-beta repeat family. Beta-transducin (G-beta) is one of the three subunits (alpha, beta, and gamma) of the guanine nucleotide-binding proteins (G proteins) which act as intermediaries in the transduction of signals generated by transmembrane receptors (Gilman, Annu. Rev. Biochem. (1987) 56:615). The alpha subunit binds to and hydrolyzes GTP; the functions of the beta and gamma subunits are less clear but they seem to be required for the replacement of GDP by GTP as well as for membrane anchoring and receptor recognition.

In higher eukaryotes, G-beta exists as a small multigene family of highly conserved proteins of about 340 amino acid residues. Structurally, G-beta consists of eight tandem repeats of about 40 residues, each containing a central Trp-Asp motif (this type of repeat is sometimes called a WD-40 repeat). Such a repetitive segment has been shown to exist in a number of other proteins including: human LIS1, a neuronal protein involved in type-1 lissencephaly; and mammalian coatomer beta' subunit (beta'-COP), a component of a cytosolic protein complex that reversibly associates with Golgi membranes to form vesicles that mediate biosynthetic protein transport.

The consensus pattern for the WD domain/G-Beta repeat family is: [LIVMSTAC]-

[LIVMFYWSTAGC]-[LIMSTAG]-[LIVMSTAGC]-x(2)-[DN]-x(2)-[LIVMWSTAC]-x-

[LIVMFSTAG]-W-[DEN]-[LIVMFSTAGCN]. r) wnt Family of Developmental Signaling Proteins. SEQ ID NO: 23, 291, 324, 330, 341, and 353 correspond to novel members of the wnt family of developmental signaling proteins. Wnt-1 (previously known as int-1), the seminal member of this family, (Nusse R.,

Trends Genet. (1988) 4:291) is a proto-oncogene induced by the integration of the mouse mammary tumor virus. It is thought to play a role in intercellular communication and seems to be a signalling molecule important in the development of the central nervous system (CNS). The sequence of wnt- 1 is highly conserved in mammals, fish, and amphibians. Wnt-

1 was found to be a member of a large family of related proteins (Nusse R., et al, Cell (1992) 69:1073; McMahon A.P., Trends Genet. (1992) 5:1; Moon R.T., BioEssays (1993) 75:91) that are all thought to be developmental regulators. These proteins are known as wnt-

2 (also known as irp), wnt-3, -3 A, -4, -5A, -5B, -6, -7 A, -7B, -8, -8B, -9 and -10. At least four members of this family are present in Drosophila; one of them, wingless (wg), is implicated in segmentation polarity. All these proteins share the following features characteristics of secretory proteins: a signal peptide, several potential N-glycosylation sites and 22 conserved cysteines that are probably involved in disulfide bonds. The Wnt proteins seem to adhere to the plasma membrane of the secreting cells and are therefore likely to signal over only few cell diameters. The consensus pattern, which is based upon a highly conserved region including three cysteines, is as follows: C-K-C-H-G-[LIVMT]-S-G-x-C.

All sequences known to belong to this family are detected by the provided consensus pattern. s) Ww/rsp5/WWP Domain-Containing Proteins. SEQ ID NOS: 188, 379 , and 395 represent polynucleotides encoding a polypeptide in the family of WW/rsp5/WWP domain- containing proteins. The WW domain (Bork et al, Trends Biochem. Sci. (1994) 79:531 ;

Andre et al, Biochem. Biophys. Res. Commun. (1994) 205:1201; Hofmann et al, FEBS Lett. (1995) 555:153; and Sudol et al, FEBS Lett. (1995) 569:67), also known as rsp5 or WWP), was originally discovered as a short conserved region in a number of unrelated proteins, among them dystrophin, the gene responsible for Ducherme muscular dystrophy. The domain, which spans about 35 residues, is repeated up to 4 times in some proteins. It has been shown (Chen et al, Proc. Natl. Acad. Sci. USA (1995) 92:7819) to bind proteins with particular proline-motifs, [AP]-P-P-[AP]-Y, and thus resembles somewhat SH3 domains. It appears to contain beta-strands grouped around four conserved aromatic positions, generally Trp. The name WW or WWP derives from the presence of these Trp as well as that of a conserved Pro. It is frequently associated with other domains typical for proteins in signal transduction processes.

Proteins containing the WW domain include:

1. Dystrophin, a multidomain cytoskeletal protein. Its longest alternatively spliced form consists of an N-terminal actin-binding domain, followed by 24 spectrin-like repeats, a cysteine-rich calcium-binding domain and a C-terminal globular domain. Dystrophins form tetramers and is thought to have multiple functions including involvement in membrane stability, transduction of contractile forces to the extracellular environment and organization of membrane specialization. Mutations in the dystrophin gene lead to muscular dystrophy of Ducherme or Becker type. Dystrophin contains one WW domain C-terminal of the spectrin-repeats.

2. Vertebrate YAP protein, which is a substrate of an unknown serine kinase. It binds to the SH3 domain of the Yes oncoprotein via a proline-rich region. This protein appears in alternatively spliced isoforms, containing either one or two WW domains. 3. IQGAP, which is a human GTPase activating protein acting on ras. It contains an N-terminal domain similar to fly muscle mp20 protein and a C-terminal ras

GTPase activator domain.

For the sensitive detection of WW domains, the profile spans the whole homology region as well as a pattern. The consensus for this family is: W-x(9,l 1)-[VFY]-[FYW]- x(6,7)-[GSTNE]-[GSTQCR]-[FYW]-x(2)-P. t) Zinc Finger, C2H2 Type. SEQ ID NO:61, 306, and 386 correspond to polynucleotides encoding novel members of the of the C2H2 type zinc finger protein family.

Zinc finger domains (Klug et al, Trends Biochem. Sci. (1987) 72:464; Evans et al, Cell

(1988) 52:1; Payre et al, FEBS Lett. (1988) 254:245; Miller et al, EMBOJ. (1985) 4:1609; and Berg, Proc. Natl. Acad. Sci. USA (1988) 55:99) are nucleic acid-binding protein structures first identified in the Xenopus transcription factor TFIIIA. These domains have since been found in numerous nucleic acid-binding proteins. A zinc finger domain is composed of 25 to 30 amino acid residues. Two cysteine or histidine residues are positioned at both extremities of the domain, which are involved in the tetrahedral coordination of a zinc atom. It has been proposed that such a domain interacts with about five nucleotides.

Many classes of zinc fingers are characterized according to the number and positions of the histidine and cysteine residues involved in the zinc atom coordination. In the first class to be characterized, called C2H2, the first pair of zinc coordinating residues are cysteines, while the second pair are histidines. A number of experimental reports have demonstrated the zinc-dependent DNA or RNA binding property of some members of this class.

Mammalian proteins having a C2H2 zipper include (number in parenthesis indicates number of zinc finger regions in the protein): basonuclin (6), BCL-6/LAZ-3 (6), erythroid krueppel-like transcription factor (3), transcription factors Spl (3), Sp2 (3), Sp3 (3) and Sp(4) 3, transcriptional repressor YY1 (4), Wilms' tumor protein (4), EGRl/Krox24 (3), EGR2/Krox20 (3), EGR3/Pilot (3), EGR4/AT133 (4), Evi-1 (10), GLI1 (5), GLI2 (4+), GLI3 (3+), HIV-EP1/ZNF40 (4), HIV-EP2 (2), KR1 (9+), KR2 (9), KR3 (15+), KR4 (14+), KR5 (11+), HF.12 (6+), REX-1 (4), ZfX (13), ZfY (13), Zfp-35 (18), ZNF7 (15), ZNF8 (7), ZNF35 (10), ZNF42/MZF-1 (13), ZNF43 (22), ZNF46/Kup (2), ZNF76 (7), ZNF91 (36), ZNF133 (3).

In addition to the conserved zinc ligand residues, it has been shown that a number of other positions are also important for the structural integrity of the C2H2 zinc fingers. (Rosenfeld et al, J. Biomol Struct. Dyn. (1993) 77:557) The best conserved position is found four residues after the second cysteine; it is generally an aromatic or aliphatic residue.

The consensus pattern for C2H2 zinc fingers is: C-x(2,4)-C-x(3)-[LIVMFYWC]- x(8)-H-x(3,5)-H. The two C's and two H's are zinc ligands. u) Zinc Finger, CCHC Class. SEQ ID NO:322 corresponds to a polynucleotide encoding a novel member of the zinc finger CCHC family. The CCHC zinc finger protein family to date has been mostly composed of retroviral gag proteins (nucleocapsid). The prototype structure of this family is from HIV. The family also contains members involved in eukaryotic gene regulation, such as C. elegans GLH-1. The consensus sequence of this family is based upon the common structure of an 18-residue zinc finger. v) Zinc-Binding Metalloprotease Domain. SEQ ID NO: 306 and 395 represent polynucleotides encoding novel members of the zinc-binding metalloprotease domain protein family. The majority of zinc-dependent metallopeptidases (with the notable exception of the carboxypeptidases) share a common pattern of primary structure (Jongeneel et al. , FEBS Lett. ( 1989) 242:211 ; Murphy et al. , FEBS Lett. ( 1991 ) 259:4; and Bode et al. , Zoology (1996) 99:237) in the part of their sequence involved in the binding of zinc, and can be grouped together as a superfamily, known as the metzincins, on the basis of this sequence similarity. Examples of these proteins include: 1) Angiotensin-converting enzyme (EC 3.4.15.1) (dipeptidyl carboxypeptidase I) (ACE), the enzyme responsible for hydrolyzing angiotensin I to angiotensin II. 2) Mammalian extracellular matrix metalloproteinases (known as matrixins) (Woessner, FASEB J. (1991) 5:2145): MMP-1 (EC 3.4.24.7) (interstitial collagenase), MMP-2 (EC 3.4.24.24) (72 Kd gelatinase), MMP-9 (EC 3.4.24.35) (92 Kd gelatinase), MMP-7 (EC 3.4.24.23) (matrylisin), MMP-8 (EC 3.4.24.34) (neutrophil collagenase), MMP-3 (EC 3.4.24.17) (stromelysin-1), MMP-10 (EC 3.4.24.22) (stromelysin-2), and MMP-11 (stromelysin-3), MMP-12 (EC 3.4.24.65) (macrophage metalloelastase). 3) Endothelin-converting enzyme 1 (EC 3.4.24.71) (ECE-1), which processes the precursor of endothelin to release the active peptide.

A signature pattern which includes the two histidine and the glutamic acid residues is sufficient to detect this superfamily of proteins, having the consensus pattern: [GSTALIVN]- x(2)-H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ]. The two H's are zinc ligands, and E is the active site residue.

Example 4: Differential Expression of Polynucleotides of the Invention : Description of Libraries and Detection of Differential Expression The relative expression levels of the polynucleotides of the invention was assessed in several libraries prepared from various sources, including cell lines and patient tissue samples. Table 4 provides a summary of these libraries, including the shortened library name (used hereafter), the mRNA source used to prepared the cDNA library, the "nickname" of the library that is used in the tables below (in quotes), and the approximate number of clones in the library. Table 4 Description of cDNA Libraries

The KM12L4 and KM12C cell lines are described in Example 1 above. The MDA-

MB-231 cell line was originally isolated from pleural effusions (Cailleau, J. Natl. Cancer. Inst. (1974) 55:661), is of high metastatic potential, and forms poorly differentiated adenocarcinoma grade II in nude mice consistent with breast carcinoma. The MCF7 cell line was derived from a pleural effusion of a breast adenocarcinoma and is non-metastatic. The MV-522 cell line is derived from a human lung carcinoma and is of high metastatic potential. The UCP-3 cell line is a low metastatic human lung carcinoma cell line; the MV- 522 is a high metastatic variant of UCP-3. These cell lines are well-recognized in the art as models for the study of human breast and lung cancer (see, e.g., Chandrasekaran et al, Cancer Res. (1979) 59:870 (MDA-MB-231 and MCF-7 ); Gastpar et al. , J Med Chem (1998) 47:4965 (MDA-MB-231 and MCF-7); Ranson et al., Br J Cancer (1998) 77:1586 (MDA- MB-231 and MCF-7); Kuang et al, Nucleic Acids Res (1998) 26:1116 (MDA-MB-231 and MCF-7); Varki et al. nt J Cancer (1987) 40:46 (UCP-3); Varki et al, Tumour Biol. (1990) 77:327; (MV-522 and UCP-3); Varki et al. , Anticancer Res. (1990) 70:637; (MV-522); Kelner et al. , Anticancer Res (1995) 75:867 (MV-522); and Zhang et al. , Anticancer Drugs (1997) 5:696 (MV522)). The samples of libraries 15-20 are derived from two different patients (UC#2, and UC#3).

Each of the libraries is composed of a collection of cDNA clones that in turn are representative of the mRNAs expressed in the indicated mRNA source. In order to facilitate the analysis of the millions of sequences in each library, the sequences were assigned to clusters. The concept of "cluster of clones" is derived from a sorting/grouping of cDNA clones based on their hybridization pattern to a panel of roughly 300 7bp oligonucleotide probes (see Drmanac et al, Genomics (1996) 7(1):29). Random cDNA clones from a tissue library are hybridized at moderate stringency to 300 7bp oligonucleotides. Each oligonucleotide has some measure of specific hybridization to that specific clone. The combination of 300 of these measures of hybridization for 300 probes equals the "hybridization signature" for a specific clone. Clones with similar sequence will have similar hybridization signatures. By developing a sorting/grouping algorithm to analyze these signatures, groups of clones in a library can be identified and brought together computationally. These groups of clones are termed "clusters". Depending on the stringency of the selection in the algorithm (similar to the stringency of hybridization in a classic library cDNA screening protocol), the "purity" of each cluster can be controlled. For example, artifacts of clustering may occur in computational clustering just as artifacts can occur in "wet-lab" screening of a cDNA library with 400 bp cDNA fragments, at even the highest stringency. The stringency used in the implementation of cluster herein provides groups of clones that are in general from the same cDNA or closely related cDNAs. Closely related clones can be a result of different length clones of the same cDNA, closely related clones from highly related gene families, or splice variants of the same cDNA. Differential expression for a selected cluster was assessed by first determining the number of cDNA clones corresponding to the selected cluster in the first library (Clones in 1^st), and the determining the number of cDNA clones corresponding to the selected cluster in the second library (Clones in 2^nd). Differential expression of the selected cluster in the first library relative to the second library is expressed as a "ratio" of percent expression between the two libraries. In general, the "ratio" is calculated by: 1) calculating the percent expression of the selected cluster in the first library by dividing the number of clones corresponding to a selected cluster in the first library by the total number of clones analyzed from the first library; 2) calculating the percent expression of the selected cluster in the second library by dividing the number of clones corresponding to a selected cluster in a second library by the total number of clones analyzed from the second library; 3) dividing the calculated percent expression from the first library by the calculated percent expression from the second library. If the "number of clones" corresponding to a selected cluster in a library is zero, the value is set at 1 to aid in calculation. The formula used in calculating the ratio takes into account the "depth" of each of the libraries being compared, i.e., the total number of clones analyzed in each library.

In general, a polynucleotide is said to be significantly differentially expressed between two samples when the ratio value is greater than at least about 2, preferably greater than at least about 3, more preferably greater than at least about 5 , where the ratio value is calculated using the method described above. The significance of differential expression is determined using a z score test (Zar, Biostatistical Analysis. Prentice Hall, Inc., USA, "Differences between Proportions," pp 296-298 (1974).

Tables 5 to 7 (inserted before the claims) show the number of clones in each of the above libraries that were analyzed for differential expression. Examples of differentially expressed polynucleotides of particular interest are described in more detail below.

Example 5: Polynucleotides Differentially Expressed in High Metastatic Potential Breast

Cancer Cells Versus Low Metastatic Breast Cancer Cells

A number of polynucleotide sequences have been identified that are differentially expressed between cells derived from high metastatic potential breast cancer tissue and low metastatic breast cancer cells. Expression of these sequences in breast cancer can be valuable in determining diagnostic, prognostic and/or treatment information. For example, sequences that are highly expressed in the high metastatic potential cells can be indicative of increased expression of genes or regulatory sequences involved in the metastatic process. A patient sample displaying an increased level of one or more of these polynucleotides may thus warrant more aggressive treatment. In another example, sequences that display higher expression in the low metastatic potential cells can be associated with genes or regulatory sequences that inhibit metastasis, and thus the expression of these polynucleotides in a sample may warrant a more positive prognosis than the gross pathology would suggest. The differential expression of these polynucleotides can be used as a diagnostic marker, a prognostic marker, for risk assessment, patient treatment and the like. These polynucleotide sequences can also be used in combination with other known molecular and/or biochemical markers.

The following table summarizes identified polynucleotides with differential expression between high metastatic potential breast cancer cells and low metastatic potential breast cancer cells.

Table 8. Differentially expressed polynucleotides: High metastatic potential breast cancer vs. low metastatic breast cancer cells

Library Library

9 H gh Breast > Low Breast Lib3 > Lib4) 2623 31 4 7.561356

42 H gh Breast > Low Breast Lib3 > Lib4) 307 196 75 2.549721

52 H gh Breast > Low Breast Lib3 > Lib4) 19 1364 525 2.534854

62 H gh Breast > Low Breast Lib3 > Lib4) 2623 31 4 7.561356

65 H gh Breast > Low Breast Lib3 > Lib4) 5749 9 0 8.780930

66 H gh Breast > Low Breast Lib3 > Lib4) 6455 6 0 5.853953

68 H gh Breast > Low Breast Lib3 > Lib4) 6455 6 0 5.853953

114 H gh Breast > Low Breast Lib3 > Lib4) 2030 32 4 7.805271

123 H gh Breast > Low Breast Lib3 > Lib4) 3389 13 2 6.341782

144 H gh Breast > Low Breast Lib3 > Lib4) 4623 12 2 5.853953

172 H gh Breast > Low Breast Lib3 > Lib4) 102 278 1 16 2.338217

178 H gh Breast > Low Breast Lib3 > Lib4) 3681 10 1 9.756589

214 H gh Breast > Low Breast Lib3 > Lib4) 3900 8 1 7.805271

219 H gh Breast > Low Breast Lib3 > Lib4) 3389 13 2 6.341782

223 H gh Breast > Low Breast Lib3 > Lib4) 1399 19 7 2.648217

258 H gh Breast > Low Breast Lib3 > Lib4) 4837 10 0 9.756589

317 H gh Breast > Low Breast Lib3 > Lib4) 1577 25 3 8.130490

Cancer Cells Versus Low Metastatic Lung Cancer Cells A number of polynucleotide sequences have been identified that are differentially expressed between cells derived from high metastatic potential lung cancer tissue and low metastatic lung cancer cells. Expression of these sequences in lung cancer tissue can be valuable in determining diagnostic, prognostic and/or treatment information. For example, sequences that are highly expressed in the high metastatic potential cells are associated can be indicative of increased expression of genes or regulatory sequences involved in the metastatic process. A patient sample displaying an increased level of one or more of these polynucleotides may thus warrant more aggressive treatment. In another example, sequences that display higher expression in the low metastatic potential cells can be associated with genes or regulatory sequences that inhibit metastasis, and thus the expression of these polynucleotides in a sample may warrant a more positive prognosis than the gross pathology would suggest.

The differential expression of these polynucleotides can be used as a diagnostic marker, a prognostic marker, for risk assessment, patient treatment and the like. These polynucleotide sequences can also be used in combination with other known molecular and/or biochemical markers.

The following table summarizes identified polynucleotides with differential expression between high metastatic potential lung cancer cells and low metastatic potential lung cancer cells:

Table 9 Differentially expressed polynucleotides: High metastatic potential lung cancer vs. low metastatic lung cancer cells

SEQ ID Differential Expression Cluster Clones in Clones in Ratio

NO. ID 1^st 2^nd Library Library

400 High Lung > Low Lung (Lib8 > Lib 9) 14929 23 16 2.008868

9 High Lung > Low Lung (Lib8 > Lib9) 2623 6 1 8.384840

34 High Lung > Low Lung (Lib8 > Lib9) 5832 5 0 6.987366

42 High Lung > Low Lung (Lib 8 > Lib9) 307 79 27 4.088903

62 High Lung > Low Lung (Lib8 > Lib9) 2623 6 1 8.384840

74 High Lung > Low Lung (Lib8 > Lib9) 6268 5 0 6.987366

106 High Lung > Low Lung (Lib8 > Lib9) 10717 8 0 1 1.17978

119 High Lung > Low Lung (Lib8 > Lib9) 8 1355 122 15.521 1 1

361 High Lung > Low Lung (Lib8 > Lib9) 1120 5 0 6.987366

369 High Lung > Low Lung (Lib8 > Lib9) 2790 6 0 8.384840

371 High Lung > Low Lung (Lib8 > Lib9) 8847 6 1 8.384840

379 High Lung > Low Lung (Lib8 > Lib9) 260 15 0 20.96210

395 High Lung > Low Lung (Lib8 > Lib9) 13538 9 1 12.57726

135 Low Lung > High Lung (Lib9 > Lib8) 36313 30 1 21.46731

154 Low Lung > High Lung (Lib9 > Lib8) 5345 27 6 3.220097

160 Low Lung > High Lung (Lib9 > Lib8) 4386 21 3 5.009039

260 Low Lung > High Lung (Lib9 > Lib8) 4141 27 4 4.830145

308 Low Lung > High Lung (Lib9 > Lib8) 15855 213 12 12.70149

323 Low Lung > High Lung (Lib9 > Lib8) 5257 25 5 3.577885

349 Low Lung > High Lung (Lib9 > Lib8) 2797 14 1 10.01807

381 Low Lung > High Lung (Lib9 > Lib8) 2428 19 2 6.797982

Exampl e 7: Polvnucleotides Differentially Expressed i n High Metastatic Potential Colon

Cancer Cells Versus Low Metastatic Colon Cancer Cells

A number of polynucleotide sequences have been identified that are differentially expressed between cells derived from high metastatic potential colon cancer tissue and low metastatic colon cancer cells. Expression of these sequences in colon cancer tissue can be valuable in determining diagnostic, prognostic and/or treatment information. For example, sequences that are highly expressed in the high metastatic potential cells can be indicative of increased expression of genes or regulatory sequences involved in the metastatic process. A patient sample displaying an increased level of one or more of these polynucleotides may thus warrant more aggressive treatment. In another example, sequences that display higher expression in the low metastatic potential cells can be associated with genes or regulatory sequences that inhibit metastasis, and thus the expression of these polynucleotides in a sample may warrant a more positive prognosis than the gross pathology would suggest. The differential expression of these polynucleotides can be used as a diagnostic marker, a prognostic marker, for risk assessment, patient treatment and the like. These polynucleotide sequences can also be used in combination with other known molecular and/or biochemical markers.

The following table summarizes identified polynucleotides with differential expression between high metastatic potential colon cancer cells and low metastatic potential colon cancer cells:

Table 11: Differentially expressed polynucleotides: High metastatic potential colon cancer vs. low metastatic colon cancer cells

SEQ ID Differential Expression Cluster Clones in Clones in Ratio

NO. ID 1^st 2^nd

Library Library

1 High Colon > Low Colon (Lib 1 > Lib2) 6660 7 0 6.489973

176 High Colon > Low Colon (Lib 1 > Lib2) 3765 19 6 2.935940

241 High Colon > Low Colon (Lib 1 > Lib2) 4275 11 2 5.099264

362 High Colon > Low Colon (Lib 1 > Lib2) 6420 8 0 7.417112

374 High Colon > Low Colon (Lib 1 > Lib2) 6420 8 0 7.417112

39 Low Colon > High Colon (Lib2 > Lib 1) 4016 14 5 3.020043

97 Low Colon > High Colon (Lib2 > Lib 1 ) 945 21 9 2.516702

134 Low Colon > High Colon (Lib2 > Lib 1 ) 2464 19 5 4.098630

317 Low Colon > High Colon (Lib2 > Lib 1) 1577 40 12 3.595289

357 Low Colon > High Colon (Lib2 > Libl ) 4309 13 4 3.505407

Example 8: Polynucleotides Differentially Expressed at Higher Levels in High Metastatic

Potential Colon Cancer Patient Tissue Versus Normal Patient Tissue

A number of polynucleotide sequences have been identified that are differentially expressed between cells derived from high metastatic potential colon cancer tissue and normal tissue. Expression of these sequences in colon cancer tissue can be valuable in determining diagnostic, prognostic and/or treatment information. For example, sequences that are highly expressed in the high metastatic potential cells are associated can be indicative of increased expression of genes or regulatory sequences involved in the advanced disease state which involves processes such as angiogenesis, dedifferentiation, cell replication, and metastasis. A patient sample displaying an increased level of one or more of these polynucleotides may thus warrant more aggressive treatment. The differential expression of these polynucleotides can be used as a diagnostic marker, a prognostic marker, for risk assessment, patient treatment and the like. These polynucleotide sequences can also be used in combination with other known molecular and/or biochemical markers.

The following table summarizes identified polynucleotides with differential expression between high metastatic potential colon cancer cells and normal colon cells:

Table 11: Differentially expressed polynucleotides: High metastatic potential colon tissue vs. normal colon tissue

SEQ ID Differential Expression Cluster Clones in Clones in Ratio

NO. ID 1^st 2^nd

Library Library

52 High Colon Metastasis Tissue > Normal 19 10 0 1 1.69918

Colon Tissue of UC#3 (Lib20 > Lib 18) 52 High Colon Metastasis Tissue > Normal 19 13 2 6.025646

Tissue in UC#2 (Lib 17 > Lib 15) 172 High Colon Metastasis Tissue > Normal 102 65 22 2.738930

Tissue in UC#2 (Lib 17 > Lib 15)

Example 9: Polynucleotides Differentially Expressed at Higher Levels in High Colon Tumor Potential Patient Tissue Versus Metastasized Colon Cancer Patient Tissue A number of polynucleotide sequences have been identified that are differentially expressed between cells derived from high tumor potential colon cancer tissue and cells derived from high metastatic potential colon cancer cells. Expression of these sequences in colon cancer tissue can be valuable in determining diagnostic, prognostic and/or treatment information associated with the transformation of precancerous tissue to malignant tissue. This information can be useful in the prevention of achieving the advanced malignant state in these tissues, and can be important in risk assessment for a patient. The following table summarizes identified polynucleotides with differential expression between high tumor potential colon cancer tissue and cells derived from high metastatic potential colon cancer cells: Table 12: Differentially expressed polynucleotides: High tumor potential colon tissue vs. metastatic colon tissue

SEQ ID Differential Expression Cluster Clones in 1^st Clones in 2 nd Ratio NO. ID Library Library

52 High Colon Tumor Tissue > Metastasis 19 69 10 5.160829 Tissue of UC#3 (Lib 19 > Lib20)

119 High Colon Tumor Tissue > Metastasis 14 10.47124 Tissue of UC#3 (Lib 19 > Lib20)

172 High Colon Tumor Tissue > Metastasis 102 43 3.216168 Tissue of UC#3 (Lib 19 > Lib20)

Example 10: Polynucleotides Differentially Expressed at Higher Levels in High Tumor Potential Colon Cancer Patient Tissue Versus Normal Patient Tissue A number of polynucleotide sequences have been identified that are differentially expressed between cells derived from high tumor potential colon cancer tissue and normal tissue. Expression of these sequences in colon cancer tissue can be valuable in determining diagnostic, prognostic and/or treatment information associated with the prevention of achieving the malignant state in these tissues, and can be important in risk assessment for a patient. For example, sequences that are highly expressed in the potential colon cancer cells are associated with or can be indicative of increased expression of genes or regulatory sequences involved in early tumor progression. A patient sample displaying an increased level of one or more of these polynucleotides may thus warrant closer attention or more frequent screening procedures to catch the malignant state as early as possible.

The following table summarizes identified polynucleotides with differential expression between high metastatic potential colon cancer cells and normal colon cells:

Table 13: Differentially expressed polynucleotides: High tumor potential colon tissue vs. normal colon tissue

SEQ ID Differential Expression Cluster Clones in Clones in Ratio NO. ID I^s' 2^nd

Library Library

52 High Colon Tumor Tissue > Normal Tissue 19 13 2 6.255508 ofUC#2 (Libl6 > Libl5) 288 High Colon Tumor Tissue > Normal Tissue 1267 7 0 6.125253 ofUC#2 (Libl6 > Libl5) 52 High Colon Tumor Tissue > Normal Tissue 19 69 0 60.37750 ofUC#3 (Libl9 > Libl8) 119 High Colon Tumor Tissue > Normal Tissue 8 14 1 12.25050 ofUC#3 (Libl9 > Libl8) 172 High Colon Tumor Tissue > Normal Tissue 102 43 7 5.375222 ofUC#3 (Libl9 > Libl8) Example 1 1 : Polynucleotides Differentially Expressed Across Multiple Libraries

A number of polynucleotide sequences have been identified that are differentially expressed between cancerous cells and normal cells across all three tissue types tested (i.e. , breast, colon, and lung). Expression of these sequences in a tissue or any origin can be valuable in determining diagnostic, prognostic and/or treatment information associated with the prevention of achieving the malignant state in these tissues, and can be important in risk assessment for a patient. These polynucleotides can also serve as non-tissue specific markers of, for example, risk of metastasis of a tumor. The following table summarizes identified polynucleotides that were differentially expressed but without tissue type- specificity in the breast, colon, and lung libraries tested.

Table 14: Polynucleotides Differentially Expressed Across Multiple Library Comparisons

SEQ ID Differential Expression Cluster Clones in Clones in Ratio NO. ID 1^st 2^nd

Library Library

9 High Breast > Low Breast (Lib3 > Lib4) 2623 31 4 7.561356 High Lung > Low Lung (Lib8 > Lib9) 2623 6 1 8.384840

39 Low Breast > High Breast (Lib4 > Lib3) 4016 6 0 6.149690 Low Colon > High Colon (Lib2 > Libl) 4016 14 5 3.020043

42 High Breast > Low Breast (Lib3 > Lib4) 307 196 75 2.549721 High Lung > Low Lung (Lib8 > Lib9) 307 79 27 4.088903

52 High Breast > Low Breast (Lib3 > Lib4) 19 1364 525 2.534854

High Colon Metastasis Tissue > Normal 19 10 0 11.69918 Colon Tissue of UC#3 (Lib20 > Libl 8) High Colon Metastasis Tissue > Normal 19 13 2 6.025646 Tissue in UC#2 (Lib 17 > Lib 15) High Colon Tumor Tissue > Metastasis 19 69 10 5.160829 Tissue of UC#3 (Lib 19 > Lib20) High Colon Tumor Tissue > Normal Tissue 19 13 2 6.255508 ofUC#2 (Libl6 > Libl5) High Colon Tumor Tissue > Normal Tissue 19 69 0 60.37750 ofUC#3 (Libl9 > Libl8)

62 High Breast > Low Breast (Lib3 > Lib4) 2623 31 4 7.561356 High Lung > Low Lung (Lib8 > Lib9) 2623 6 1 8.384840

74 High Lung > Low Lung (Lib8 > Lib9) 6268 5 0 6.987366

Low Breast > High Breast (Lib4 > Lib3) 6268 18 3 6.149690

1 19 High Colon Tumor Tissue > Metastasis 8 14 1 10.47124

Tissue of UC#3 (Lib 19 > Lib20) High Colon Tumor Tissue > Normal Tissue 14 1 12.25050 ofUC#3 (Libl9 > Libl8) High Lung > Low Lung (Lib8 > Lib9) 8 1355 122 15.521 1 1

172 High Breast > Low Breast (Lib3 > Lib4) 102 278 1 16 2.338217

High Colon Metastasis Tissue > Normal 102 65 22 2.738930 Tissue in UC#2 (Lib 17 > Lib 15)

High Colon Tumor Tissue > Metastasis 102 43 10 3.216168 in Ratio

Library Library

Tissue of UC#3 (Libl 9 > Lib20)

High Colon Tumor Tissue > Normal Tissue 10 022 43 7 5.375222 ofUC#3 (Libl9 > Libl8)

317 High Breast > Low Breast (Lib3 > Lib4) 1577 25 3 8.130490

Low Colon > High Colon (Lib2 > Libl) 1577 40 12 3.595289

379 High Breast > Low Breast (Lib3 > Lib4) 260 27 2 13.17139

High Lung > Low Lung (Lib8 > Lib9) 260 15 0 20.96210

Example 12: Polynucleotides Exhibiting Colon-Specific Expression

The cDNA libraries described herein were also analyzed to identify those polynucleotides that were specifically expressed in colon cells or tissue, i.e., the polynucleotides were identified in libraries prepared from colon cell lines or tissue, but not in libraries of breast or lung origin. The polynucleotides that were expressed in a colon cell line and/or in colon tissue, but were present in the breast or lung cDNA libraries described herein, are shown in Table 15.

Table 15 Polynucleotides specifically expressed in colon cells.

SEQ IE » Cluster Clones in Clones in SEQ ID Cluster Clones in Clones in

NO. 1^st Library 2^nd Library NO. 1^st Library 2^nd Library

5 36535 2 0 229 39648 2 0

13 27250 2 0 231 85064 1 0

19 16283 3 0 234 39391 2 0

24 16918 4 0 236 39498 2 0

26 40108 2 0 242 22113 3 0

32 32663 1 1 247 19255 2 0

43 39833 2 0 252 22814 3 0

47 18957 3 0 253 39563 2 0

48 39508 2 0 254 39420 2 0

56 7005 8 2 257 39412 2 0

58 18957 3 0 261 38085 2 0

59 18957 3 0 265 40054 1 0

60 16283 3 0 266 39423 2 0

64 13238 4 1 267 39453 2 0

70 39442 2 0 270 78091 1 0

71 17036 4 0 276 39168 2 0

73 7005 8 2 277 39458 2 0

83 11476 6 0 278 14391 3 1

86 39425 2 0 279 39195 2 0

94 21847 2 1 282 12977 5 0

100 16731 3 1 284 14391 3 1

101 12439 4 0 290 16347 4 0

113 17055 4 0 293 39478 2 0

120 67907 1 0 294 39392 2 0

121 12081 4 0 297 39180 2 0

124 39174 2 0 299 6867 7 3 SEQ ID Cluster Clones in Clones in SEQ ID Cluster Clones in Clones in

NO. 1^st Library 2^nd Library NO. JS1 Library 2 Library

126 8210 2 6 301 41633 1 1

128 40455 2 0 302 23218 3 0

139 22195 3 0 303 39380 2 0

143 86859 1 0 309 84328 1 0

150 8672 4 4 314 14367 3 0

153 16977 4 0 320 39886 2 0

156 17036 4 0 324 9061 5 2

159 40044 2 0 327 16653 3 1

161 40044 2 0 328 16985 4 0

163 22155 3 0 329 12977 5 0

166 15066 4 0 330 9061 5 2

170 11465 5 0 333 16392 3 0

176 3765 19 6 342 39486 2 0

181 86110 1 0 344 6874 6 3

182 39648 2 0 345 6874 6 3

185 17076 4 0 353 11494 4 0

186 22794 2 0 354 17062 -> 0

187 39171 2 0 355 16245 4 0

194 40455 2 0 356 83103 1 0

199 16317 3 0 358 13072 4 1

210 39186 2 0 366 14364 1 0

211 40122 2 0 368 84182 1 0

218 26295 2 0 372 56020 1 0

222 4665 5 9 389 7514 5 3

226 82498 1 0 391 7570 5 3

227 35702 2 0 393 23210 3 0

In addition to the above, SEQ ID NOS:159 and 161 were each present in one clone in each of Lib 16 (Normal Colon Tumor Tissue), and SEQ ID NOS:344 and 345 were each present in one clone in Lib 17 (High Colon Metastasis Tissue). No clones corresponding to the colon-specific polynucleotides in the table above were present in any of Libraries 3, 4, 8, or 9. The polynucleotide provided above can be used as markers of cells of colon origin, and find particular use in reference arrays, as described above.

Example 13: Identification of Contiguous Sequences Having a Polynucleotide of the Invention

The novel polynucleotides were used to screen publicly available and proprietary databases to determine if any of the polynucleotides of SEQ ID NOS: 1-404 would facilitate identification of a contiguous sequence, e.g., the polynucleotides would provide sequence that would result in 5' extension of another DNA sequence, resulting in production of a longer contiguous sequence composed of the provided polynucleotide and the other DNA sequence(s). Contiging was performed using the AssemblyLign program with the following parameters: 1) Overlap: Minimum Overlap Length: 30; % Stringency: 50; Minimum

Repeat Length: 30; Alignment: gap creation penalty: 1.00, gap extension penalty: 1.00; 2) Consensus: % Base designation threshold: 80.

Using these parameters, 44 polynucleotides provided contiged sequences. These contiged sequences are provided as SEQ ID NOS:801-844. The contiged sequences can be correlated with the sequences of SEQ ID NOS: 1-404 upon which the contiged sequences are based by identifying those sequences of SEQ ID NOS: 1-404 and the contiged sequences of SEQ ID NOS: 801-844 that share the same clone name in Table 1. It should be noted that of these 44 sequences that provided a contiged sequence, the following members of that group of 44 did not contig using the overlap settings indicated in parentheses (Stringency/Overlap): SEQ ID NO:804 (30%/10); SEQ ID NO:810 (20%/ 20); SEQ ID NO:812 (30%/10); SEQ ID NO:814 (40%/20); SEQ ID NO:816 (30%/10); SEQ ID NO:832 (30%/10); SEQ ID NO:840 (20%/20); SEQ ID NO:841 (40%/20). To generalize, the indicated polynucleotides did not contig using a minimum 20% stringency, 10 overlap. There was a corresponding increase in the number of degenerate codons in these sequences.

The contiged sequences (SEQ ID NO:801-844) thus represent longer sequences that encompass a polynucleotide sequence of the invention. The contiged sequences were then translated in all three reading frames to determine the best alignment with individual sequences using the BLAST programs as described above for SEQ ID NOS: 1-404 and the validation sequences SEQ ID NOS:405-800. Again the sequences were masked using the XBLAST profram for masking low complexity as described above in Example 1 (Table 2). Several of the contiged sequences were found to encode polypeptides having characteristics of a polypeptide belonging to a known protein families (and thus represent new members of these protein families) and/or comprising a known functional domain (Table 16). Thus the invention encompasses fragments, fusions, and variants of such polynucleotides that retain biological activity associated with the protein family and/or functional domain identified herein. Table 16. Profile hits using contiged sequences

All stop/start sequences are provided in the forward direction.

The profiles for the ATPases (AAA) and protein kinase families are described above in Example 2. The homeobox and MAP kinase kinase protein families are described further below.

Homeobox domain. The 'homeobox' is a protein domain of 60 amino acids (Gehring In: Guidebook to the Homeobox Genes. Duboule D., Ed., ppl-10, Oxford University Press, Oxford, (1994); Buerglin In: Guidebook to the Homeobox Genes, pp25-72, Oxford

University Press, Oxford, (1994); Gehring Trends Biochem. Sci. (1992) 7:277-280; Gehring et alAnnu. Rev. Genet. (1986) 20:147-173; Schofield Trends Neurosci. (1987) 70:3-6; http://copan.bioz.unibas.ch/ homeo.html) first identified in number of Drosophila homeotic and segmentation proteins. It is extremely well conserved in many other animals, including vertebrates. This domain binds DNA through a helix-turn-helix type of structure. Several proteins that contain a homeobox domain play an important role in development. Most of these proteins are sequence-specific DNA-binding transcription factors. The homeobox domain is also very similar to a region of the yeast mating type proteins. These are sequence-specific DNA-binding proteins that act as master switches in yeast differentiation by controlling gene expression in a cell type-specific fashion.

A schematic representation of the homeobox domain is shown below. The helix- turn-helix region is shown by the symbols 'H' (for helix), and 't' (for turn).

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxHHHHHHHHtttHHHHHHHHHxxxxxxxxxx 1 60 The pattern detects homeobox sequences 24 residues long and spans positions 34 to 57 of the homeobox domain. The consensus pattern is as follows: [LIVMFYG]-[ASLVR]-x(2)- [LIVMSTACN]-x-[LIVM]-x(4)-[LIV]-[RKNQESTAIY]-[LIVFSTNKH]-W-[FYVC]-x- [NDQTAH]-x(5)-[RKNAIMW] . MAP kinase kinase (MAPKK). MAP kinases (MAPK) are involved in signal transduction, and are important in cell cycle and cell growth controls. The MAP kinase kinases (MAPKK) are dual-specificity protein kinases which phosphorylate and activate MAP kinases. MAPKK homologues have been found in yeast, invertebrates, amphibians, and mammals. Moreover, the MAPKK/MAPK phosphorylation switch constitutes a basic module activated in distinct pathways in yeast and in vertebrates. MAPKK regulation studies have led to the discovery of at least four MAPKK convergent pathways in higher organisms. One of these is similar to the yeast pheromone response pathway which includes the stel 1 protein kinase. Two other pathways require the activation of either one or both of the serine/threonine kinase-encoded oncogenes c-Raf-1 and c-Mos. Additionally, several studies suggest a possible effect of the cell cycle control regulator cyclin-dependent kinase 1 (cdc2) on MAPKK activity. Finally, MAPKKs are apparently essential transducers through which signals must pass before reaching the nucleus. For review, see, e.g., Biologique Biol Cell (1993) 79:193-207; Nishida et al, Trends Biochem Sci (1993) 75:128-31; Ruderman Curr Opin Cell Biol (1993) 5:207-13; Dhanasekaran et al, Oncogene (1998) 77:1447-55; Kiefer et al, Biochem Soc Trans (1997) 25:491-8; and Hill, Ce/7 Signal (1996) 5:533-44. Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such specific embodiments and equivalents are intended to be encompassed by the following claims. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Deposit Information:

The following materials were deposited with the American Type Culture Collection: CMCC = (Chiron Master Culture Collection)

Cell Lines Deposited with ATCC

CDNA Library Deposits cDNA Library ESI - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001395A:C03 4016 79.Al .sp6:130016.Seq

M00001395A:C03 4016 RTAOOOOO 1 18A.C.4.1

M00001449A:D12 3681 RTA00000131A.g. l5.2

M00001449A:D12 3681 79.El.sp6:130064.Seq

M00001452A:D08 1120 79.C2.sp6:130041.Seq

M00001452A:D08 1120 RTAOOOOO 1 18A.p.15.3

M00001513A:B06 4568 79.D4.sp6: 130055.Seq

M00001513A:B06 4568 RTA00000122A.d.l5.3

M00001517A:B07 4313 79.F4.sp6: 130079.Seq

M00001517A:B07 4313 RTAOOOOO 122A.n.3.1

M00001533A:C11 2428 RTAOOOOO 123 A.1.21.1

M00001533A:C1 1 2428 79.A5.sp6:130020.Seq

M00001533A:C1 1 2428 RTA00000123A.1.21.1.Seq_THC205063

M00001542A:A09 22113 79.F5.sp6:130080.Seq

M00001542A:A09 221 13 RTA00000125A.C.7.1 cDNA Library ES2 - ATCC#

Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001343C.F10 2790 80.El .sp6:130256.Seq

M00001343C:F10 2790 RTAOOOOO 177AF.e.2.1.Seq_THC229461

M00001343C:F10 2790 RTAOOOOO 177AF.e.2.1

M00001343D:H07 23255 100.Cl .sp6:131446.Seq

M00001343D:H07 23255 RTAOOOOO 177AF.e.14.3.Seq_THC228776

M00001343D:H07 23255 80.Fl .sp6: 130268.Seq

M00001343D:H07 23255 RTAOOOOO 177AF.e.14.3

M00001345A:E01 6420 172.El .sp6:133925.Seq

M00001345A:E01 6420 RTAOOOOO 177AF.f.10.3

M00001345A:E01 6420 RTAOOOOO 177AF.f.10.3.Seq_THC226443

M00001345A:E01 6420 80.Gl .sp6:130280.Seq

M00001347A:B10 13576 80.D2.sp6:130245.Seq

M00001347A:B10 13576 100.El .sp6:131470.Seq

M00001347A:B10 13576 RTAOOOOO 177AF.g.16.1

M00O01353A:G12 8078 80.E3.sp6:130258.Seq

M00001353A:G12 8078 RTAOOOOO 177AR.1.13.1

M00001353A:G12 8078 172.C3.sp6:133903.Seq

M00001353D:D10 14929 RTAOOOOO 177AF.m.1.2

M00001353D:D10 14929 80.F3.sp6:130270.Seq

M00001353D:D10 14929 172.D3.sp6:133915.Seq

M00001361A.A05 4141 80.B4.sp6:130223.Seq

M00001361A:A05 4141 RTAOOOOO 177AF.p.20.3

M00001362B:D10 5622 80.D4.sp6:130247.Seq

M00001362B:D10 5622 RTAOOOOO 178AF.a.1 1.1 cDNA Library ES3 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001362C:H1 1 945 RTAOOOOO 178AR.a.20.1

M00001362C:H1 1 945 100.E4.sp6:131473.Seq

M00001362C:H1 1 945 80.E4.sp6:130259.Seq

M00001362C:H1 1 945 180.C2.sp6: 135940.Seq

M00001376B:G06 17732 RTAOOOOO 178AR.i.2.2

M00001376B:G06 17732 80.B5.sp6:130224.Seq

M00001387A:C05 2464 80.D6.sp6:130249.Seq

M00001387A:C05 2464 RTAOOOOO 178AF.n.18.1

M00001412B:B10 8551 RTAOOOOO 179AF.p.21.1

M00001412B:B10 8551 80.G7.sp6:130286.Seq

M00001415A:H06 13538 80.B8.sp6:130227.Seq

M00001415A:H06 13538 RTAOOOOO 180AF.a.24.1

M00001416B:H1 1 8847 80.C8.sp6: 130239.Seq

M00001416B:H1 1 8847 RTAOOOOO 180AF.b.16.1

M00001429D:D07 40392 RTAOOOOO 180AF.J.8.1

M00001429D:D07 40392 80.H9.sp6:130300.Seq

M00001448D:H01 36313 80.Al l .sp6: 130218.Seq

M00001448D:H01 36313 RTA00000181AF.e.23.1 cDNA Library ES4 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001463C:B1 1 19 RTAOOOOO 182AF.b.7.1

M00001463C:B11 19 89.Dl .sp6:130703.Seq

M00001470A:B10 1037 89.F2.sp6:130728.Seq

M00001470A:B10 1037 RTA00000121A.f.8.1

M00001497A:G02 2623 89.F3.sp6:130729.Seq

M00001497A:G02 2623 RTAOOOOO 183 AF.a.6.1

M00001500A:E11 2623 RTAOOOOO 183AF.b.14.1

M00001500A:E11 2623 89.A4.sp6:130670.Seq

M00001501D:C02 9685 RTAOOOOO 183 AF.c.1 1.1.Seq_THC 109544

M00001501D:C02 9685 RTAOOOOO 183 AF.c.1 1.1

M00001501D:C02 9685 89.C4.sp6:130694.Seq

M00001504C:H06 6974 89.F4.sp6:130730.Seq

M00001504C:H06 6974 RTAOOOOO 183 AF.d.9.1

M00001504C:H06 6974 RTAOOOOO 183AF.d.9.1.Seq_THC223129

M00001504D:G06 6420 173.F5.SP6:134133.Seq

M00001504D:G06 6420 89.G4.sp6:130742.Seq

M00001504D:G06 6420 RTAOOOOO 183 AF.d.11.1.Seq_THC226443

M00001504D:G06 6420 RTAOOOOO 183 AF.d.1 1.1

M00001528A:C04 35555 89.B6.sp6:130684.Seq

M00001528A:C04 7337 RTA00000123A.b.l7.1

M00001528A:C04 35555 184.A5.sp6: 135530.Seq cDNA Library ES5 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001537B:G07 3389 RTAOOOOO 183AF.m.19.1

M00001537B:G07 3389 89A8.sp6:130674.Seq

M00001541A:D02 3765 89.C8.sp6:130698.Seq

M00001541A:D02 3765 RTA00000135A.d.l . l

M00001544B:B07 6974 89.A9.sp6:130675.Seq

M00001544B:B07 6974 RTA00000184AF.a. l5.1

M00001546A:G1 1 1267 89.D9.sp6:13071 1.Seq

M00001546A:G1 1 1267 RTA00000125A.o.5.1

M00001549B:F06 4193 89.G9.sp6:130747.Seq

M00001549B:F06 4193 RTAOOOOO 184 AF.e.13.1

M00001556A:F1 1 1577 173.C9.SP6: 134101.Seq

M00001556A:F1 1 1577 89.Fl l .sp6: 130737.Seq

M00001556A:F1 1 1577 RTA00000184AF.i.23.1

M00001556B:C08 4386 RTAOOOOO 184AF.J.4.1

M00001556B:C08 4386 89.Hl l .sp6:130761.Seq cDNA Library ES6 - • ATCC#

Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001563B.F06 102 RTA00000184AF.O.5.1

M00001563B:F06 102 90.Bl .sp6: 130871. Seq

M00001571C:H06 5749 90.El .sp6:130907.Seq

M00001571C:H06 5749 RTAOOOOO 185AF.a.19.1

M00001594B:H04 260 90.D2.sp6:130896.Seq

M00001594B:H04 260 RTA00000185AR.i.l2.2

M00001597C:H02 4837 90.E2.sp6: 130908.Seq

M00001597C:H02 4837 RTAOOOOO 185AR.k.3.2

M00001624C:F01 4309 90.C4.sp6:130886.Seq

M00001624C:F01 4309 RTA00000186AF.e.22.1

M00001679A:A06 6660 90.F6.sp6: 130924.Seq

M00001679A:A06 6660 122.B5.sp6:132089.Seq

M00001679A:A06 6660 RTAOOOOO 187AF.h.15.1

M00003759B:B09 697 90.G8.sp6:130938.Seq

M00003759B:B09 697 RTAOOOOO 188AF.d.6.1

M00003759B:B09 697 RTAOOOOO 188AF.d.6.1.Seq_THC 178884

M00003844C:B11 6539 176.D9.sp6:134556.Seq

M00003844C:B11 6539 RTAOOOOO 189AF.d.22.1

M00003844C:B11 6539 90.B10.sp6:130880.Seq

M00003857A:G10 3389 90.Al l .sp6:130869.Seq

M00003857A:G10 3389 RTAOOOOO 189AF.g.3.1 cDNA Library ES7 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00003914C:F05 3900 99.El .sp6: 131278.Seq

M00003914C:F05 3900 RTAOOOOO 190AF.g.13.1

M00003922A:E06 23255 RTAOOOOO 190AF.J.4.1

M00003922A:E06 23255 99.Fl .sp6:131290.Seq

M00003922A:E06 23255 RTA00000190AF.j.4.1.Seq_THC228776

M00003983A:A05 9105 99.C3.sp6:131256.Seq

M00003983A:A05 9105 RTA00000191AF.a.21.2

M00004028D:A06 6124 RTA00000191AR.e.2.3

M00004028D:A06 6124 99.D3.sp6: 131268.Seq

M00004031A:A12 9061 RTA00000191AR.e.l l .2

M00004031A:A12 9061 RTA00000191AR.e.l l .3

M00004087D:A01 6880 RTA00000191AF.m.20.1

M00004087D:A01 6880 99.A5.sp6:131234.Seq

M00004108A:E06 4937 99.E5.sp6: 131282.Seq

M00004108A:E06 4937 RTA00000191AF.p.21.1

M000041 14C:F1 1 13183 123.D5.sp6:132305.Seq

M00004114C:F11 13183 RTAOOOOO 192AF.a.24.1

M000041 14C:F11 13183 99.G5.sp6:131306.Seq cDNA Library ES8 ^{■ ■} ATCC#

Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00004146C:C1 1 5257 99.B6.sp6: 131247.Seq

M00004146C:C1 1 5257 177.F5.sp6:134768.Seq

M00004146C:C1 1 5257 RTAOOOOO 192 AF.f.3.1

M00004146C:C1 1 5257 RTA00000192AF.f.3.1.Seq_THC213833

M00004157C:A09 6455 RTAOOOOO 192AF.g.23.1

M00004157C:A09 6455 99.D6.sp6: 131271.Seq

M00004157C:A09 6455 123.E7.sp6:132319.Seq

M00004172C:D08 11494 RTAOOOOO 192AF.J.6.1

M00004172C:D08 11494 99.G6.sp6: 131307.Seq

M00004172C:D08 11494 177.E6.sp6:134757.Seq

M00004229B:F08 6455 RTAOOOOO 193 AF.b.9.1

M00004229B:F08 6455 99.C8.sp6:131261.Seq cDNA Library ES9 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001466A:E07 4275 RTA00000120A.J.14.1

M00001531A:H11 89.F6.sp6:130732.Seq

M00001531A:H1 1 RTA00000123A.g.l9.1

M00001551A:B10 6268 79.G9.sp6:130096.Seq

M00001551A:B10 6268 184.C12.sp6:135561.Seq

M00001551A:B10 6268 RTAOOOOO 126A.0.23.1

M00001552A:B12 307 RTA00000136A.O.4.2

M00001552A:B12 307 79.C7.sp6: 130046.Seq

M00001556A:H01 15855 RTAOOOOO 184AF.J.1.1

M00001586C:C05 4623 RTAOOOOO 185 AF.f.4.1

M00001604A:B10 1399 79.G8.sp6: 130095.Seq

M00001604A:B10 1399 RTAOOOOO 129A.0.10.1

M00003879B:C11 5345 RTAOOOOO 189AF.1.19.1

M00003879B:C1 1 5345 90.B12.sp6:130882.Seq cDNA Library ES10 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001358C:C06 RTAOOOOO 177AF.0.4.3

M00001388D:G05 5832 80.F6.sp6:130273.Seq

M00001388D:G05 5832 RTAOOOOO 178AF.o.23.1

M00001394A:F01 6583 RTAOOOOO 179AF.d.13.1

M00001394A:F01 6583 172.B8.sp6: 133896.Seq

M00001394A:F01 6583 80.H6.sp6:130297.Seq

M00001429A:H04 2797 RTAOOOOO 180AF.i.19.1

M00001447A:G03 10717 RTAOOOOO 181 AF.d.10.1

M00001448D:C09 8 80.H10.sp6: 130301.Seq

M00001448D:C09 8 RTA00000181AF.e.l7.1

M00001448D:C09 8 100.Bl l .sp6:131444.Seq

M00001454D:G03 689 RTA00000181AR.1.22.1 cDNA Library ESI 1 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00003975A:G1 1 12439 RTAOOOOO 190AF.O.24.1

M00003978B:G05 5693 RTAOOOOO 190AF.p.17.2.Seq_THC 173318

M00003978B:G05 5693 RTAOOOOO 190AF.p.17.2

M00004059A:D06 5417 RTA00000191AF.h.l9.1

M00004068B:A01 3706 99.C4.sp6:131257.Seq

M00004068B:A01 3706 RTA00000191AF.i.l7.2

M00004205D:F06 99.E7.sp6:131284.Seq

M00004205D:F06 177.G7.sp6:134782.Seq

M00004205D:F06 RTAOOOOO 192AF.0.11.1

M00004212B:C07 2379 RTAOOOOO 192AF.p.8.1

M00004223A:G10 16918 RTAOOOOO 193AF.a.16.1 cDNA Library ESI 2 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00004223B:D09 7899 RTAOOOOO 193AF.a.17.1

M00004249D:G12 RTAOOOOO 193 AF.c.22.1

M00004251C:G07 RTAOOOOO 193 AF.d.2.1

M00004372A:A03 2030 RTAOOOOO 193 AF.m.20.1

cDNA Library ESI 3 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001340B:A06 17062 80.Al .sp6:130208.Seq

M00001340B:A06 17062 RTA00000177AF.b.8.4

M00001340D:F10 1 1589 80.Bl .sp6:130220.Seq

M00001340D:F10 11589 RTAOOOOO 177AF.b.17.4

M00001341A:E12 4443 80.Cl.sp6:130232.Seq

M00001341A:E12 4443 RTAOOOOO 177AF.b.20.4

M00001342B:E06 39805 80.Dl.sp6:130244.Seq

M00001342B:E06 39805 RTAOOOOO 177AF.C.21.3

M00001346A:F09 5007 RTA00000177AF.g.2.1

M00001346A:F09 5007 80.Hl.sp6:130292.Seq

M00001346D:G06 5779 RTAOOOOO 177AF.g.14.3

M00001346D:G06 5779 RTAOOOOO 177AF.g.14.1

M00001348B:B04 16927 80.E2.sp6:130257.Seq

M00001348B:B04 16927 RTA00000177AF.h.9.3

M00001348B:G06 16985 RTAOOOOO 177AF.h.10.1

M00001348B:G06 16985 80.F2.sp6:130269.Seq

M00001349B:B08 3584 RTAOOOOO 177AF.h.20.1

M00001349B:B08 3584 80.G2.sp6:130281.Seq

M00001350A:H01 7187 100.C2.sp6:131447.Seq

M00001350A:H01 7187 80.A3.sp6:130210.Seq

M00001350A:H01 7187 RTA00000177AF.i.8.2

M00001352A:E02 16245 RTAOOOOO 177AF.k.9.3

M00001352A:E02 16245 172.D2.sp6:133914.Seq

M00001352A:E02 16245 80.D3.sp6:130246.Seq

M00001355B:G10 14391 RTAOOOOO 177AF.m.17.3

M00001355B.G10 14391 80.G3.sp6:130282.Seq

M00001355B:G10 14391 172.H3.sp6:133963.Seq

M00001355B:G10 14391 100.E3.sp6:131472.Seq

M00001361D:F08 2379 80.C4.sp6:130235.Seq

M00001361D:F08 2379 RTAOOOOO 178AF.a.6.1

M00001365C:C10 40132 RTAOOOOO 178AF.C.7.1

M00001365C:C10 40132 80.F4.sp6:130271.Seq

M00001368D:E03 80.G4.sp6:13O283.Seq

M00001368D:E03 RTAOOOOO 178AF.d.20.1

M00001370A:C09 6867 80.H4.sp6:130295.Seq

M00001370A:C09 6867 RTAOOOOO 178AF.e.12.1

M00001371C:E09 7172 100.A5.sp6:131426.Seq

M00001371C:E09 7172 RTAOOOOO 178AF.f.9.1

M00001371C:E09 7172 80.A5.sp6:130212.Seq

M00001378B:B02 39833 80.C5.sp6:130236.Seq

M00001378B:B02 39833 RTAOOOOO 178AF.i.23.1

M00001379A:A05 1334 80.D5.sp6:130248.Seq

M00001379A:A05 1334 RTA00000178AF.J.7.1

M00001380D:B09 39886 RTA00000178AF.J.24.1

M00001380D:B09 39886 80.E5.sp6:130260.Seq

M00001381D:E06 80.F5.sp6: 130272.Seq

M00001381D:E06 RTAOOOOO 178AF.k.16.1

M00001382C:A02 22979 80.G5.sp6:130284.Seq

M00001382C:A02 22979 RTA00000178AF.k.22.1

M00001384B:A1 1 80.B6.sp6:130225.Seq

M00001384B:A1 1 RTAOOOOO 178AF.m.13.1

M00001386C:B12 5178 80.C6.sp6: 130237.Seq cDNA Library ESI 3 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001386C:B12 5178 RTAOOOOO 178AF.n.10.1

M00001387B:G03 7587 80.E6.sp6:130261.Seq

M00001387B:G03 7587 RTAOOOOO 178AF.n.24.1

M00001389A:C08 16269 RTAOOOOO 178AF.p.1.1

M00001389A:C08 16269 80.G6.sp6: 130285.Seq

M00001396A:C03 4009 172.D8.sp6: 133920.Seq

M00001396A:C03 4009 80.A7.sp6: 130214.Seq

M00001396A:C03 4009 RTAOOOOO 179AF.e.20.1

M00001400B:H06 172.B9.sp6:133897.Seq

M00001400B:H06 80.B7.sp6:130226.Seq

M00001400B:H06 RTAOOOOO 179 AF.j.13.1

M00001400B:H06 RTA00000179AF.j.l3.1.Seq_THC105720

M00001402A:E08 39563 80.C7.sp6:130238.Seq

M00001402A:E08 39563 RTAOOOOO 179AF.k.20.1

M00001407B:D1 1 5556 RTAOOOOO 179AF.n.10.1

M00001407B:D1 1 5556 80.D7.sp6: 130250.Seq

M00001410A:D07 7005 180.H5.sp6:136003.Seq

M00001410A:D07 7005 RTAOOOOO 179AF.0.22.1

M00001410A:D07 7005 80.F7.sp6: 130274.Seq

M00001414A.B01 RTAOOOOO 180AF.a.9.1

M00001414A:B01 80.H7.sp6: 130298.Seq

M00001414C:A07 80.A8.sp6:130215.Seq

M00001414C:A07 RTAOOOOO 180AF.a.11.1

M00001416A:H01 7674 79.Cl.sp6:130040.Seq

M00001416A:H01 7674 RTAOOOOO 118A.g.9.1

M00001417A:E02 36393 RTA00000180AF.C.2.1

M00001417A:E02 36393 80.D8.sp6: 130251.Seq

M00001423B:E07 15066 RTA00000180AF.e.24.1

M00001423B:E07 15066 80.H8.sp6:130299.Seq

M00001424B:G09 10470 80A9.sp6:130216.Seq

M00001424B:G09 10470 RTAOOOOO 180AF.f.18.1

M00001425B:H08 22195 RTAOOOOO 180AF.g.7.1

M00001425B:H08 22195 80.B9.sp6: 130228.Seq

M00001426B:D12 RTAOOOOO 180AF.g.22.1

M00001426B.D12 80.C9.sp6:130240.Seq

M00001426D:C08 4261 80.D9.sp6: 130252.Seq

M00001426D:C08 4261 RTAOOOOO 180AF.h.5.1

M00001428A:H10 84182 100.G9.sp6:131502.Seq

M00001428A:H10 84182 RTAOOOOO 180AF.h.19.1

M00001428A:H10 84182 80.E9.sp6:130264.Seq

M00001449A:A12 5857 80.Bl l .sp6:130230.Seq

M00001449A:A12 5857 RTA00000118A.g.l4.1

M00001449A:B12 41633 80.Cl l .sp6:130242.Seq

M00001449A:B12 41633 RTA00000118A.g.l6.1

M00001449A:G10 36535 RTA00000181AF.f.5.1

M00001449A:G10 36535 80.Dl l .sp6:130254.Seq

M00001449A:G10 36535 100.Dl l .sp6:131468.Seq

M00001449C:D06 86110 RTA00000181AF.f.l2.1

M00001449C:D06 861 10 80.El l .sp6:130266.Seq

M00001450A:A02 39304 RTA00000118A.j.21.1.Seq_THC151859

M00001450A:A02 39304 RTAOOOOO 1 18A.J.21.1

M00001450A:A02 39304 79.Fl.sp6:130076.Seq cDNA Library ESI 3 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001450A:A02 39304 180.G9.sp6:135995.Seq

M00001450A:A1 1 32663 80.Fl l .sp6: 130278.Seq

M00001450A:A1 1 32663 RTAOOOOO 1 18A.1.8.1

M00001450A:B12 82498 100.Fl l .sp6:131492.Seq

M00001450A:B12 82498 RTAOOOOO 1 18A.m.10.1

M00001450A:B12 82498 79.Gl .sp6:130088.Seq

M00001450A:D08 27250 80.Gl l .sp6:130290.Seq

M00001450A:D08 27250 180.B10.sp6: 135936.Seq

M00001450A:D08 27250 RTA00000181AF.g.l0.1

M00001452A:B04 84328 RTAOOOOO 118A.p.10.1

M00001452A:B04 84328 79.A2.sp6:130017.Seq

M00001452A:B12 86859 RTA00000118A.p.8.1

M00001452A:B12 86859 79.B2.sp6:130029.Seq

M00001452A:F05 85064 RTA00000131A.m.23.1

M00001452A:F05 85064 79.D2.sp6:130053.Seq

M00001452C:B06 16970 80.Hl l .sp6:130302.Seq

M00001452C:B06 16970 100.C12.sp6:131457.Seq

M00001452C:B06 16970 RTA00000181AR.i.l8.2

M00001453A:E1 1 16130 80.A12.sp6:130219.Seq

M00001453A:E11 16130 100.D12.sp6:131469.Seq

M00001453A:E1 1 16130 RTAOOOOO 1 19A.C.13.1

M00001453C:F06 16653 80.B12.sp6:130231.Seq

M00001453C:F06 16653 RTA00000181AF.k.5.3

M00001454A:A09 83103 RTAOOOOO 119A.e.24.2

M00001454A:A09 83103 79.G2.sp6:130089.Seq

M00001454B:C12 7005 121.Dl.sp6:131917.Seq

M00001454B:C12 7005 RTA00000181AF.k.24.1

M00001454B:C12 7005 80.C12.sp6:130243.Seq

M00001455B:E12 13072 80.F12.sp6:130279.Seq

M00001455B:E12 13072 RTA00000181AR.m.5.2

M00001460A:F06 2448 89.Al .sp6:13066JSeq

M00001460A:F06 2448 RTAOOOOO 1 19A.J.21.1

M00001461A:D06 1531 89.Cl .sp6:130691.Seq

M00001461A:D06 1531 RTAOOOOO 1 19A.0.3.1

M00001465A:B11 10145 79.F3.sp6:130078.Seq

M00001465A:B11 10145 RTAOOOOO 120A.g.12.1

M00001467A:B07 38759 89.Fl.sp6:130727.Seq

M00001467A:B07 38759 RTAOOOOO 120A.m.12.3

M00001467A:D04 39508 RTAOOOOO 120A.O.2.1

M00001467A:D04 39508 89.Gl .sp6:130739.Seq

M00001467A:E10 39442 89A2.sp6:130668.Seq

M00001467A:E10 39442 RTAOOOOO 120A.O.21.1

M00001468A:F05 7589 RTAOOOOO 120A.p.23.1

M00001468A:F05 7589 89.B2.sp6:130680.Seq

M00001469A:A01 RTA00000121A.C.10.1

M00001469A:A01 89.C2.sp6:130692.Seq

M00001469A:C10 12081 89.D2.sp6:130704.Seq

M00001469A:C10 12081 RTAOOOOO 133A.d.14.2

M00001469A:H12 19105 89.E2.sp6:130716.Seq

M00001469A:H12 19105 RTAOO0OO133A.e.l5.1

M00001470A:C04 39425 89.G2.sp6:130740.Seq

M00001470A:C04 39425 RTA00000133A.f.l.l cDNA Library ESI 3 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001471A:B01 39478 89.H2.sp6: 130752.Seq

M00001471A:B01 39478 RTA00000133A.i.5.1

M00001487B:H06 RTA00000182AF.1.15.1

M00001487B:H06 89.B3.sp6:130681.Seq

M00001488B:F12 RTAOOOOO 182AF.1.20.1

M00001488B:F12 89.C3.sp6:130693.Seq

M00001494D:F06 7206 RTAOOOOO 182AF.o.15.1

M00001494D:F06 7206 89.E3.sp6: 130717.Seq

M00001499B:A1 1 10539 RTAOOOOO 183 AF.a.24.1

M00001499B:A1 1 10539 89.G3.sp6:130741.Seq

M00001499B:A1 1 10539 173.B5.SP6:134085.Seq

M00001500A:C05 5336 RTA00000183AF.b.l3.1

M00001500A:C05 5336 89.H3.sp6: 130753.Seq

M00001504A:E01 RTAOOOOO 183 AF.c.24.1

M00001504A:E01 89.D4.sp6:130706.Seq

M00001504A:E01 RTAOOOOO 183 AF.c.24.1.Seq_THC 125912

M00001504C:A07 10185 RTAOOOOO 183 AF.d.5.1

M00001504C:A07 10185 89.E4.sp6: 130718.Seq

M00001505C:C05 89.H4.sp6: 130754.Seq

M00001505C:C05 RTAOOOOO 183 AF.e.1.1

M00001506D:A09 89.A5.sp6:130671.Seq

M00001506D:A09 RTAOOOOO 183 AF.e.23.1

M00001506D:A09 121.G6.sp6:131958.Seq

M00001507A:H05 39168 RTA00000121A.1.10.1

M00001507A:H05 39168 89.B5.sp6:130683.Seq

M00001535A:F10 39423 79.C5.sp6:130044.Seq

M00001535A:F10 39423 RTA00000134A.k.22.1

M00001541A:H03 39174 79.E5.sp6:130068.Seq

M00001541A:H03 39174 RTAOOOOO 124A.n.13.1

M00001544A:G02 19829 79.H5.sp6: 130104.Seq

M00001544A:G02 19829 RTAOOOOO 125 A.h.24.4

M00001545A:D08 13864 RTAOOOOO 125 A.m.9.1

M00001545A:D08 13864 79.B6.sp6: 130033.Seq

M00001551A:F05 39180 RTA00000126A.n.8.2

M00001551A:F05 39180 79.A7.sp6:130022.Seq

M00001552A:D1 1 39458 RTAOOOOO 126A.p.15.2

M00001552A:D11 39458 79.D7.sp6:130058.Seq

M00001557A:F03 39490 RTAOOOOO 128A.b.4.1

cDNA Library ES 14 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M0000151 1A:H06 39412 RTA00000133A.k.l7.1

M00001511A:H06 39412 89.C5.sp6:130695.Seq

M00001512A:A09 39186 89.D5.sp6:130707.Seq

M00001512A:A09 39186 RTA00000121A.p.l5.1

M00001512D:G09 3956 89.E5.sp6:130719.Seq

M00001512D:G09 3956 173.H5.SP6: 134157.Seq

M00001512D:G09 3956 RTAOOOOO 183 AF.g.3.1

M00001513B:G03 RTA00000183AF.g.9.1

M00001513B:G03 89.F5.sp6:130731.Seq

M00001513B:G03 RTAOOOOO 183 AF.g.9.1.Seq_THC 198280

M00001513C:E08 14364 RTAOOOOO 183AF.g.12.1

M00001513C:E08 14364 89.G5.sp6: 130743.Seq

M00001514C:D1 1 40044 RTA00000183AF.g.22.1

M00001514C:D1 1 40044 RTA00000183AF.g.22.1.Seq_THC232899

M00001514C:D1 1 40044 89.H5.sp6:130755.Seq

M00001518C:B1 1 8952 89.A6.sp6:130672.Seq

M00001518C:B1 1 8952 RTAOOOOO 183AF.h.15.1

M00001528B:H04 8358 89.D6.sp6:130708.Seq

M00001528B:H04 8358 RTAOOOOO 183 AF.i.5.1

M00001531A:D01 38085 RTA00000123A.e.l5.1

M00001531A:D01 38085 89.E6.sp6:130720.Seq

M00001534A:C04 16921 RTAOOOOO 183 AF.k.6.1

M00001534A:C04 16921 89.H6.sp6:130756.Seq

M00001534A:D09 5097 RTAOOOOO 134A. 1.1

M00001534A:D09 5097 RTAOOOOO 134A.k.l .l .Seq_THC215869

M00001534C:A01 41 19 RTAOOOOO 183AF.k.16.1

M00001534C:A01 41 19 89.C7.sp6:130697.Seq

M00001535A:C06 20212 89.E7.sp6:130721.Seq

M00001535A:C06 20212 RTA00000134A.1.22.1.Seq_THC128232

M00001535A:C06 20212 RTA00000134A.1.22.1

M00001536A:B07 2696 RTAOOOOO 134A.m.13.1

M00001536A:B07 2696 89.F7.sp6:130733.Seq

M00001537A:F12 39420 89.H7.sp6:130757.Seq

M00001537A:F12 39420 RTAOOOOO 134A.0.23.1

M00001540A:D06 8286 89.B8.sp6:130686.Seq

M00001540A:D06 8286 RTAOOOOO 183 AF.o.1.1

M00001542A:E06 39453 89.E8.sp6:130722.Seq

M00001542A:E06 39453 RTAOOOOO 135A.g.11.1

M00001544A:E06 RTAOOOOO 184 AF.a.8.1

M00001544A:E06 173.G7.SP6:134147.Seq

M00001544A:E06 89.H8.sp6:130758.Seq

M00001545A:B02 89.B9.sp6:130687.Seq

M00001545A:B02 RTA00000135A.1.2.2

M00001548A:E10 5892 89.E9.sp6:130723.Seq

M00001548A:E10 5892 RTAOOOOO 184AF.d.11.1

M00001548A:E10 5892 RTA00000184AF.d.l l .l .Seq_THC161896

M00001549C:E06 16347 89.H9.sp6:130759.Seq

M00001549C:E06 16347 RTAOOOOO 184AF.e.15.1

M00001550A:A03 7239 89.A10.sp6:130676.Seq

M00001550A:A03 7239 RTAOOOOO 126A.m.4.2

M00001550A:G01 5175 RTA00000184AF. .1

M00001550A:G01 5175 89.B10.sp6:130688.Seq cDNA Library ESI 4 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001551A:G06 22390 RTAOOOOO 136A.j.13.1

M00001551A:G06 22390 89.C10.sp6:130700.Seq

M00001551C:G09 3266 RTAOOOOO 184AR.g.1.1

M00001551C:G09 3266 89.D10.sp6:130712.Seq

M00001553A:H06 8298 RTAOOOOO 127A.d.19.1

M00001553A:H06 8298 89.G10.sp6:130748.Seq

M00001553B:F12 4573 89.H10.sp6:130760.Seq

M00001553B:F12 4573 RTA00000184AF.h.9.1

M00001555A:B02 39539 RTAOOOOO 127A...21.1

M00001555A.B02 39539 89.Bl l .sp6:130689.Seq

M00001555A:C01 39195 89.Cl l .sp6: 130701.Seq

M00001555A:C01 39195 RTA00000137A.C.16.1

M00001555D:G10 4561 RTAOOOOO 184AF.i.21.1

M00001555D:G10 4561 89.Dl l .sp6:130713.Seq

M00001556A:C09 9244 89.El l .sp6:130725.Seq

M00001556A:C09 9244 RTA00000127A.1.3.1

M00001556B:G02 1 1294 RTAOOOOO 184AF.J.6.1

M00001556B:G02 11294 89A12.sp6:130678.Seq

M00001557B:H10 5192 173.E9.SP6:134125.Seq

M00001557B:H10 5192 RTAOOOOO 184AF.k.2.1

M00001557B:H10 5192 89.D12.sp6:130714.Seq

M00001557D:D09 8761 RTAOOOOO 184AF.k.12.1

M00001557D:D09 8761 89.E12.sp6:130726.Seq

M00001558B:H1 1 7514 RTAOOOOO 184AF. 21.1

M00001558B:H1 1 7514 89.G12.sp6:130750.Seq

M00001559B:F01 89.H12.sp6:130762.Seq

M00001559B:F01 RTAOOOOO 184AF.1.11.1

M00001560D:F10 6558 90.Al.sp6:130859.Seq

M00001560D:F10 6558 RTAOOOOO 184AF.m.21.1

M00001566B:D1 1 RTAOOOOO 184AF.p.3.1

M00001566B:D1 1 90.Dl.sp6:130895.Seq

M00001583D:A10 6293 RTAOOOOO 185 AF.e.11.1

M00001583D:A10 6293 90.A2.sp6:130860.Seq

M00001590B:F03 RTAOOOOO 185 AF.g.11.1

M00001590B:F03 90.C2.sp6:130884.Seq

M00001597D:C05 10470 RTAOOOOO 185 AF.k.6.1

M00001597D:C05 10470 90.F2.sp6:130920.Seq

M00001598A:G03 16999 90.G2.sp6:130932.Seq

M00001598A:G03 16999 RTAOOOOO 185 AF.k.9.1

M00001601A:D08 22794 RTA00000138A.b.5.1

M00001601A:D08 22794 90.H2.sp6: 130944.Seq

M00001607A:E1 1 1 1465 RTAOOOOO 185AF.m.19.1

M00001607A:E1 1 1 1465 90A3.sp6:130861.Seq

M00001608A:B03 7802 RTAOOOOO 185 AF.n.5.1

M00001608A:B03 7802 90.B3.sp6:130873.Seq

M00001608B:E03 22155 RTAOOOOO 185 AF.n.9.1

M00001608B:E03 22155 90.C3.sp6:130885.Seq

M00001608D:A11 RTAOOOOO 185AF.n.12.1

M00001608D:A1 1 90.D3.sp6:130897.Seq

M00001614C:F10 13157 RTAOOOOO 186AF.a.6.1

M00001614C:F10 13157 90.E3.sp6: 130909.Seq

M00001617C:E02 17004 RTAOOOOO 186AF.b.21.1 cDNA Library ESI 4 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001617C:E02 17004 90.F3.sp6:130921.Seq

M00001619C.F12 40314 90.G3.sp6:130933.Seq

M00001619C:F12 40314 RTAOOOOO 186AF.C.15.1

MQ0001621C:C08 40044 RTAOOOOO 186AF.d.1.1

M00001621C:C08 40044 RTAOOOOO 186AF.d.1.1.Seq_THC232899

M00001621C:C08 40044 90.H3.sp6:130945.Seq

M00001621C:C08 40044 122.El .sp6:132121.Seq

M00001623D:F10 13913 RTA00000186AF.e.6.1

M00001623D:F10 13913 90.A4.sp6:130862.Seq

M00001632D:H07 RTAOOOOO 186AF.h.14.1.Seq_THC 1 12525

M00001632D:H07 RTAOOOOO 186AF.h.14.1

M00001632D:H07 90.E4.sp6:130910.Seq

M00001632D:H07 176.A3.sp6:134514.Seq

M00001644C:B07 39171 RTA00000186AF.1.7.1

M00001644C:B07 39171 90.F4.sp6:130922.Seq

M00001644C:B07 39171 217.A12.sp6: 139369.Seq

M00001645A:C12 19267 RTA00000186AF.1.12.1.Seq_THC178183

M00001645A:C12 19267 176.G3.sp6:134586.Seq

M00001645A:C12 19267 RTA00000186AF.1.12.1

M00001645A:C12 19267 90.G4.sp6:130934.Seq

M00001648C:A01 4665 90.H4.sp6: 130946.Seq

M00001648C:A01 4665 RTA00000186AF.m.3.1

M00001657D:C03 23201 RTAOOOOO 187AF.a.14.1

M00001657D:C03 23201 90.B5.sp6:130875.Seq

M00001657D:F08 76760 90.C5.sp6:130887.Seq

M00001657D:F08 76760 RTAOOOOO 187AF.a.15.1

M00001662C:A09 23218 RTAOOOOO 187 AR.c.5.2

M00001662C:A09 23218 90.D5.sp6:130899.Seq

M00001663A:E04 35702 90.E5.sp6:130911.Seq

M00001663A:E04 35702 RTA00000187AR.C.15.2

M00001669B:F02 6468 90.F5.sp6:130923.Seq

M00001669B:F02 6468 RTAOOOOO 187AF.d.15.1

M00001670C:H02 14367 90.G5.sp6:130935.Seq

M00001670C:H02 14367 RTA00000187AF.e.8.1

M00001673C:H02 7015 90.H5.sp6:130947.Seq

M00001673C:H02 7015 RTAOOOOO 187AF.L 18.1

M00001675A:C09 8773 RTAOOOOO 187AF.f.24.1

M00001675A:C09 8773 90.A6.sp6:130864.Seq

M00001675A:C09 8773 RTAOOOOO 187AFT.24.1.Seq_THC220002

M00001676B:F05 11460 RTAOOOOO 187AF.g.12.1

M00001676B:F05 11460 90.B6.sp6:130876.Seq

M00001676B:F05 11460 219.F2.sp6:139035.Seq

M00001677D:A07 7570 90.D6.sp6:130900.Seq

M00001677D:A07 7570 RTA00000187AF.g.24.1

M00001677D:A07 7570 RTA00000187AF.g.24.1.Seq_THC168636

M00001678D:F12 4416 90.E6.sp6:130912.Seq

M00001678D:F12 4416 RTAOOOOO 187AF.h.13.1

M00001679A:F10 26875 RTAOOOOO 187AF.L 1.1

M00001679A:F10 26875 90.A7.sp6:130865.Seq

M00001679B.F01 6298 90.B7.sp6:130877.Seq

M00001679B:F01 6298 RTA00000187AR.i.l0.2

M00001680D:F08 10539 90.F7.sp6:130925.Seq cDNA Library ESI 4 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001680D:F08 10539 219.F6.sp6:139039.Seq

M00001680D:F08 10539 RTA00000187AF.1.7.1

M00001682C:B12 17055 90.G7.sp6: 130937.Seq

M00001682C:B12 17055 RTA00000187AF.m.3.1

M00001682C:B12 17055 176.D6.sp6:134553.Seq

M00001688C:F09 5382 90.A8.sp6:130866.Seq

M00001688C:F09 5382 RTAOOOOO 187AF.m.23.2

M00001693C:G01 4393 RTA00000187AF.n.l7.1

M00001693C:G01 4393 90.B8.sp6:130878.Seq

M00001716D:H05 67252 RTA00000187AF.O.6.1

M00001716D:H05 67252 90.C8.sp6: 130890.Seq

M00003741D:C09 40108 90.D8.sp6:130902.Seq

M00003741D:C09 40108 RTAOOOOO 187AF.0.24.1

M00003747D:C05 11476 RTAOOOOO 187AF.p.19.1

M00003747D:C05 11476 90.E8.sp6: 130914.Seq

M00003747D:C05 11476 RTAOOOOO 187AF.p.19.1.Seq_THC 108482

M00003747D:C05 1 1476 219.H8.sp6:139065.Seq

M00003754C:E09 90.F8.sp6: 130926.Seq

M00003754C:E09 RTAOOOOO 188AF.b.12.1

M00003761D:A09 RTAOOOOO 188AF.d.1 1.1

M00003761D:A09 90.H8.sp6:130950.Seq

M00003761D:A09 RTAOOOOO 188 AF.d.1 1.1. Seq_THC212094

M00003762C:B08 17076 RTAOOOOO 188AF.d.21.1.Seq_THC208760

M00003762C:B08 17076 90A9.sp6:130867.Seq

M00003762C:B08 17076 RTAOOOOO 188AF.d.21.1

M00003763A:F06 3108 RTA00000188AF.d.24.1

M00003763A:F06 3108 90.B9.sp6:130879.Seq

M00003774C:A03 67907 RTA00000188AF.g.l l .l .Seq_THC123222

M00003774C:A03 67907 RTAOOOOO 188AF.g.1 1.1

M00003774C:A03 67907 90.C9.sp6:130891.Seq

M00003784D:D12 RTAOOOOO 188AF.Ϊ.8.1

M00003784D:D12 90.D9.sp6: 130903.Seq

M00003839A:D08 7798 RTAOOOOO 189 AF.c.18.1

M00003839A:D08 7798 90.A10.sp6:130868.Seq

M00003851B:D08 90.D10.sp6:130904.Seq

M00003851B:D08 RTAOOOOO 189AF J.1

M00003851B:D10 13595 90.E10.sp6:130916.Seq

M00003851B:D10 13595 RTA00000189AF.f.8.1

M00003853A:D04 5619 90.F10.sp6:130928.Seq

M00003853A:D04 5619 RTAOOOOO 189AF.f.17.1

M00003853A:F12 10515 90.G10.sp6:130940.Seq

M00003853A:F12 10515 RTAOOOOO 189AF.L 18.1

M00003856B:C02 4622 90.H10.sp6:130952.Seq

M00003856B:C02 4622 RTAOOOOO 189AF.g.1.1

M00003857A:H03 4718 90.Bl l .sp6:130881.Seq

M00003857A:H03 4718 RTAOOOOO 189AF.g.5.1.Seq_THC 196102

M00003857A:H03 4718 RTAOOOOO 189AF.g.5.1 cDNA Library ESI 5 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00003867A:D10 90.Cl l .sp6: 130893.Seq

M00003867A:D10 RTAOOOOO 189AF.h.17.1

M00003871C:E02 4573 RTAOOOOO 189AFJ.12.1

M00003875C:G07 8479 90.Gl l .sp6: 130941. Seq

M00003875C:G07 8479 RTA00000189AF.J.22.1

M00003875D:D1 1 90.Hl l .sp6: 130953.Seq

M00003875D:D1 1 RTAOOOOO 189 AF.j.23.1

M00003876D:E12 7798 90A12.sp6: 130870.Seq

M00003876D:E12 7798 RTA00000189AF.k.l2.1

M00003906C:E10 9285 90.H12.sp6: 130954.Seq

M00003906C:E10 9285 RTAOOOOO 190AF.d.7.1

M00003907D:A09 39809 99Al .sp6:131230.Seq

M00003907D:A09 39809 RTAOOOOO 190AF.e.3.1.Seq_THC 150217

M00003907D:A09 39809 RTAOOOOO 190AF.e.3.1

M00003907D:H04 16317 99.Bl .sp6:131242.Seq

M00003907D:H04 16317 RTAOOOOO 190AF.e.6.1

M00003909D:C03 8672 RTAOOOOO 190AF.f.1 1.1

M00003909D:C03 8672 99.Cl .sp6:131254.Seq

M00003968B:F06 24488 RTA00000190AF.n.l6.1

M00003968B:F06 24488 99.C2.sp6:131255.Seq

M00003970C:B09 40122 RTAOOOOO 190AF.n.23.1

M00003970C:B09 40122 RTAOOOOO 190AF.n.23.1.Seq_THC 109227

M00003970C:B09 40122 99.D2.sp6:131267.Seq

M00003974D:E07 23210 RTAOOOOO 190AF.O.20.1

M00003974D:E07 23210 RTA00000190AF.o.20.1.Seq_THC207240

M00003974D:E07 23210 99.E2.sp6:131279.Seq

M00003974D:H02 23358 RTAOOOOO 190AF.O.21.1.Seq_THC207240

M00003974D:H02 23358 RTAOOOOO 190 AF.o.21.1

M00003974D:H02 23358 99.F2.sp6:131291.Seq

M00003981A:E10 3430 99.A3.sp6:131232.Seq

M00003981A:E10 3430 RTA00000191AF.a.9.1

M00003982C:C02 2433 RTA00000191AF.a.l5.2

M00003982C:C02 2433 99.B3.sp6:131244.Seq

M00003982C:C02 2433 RTA00000191AF.a.l5.2.Seq_THC79498

M00004028D:C05 40073 RTA00000191AF.e.3.1

M00004028D:C05 40073 99.E3.sp6:131280.Seq

M00004035C:A07 37285 99.H3.sp6:131316.Seq

M00004035C:A07 37285 RTA00000191AF.f.l l .l

M00004035D:B06 17036 RTA00000191AF.f.l3.1

M00004035D:B06 17036 99A4.sp6:131233.Seq

M00004072A:C03 RTA00000191AF.J.9.1

M00004072A:C03 99.D4.sp6:131269.Seq

M00004081C:D10 15069 99.F4.sp6:131293.Seq

M00004081C:D10 15069 RTA00000191AF.1.6.1

M00004086D:G06 9285 99.H4.sp6:131317.Seq

M00004086D:G06 9285 RTA00000191AF.m.l8.1

M00004105C:A04 7221 99.D5.sp6:131270.Seq

M00004105C:A04 7221 RTA00000191AF.p.9.1

M00004171D:B03 4908 RTAOOOOO 192AFJ.2.1

M00004171D:B03 4908 99.F6.sp6:131295.Seq

M00004185C:C03 1 1443 RTA00000192AF.1.13.2

M00004185C:C03 1 1443 123A8.sp6:132272.Seq cDNA Library ES 15 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00004185C:C03 1 1443 99.A7.sp6: 131236.Seq

M00004191D:B1 1 RTAOOOOO 192AF.m.12.1

M00004191D:B1 1 99.B7.sp6: 131248.Seq

M00004191D:B1 1 123.C8.sp6:132296.Seq

M00004197D:H01 8210 99.C7.sp6:131260.Seq

M00004197D:H01 8210 123.E8.sp6:132320.Seq

M00004197D:H01 8210 RTAOOOOO 192AF.n.13.1

M00004203B:C12 1431 1 99.D7.sp6: 131272.Seq

M00004203B:C12 1431 1 RTAOOOOO 192 AF.o.2.1

M00004214C:H05 11451 177.D8.sp6:134747.Seq

M00004214C:H05 1 1451 RTAOOOOO 192AF.p.17.1

M00004223D:E04 12971 RTAOOOOO 193 AF.a.20.1

M00004223D:E04 12971 99.B8.sp6: 131249.Seq

M00004269D:D06 4905 99.H8.sp6:131321.Seq

M00004269D:D06 4905 RTAOOOOO 193AF.e.14.1

M00004295D:F12 16921 99.D9.sp6:131274.Seq

M00004295D:F12 16921 RTA00000193AF.h.l5.1

M00004296C:H07 13046 99.E9.sp6: 131286.Seq

M00004296C:H07 13046 RTA00000193AF.h.l9.1

M00004307C:A06 9457 RTAOOOOO 193AF.i.14.2

M00004307C:A06 9457 99.F9.sp6:131298.Seq

M00004307C:A06 9457 123.Dl l .sp6:13231 1.Seq

M00004312A:G03 26295 RTA00000193AF.i.24.2

M00004312A:G03 26295 99.G9.sp6: 131310.Seq

M00004312A:G03 26295 RTAOOOOO 193AF.i.24.2.Seq_THC 197345

M00004318C:D10 21847 RTA00000193AFJ.9.1

M00004318C:D10 21847 99.H9.sp6: 131322.Seq

M00004359B:G02 RTA00000193AF.m.5.1.Seq_THC173318

M00004359B:G02 RTAOOOOO 193 AF.m.5.1

M00004505D:F08 RTAOOOOO 194AF.b.19.1

M00004505D:F08 99.H10.sp6:131323.Seq

M00004692A:H08 99.Bl l .sp6:131252.Seq

M00004692A:H08 RTA00000194AF.C.24.1

M00004692A:H08 377.F4.sp6: 141957.Seq

M00005180C:G03 RTAOOOOO 194 AF.f.4.1

cDNA Library ES 16 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001346D:E03 6806 RTAOOOOO 177AF.g.13.3

M00001350A:B08 80.H2.sp6: 130293.Seq

M00001350A:B08 RTA00000177AF.i.6.2

M00001357D:D1 1 4059 RTAOOOOO 177AF.n.l 8.3. Seq_THC 123051

M00001357D:D1 1 4059 RTAOOOOO 177AF.n.18.3

M00001409C:D12 9577 RTAOOOOO 179AF.0.17.1

M00001409C:D12 9577 80.E7.sp6:130262.Seq

M00001418B:F03 9952 RTAOOOOO 180AF.C.20.1

M00001418B:F03 9952 RTAOOOOO 180AF.C.20.1.Seq_THC 162284

M00001418B:F03 9952 80.E8.sp6: 130263.Seq

M00001418D:B06 8526 RTAOOOOO 180AF.d.1.1

M00001421C:F01 9577 RTAOOOOO 180AF.d.23.1

M00001421C:F01 9577 80.G8.sp6:130287.Seq

M00001429B:A1 1 4635 RTAOOOOO 180AF120.1

M00001432C:F06 RTAOOOOO 180AF. 24.1

M00001439C:F08 40054 RTAOOOOO 180AF.p.10.1

M00001442C:D07 16731 RTA00000181AF.a.20.1

M00001442C:D07 16731 80.C10.sp6:130241.Seq

M00001443B:F01 80.D10.sp6:130253.Seq

M00001443B:F01 RTA00000181AF.b.7.1

M00001445A:F05 13532 80.E10.sp6:130265.Seq

M00001445A:F05 13532 RTA00000181AF.C.4.1

M00001446A:F05 7801 RTA00000181AF.C.21.1

M00001455A:E09 13238 RTA00000181AF.m.4.1

M00001455A:E09 13238 RTA00000181AF.m.4.1.Seq_THC140691

M00001460A:F12 39498 RTA00000119A.J.20.1

M00001481D:A05 7985 RTAOOOOO 182 AR.j .2.1

M00001490B:C04 18699 RTAOOOOO 182AF.m.16.1

M00001490B:C04 18699 89.D3.sp6: 130705.Seq

M00001500C:E04 9443 89.B4.sp6: 130682.Seq

M00001500C:E04 9443 RTAOOOOO 183 AF.c.1.1

M00001532B:A06 3990 89.G6.sp6:130744.Seq

M00001532B:A06 3990 RTAOOOOO 183 AF.j.11.1

M00001534A:F09 5321 89.B7.sp6:130685.Seq

M00001534A:F09 5321 RTAOOOOO 183AF. 8.1

M00001535A:B01 7665 RTAOOOOO 134A.1.19.1

M00001536A:C08 39392 89.G7.sp6:130745.Seq

M00001536A:C08 39392 RTAOOOOO 134A.m.16.1

M00001541A:F07 22085 RTA00000135A.e.5.2

M00001542B:B01 RTAOOOOO 183 AF.p.4.1

M00001542B:B01 89.F8.sp6: 130734.Seq

M00001544A:E03 12170 RTAOOOOO 125A.h.18.4

M00001545A:C03 19255 RTA00000135A.m.l8.1

M00001545A:C03 19255 184.B10.sp6:135547.Seq

M00001545A:C03 19255 89.C9.sp6:130699.Seq

M00001548A:H09 1058 RTA00000126A.e.20.3.Seq_THC217534

M00001548A:H09 1058 RTAOOOOO 126A.e.20.3

M00001548A:H09 1058 79.F6.sp6: 13008 l .Seq

M00001549A:B02 4015 RTAOOOOO 136A.e.12.1

M00001549A:B02 4015 79.G6.sp6:130093.Seq

M00001549A:D08 10944 RTAOOOOO 126A.h.17.2

M00001552B:D04 5708 RTA00000184AF.g.l2.1 cDNA Library ES 16 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M00001552B.D04 5708 89.E10.sp6:130724.Seq

M00001552D.A01 89.F10.sp6:130736.Seq

M00001552D.A01 RTAOOOOO 184AF.g.22.1

M00001553D:D10 22814 RTAOOOOO 184AF.h.14.1

M00001553D:D10 22814 89.Al l .sp6:130677.Seq

M00001558A:H05 RTAOOOOO 128A.C.20.1

M00001558A:H05 89.F12.sp6.130738.Seq

M00001561A:C05 39486 RTA00000128A.m.22.2

M00001561A:C05 39486 79.B8.sp6:130035.Seq

M00001564A:B12 5053 RTA00000184AF.O.12.1

M00001578B:E04 23001 RTA00000185AF.C.24.1

M00001579D:C03 6539 90.Gl .sp6:130931.Seq

M00001579D:C03 6539 173A12.SP6: 134080.Seq

M00001579D:C03 6539 RTAOOOOO 185 AF.d.1 1.1

M00001582D:F05 RTA00000185AF.d.24.1

M00001587A:B1 1 39380 RTAOOOOO 129A.e.24.1

M00001587A:B1 1 39380 79.E8.sp6:130071.Seq

M00001604A:F05 39391 RTAOOOOO 138A.C.3.1

M00001604A:F05 39391 79.A9.sp6:130024.Seq

M00001624A:B06 3277 RTAOOOOO 138A.1.5.1

M00001624A:B06 3277 217.El .sp6: 139406.Seq

M00001624A:B06 3277 90.B4.sp6:130874.Seq

M00001630B:H09 5214 90.D4.sp6:130898.Seq

M00001630B:H09 5214 122.C2.sp6:132098.Seq

M00001630B:H09 5214 RTAOOOOO 186AF.g.11.1

M00001651A:H01 RTAOOOOO 186AF.n.7.1

M00001651A:H01 90.A5.sp6:130863.Seq

M00001677C:E10 14627 RTA00000187AF.g.23.1

M00001679C:F01 78091 90.C7.sp6:130889.Seq

M00001679C:F01 78091 RTAOOOOO 187AF.J.6.1

M00001679C:F01 78091 176.G5.sp6:134588.Seq

M00001686A:E06 4622 RTAOOOOO 187AF.m.15.2

M00003796C:D05 5619 RTAOOOOO 188AF.1.9.1.Seq_THC 167845

M00003796C:D05 5619 RTA00000188AF.1.9.1

M00003826B:A06 1 1350 RTAOOOOO 189AF.a.24.2

M00003826B:A06 1 1350 90.F9.sp6: 130927.Seq

M00003833A:E05 21877 RTAOOOOO 189AF.b.21.1

M00003837D:A01 7899 90.H9.sp6:130951.Seq

M00003837D:A01 7899 RTAOOOOO 189AF.C.10.1

M00003846B:D06 6874 RTAOOOOO 189AF.e.9.1

M00003846B:D06 6874 90.C10.sp6:130892.Seq

M00003879B:D10 31587 RTA00000189AF.1.20.1

M00003879B:D10 31587 90.C12.sp6:130894.Seq

M00003879D:A02 14507 90.D12.sp6:130906.Seq

M00003879D:A02 14507 RTAOOOOO 189AR.1.23.2

M00003891C:H09 90.G12.sp6:130942.Seq

M00003891C:H09 RTAOOOOO 189AF.p.8.1

M00003912B:D01 12532 99.Dl .sp6:131266.Seq

M00003912B:D01 12532 RTAOOOOO 190AF.g.2.1

M00004072B:B05 17036 RTA00000191AF.J.10.1

M00004081C:D12 14391 RTA00000191AF.1.7.1

M000041 11D:A08 6874 RTAOOOOO 192AF.a.14.1 cDNA Library ES 16 - ATCC# Deposit Date - December 22, 1998

Clone Name Cluster ID Sequence Name

M000041 1 1D:A08 6874 99.F5.sp6:131294.Seq

M00004121B:G01 177.H4.sp6:134791.Seq

M00004121B:G01 99.H5.sp6:131318.Seq

M00004121B:G01 RTA00000192AF.C.2.1

M00004138B:H02 13272 99.A6.sp6:131235.Seq

M00004138B.H02 13272 RTAOOOOO 192AF.e.3.1

M00004151D:B08 16977 RTAOOOOO 192AF.g.3.1

M00004169C:C12 5319 99.E6.sp6: 131283. Seq

M00004169C:C12 5319 RTA00000192AF.i.l2.1

M00004169C:C12 5319 123.F7.sp6:132331.Seq

M00004183C:D07 16392 RTAOOOOO 192AF.1.1.1

M00004183C:D07 16392 RTAOOOOO 192AF.1.1.1.Seq_THC202071

M00004230B:C07 7212 RTAOOOOO 193AF.b.14.1

M00004230B:C07 7212 99.D8.sp6:131273.Seq

M00004249D:F10 RTAOOOOO 193 AF.c.21.1.Seq _THC222602

M00004249D:F10 RTAOOOOO 193 AF.c.21.1

M00004275C:C1 1 16914 99A9.sp6:131238.Seq

M00004275C:C1 1 16914 RTAOOOOO 193 AF.f.5.1

M00004283B:A04 14286 RTA00000193AF.f.22.1

M00004285B:E08 56020 RTAOOOOO 193 AF.g.2.1

M00004327B:H04 RTA00000193AFJ.20.1

M00004377C:F05 2102 RTA00000193AF.n.7.1

M00004384C:D02 RTA00000193AF.n.l5.1

M00004384C:D02 RTAOOOOO 193 AF.n.15.1.Seq_THC215687

M00004461A:B08 RTAOOOOO 194AR.a.10.2

M00004461A:B09 RTAOOOOO 194AF.a.11.1

M00004691D:A05 RTA00000194AF.C.23.1

M00004896A:C07 RTAOOOOO 194 AF.d.13.1

The above material has been deposited with the American Type Culture Collection, Rockville, Maryland, under the accession number indicated. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. The deposit will be maintained for a period of 30 years following issuance of this patent, or for the enforceable life of the patent, whichever is greater. Upon issuance of the patent, the deposit will be available to the public from the ATCC without restriction.

This deposit is provided merely as convenience to those of skill in the art, and is not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained within the deposited material, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the written description of sequences herein. A license may be required to make, use, or sell the deposited material, and no such license is granted hereby.

Retrieval of Individual Clones from Deposit of Pooled Clones Where the ATCC deposit is composed of a pool of cDNA clones, the deposit was prepared by first transfecting each of the clones into separate bacterial cells. The clones were then deposited as a pool of equal mixtures in the composite deposit. Particular clones can be obtained from the composite deposit using methods well known in the art. For example, a bacterial cell containing a particular clone can be identified by isolating single colonies, and identifying colonies containing the specific clone through standard colony hybridization techniques, using an oligonucleotide probe or probes designed to specifically hybridize to a sequence of the clone insert (e.g., a probe based upon unmasked sequence of the encoded polynucleotide having the indicated SEQ ID NO). The probe should be designed to have a T_m of approximately 80°C (assuming 2°C for each A or T and 4°C for each G or C). Positive colonies can then be picked, grown in culture, and the recombinant clone isolated. Alternatively, probes designed in this manner can be used to PCR to isolate a nucleic acid molecule from the pooled clones according to methods well known in the art, e.g., by purifying the cDNA from the deposited culture pool, and using the probes in PCR reactions to produce an amplified product having the corresponding desired polynucleotide sequence.

Table 1. Seq uence identification numbers, cluster ID, sequence name, and clone name

SEQ ID NO: Cluster ID Sequence Name Clone Name

1 4635 RTAOOOOO 180AF.i.20.1 M00001429B.A1 1

2 RTAOOOOO 185AF.n.12.1 M00001608D:A1 1

3 4622 RTA00000187AF.m.l 5.2 M00001686A.E06

4 3706 RTA00000191AF.i.l7.2 M00004068BA01

5 36535 RTA00000181AF.f.5.1 M00001449A:G10

6 3990 RTAOOOOO 183 AF.j.11.1 M00001532B:A06

7 5319 RTA00000192AF.U2.1 M00004169C:C12

8 36393 RTAOOOOO 180AF.C.2.1 M00001417A:E02

9 2623 RTAOOOOO 183 AF.a.6.1 M00001497A:G02

10 7587 RTAOOOOO 178AF.n.24.1 M00001387B:G03

1 1 7065 RTA00000137A.g.6.1 M00001557A:D02

12 10539 RTA00000187AF.1.7.1 M00001680D:F08

13 27250 RTA00000181AF.g.l0.1 M00001450A:D08

14 5556 RTA00000179AF.n.l0.1 M00001407B:D1 1

15 RTAOOOOO 192AF.m.12.1 M00004191D:B1 1

16 8761 RTAOOOOO 184AF.k.12.1 M00001557D:D09

17 4622 RTAOOOOO 189AF.g.1.1 M00003856B:C02

18 11460 RTAOOOOO 187AF.g.12.1 M00001676B:F05

19 16283 RTAOOOOO 120A.O.20.1 M00001467A:D08

20 3430 RTAOOOOO 191 AF.a.9.1 M00003981A:E10

21 7065 RTAOOOOO 184AF.J.21.1 M00001557A:D02

22 RTAOOOOO 182AF.1.20.1 M00001488B:F12

23 RTAOOOOO 123A.g.19.1 M00001531A:H1 1

24 16918 RTAOOOOO 193AF.a.16.1 M00004223A:G10

25 16914 RTA00000193AF.f.5.1 M00004275C:C1 1

26 40108 RTA00000187AF.O.24.1 M00003741D:C09

27 14286 RTAOOOOO 193 AF.f.22.1 M00004283B:A04

28 17004 RTAOOOOO 186AF.b.21.1 M00001617C:E02

29 RTAOOOOO 180AF.g.22.1 M00001426B:D12

30 13272 RTAOOOOO 192AF.e.3.1 M00004138B:H02

31 RTAOOOOO 194AF.f.4.1 M00005180C:G03

32 32663 RTA000001 18A.1.8.1 M00001450A.A1 1

33 RTAOOOOO 180AF.a.9.1 M00001414A:B01

34 5832 RTAOOOOO 178AF.o.23.1 M00001388D:G05

35 7801 RTA00000181AF.C.21.1 M00001446A:F05

36 76760 RTA00000187AF.a.l5.1 M00001657D:F08

37 40132 RTAOOOOO 178AF.C.7.1 M00001365C:C10

38 RTAOOOOO 183 AF.e.1.1 M00001505C:C05

39 4016 RTAOOOOO 1 18A.C.4.1 M00001395A:C03

40 5382 RTAOOOOO 187AF.m.23.2 M00001688C:F09 SEQ ID NO: Cluster ID Sequence Name Clone Name

41 5693 RTAOOOOO 190AF.p.17.2 M00003978B:G05

42 307 RTAOOOOO 136A.0.4.2 M00001552A.-B12

43 39833 RTA00000178AF.i.23.1 M00001378B:B02 44 RTAOOOOO 193 AF.m.5.1 M00004359B:G02

45 5325 RTA00000191AF.O.6.1 M00004093D:B12

46 5325 RTA00000191AF.O.6.2 M00004093D:B12

47 18957 RTAOOOOO 190AR.m.9.1 M00003958A:H02

48 39508 RTA00000120A.O.2.1 M00001467A:D04

49 22390 RTAOOOOO 136A.J.13.1 M00001551A:G06

50 12170 RTAOOOOO 125A.h.18.4 M00001544A:E03

51 4393 RTAOOOOO 187AF.n.17.1 M00001693C:G01

52 19 RTAOOOOO 182AF.b.7.1 M00001463C:B11 53 RTAOOOOO 193 AF.c.21.1 M00004249D:F10

54 7899 RTAOOOOO 189AF.C.10.1 M00003837DA01

55 40073 RTA00000191AF.e.3.1 M00004028D:C05

56 7005 RTAOOOOO 179AF.0.22.1 M00001410A:D07 57 RTAOOOOO 187AF.h.22.1 M00001679A:F06

58 18957 RTAOOOOO 190AF.m.9.2 M00003958A:H02

59 18957 RTAOOOOO 183 AF.h.23.1 M00001528A:F09

60 16283 RTAOOOOO 182AF.C.22.1 M00001467A:D08 1 6974 RTAOOOOO 183 AF.d.9.1 M00001504C:H06 2 2623 RTAOOOOO 183AF.b.14.1 M00001500A:E1 1

63 9105 RTA00000191AF.a.21.2 M00003983A:A05

64 13238 RTA00000181AF.m.4.1 M00001455A:E09 5 5749 RTAOOOOO 185AF.a.19.1 M00001571C:H06 6 6455 RTAOOOOO 193 AF.b.9.1 M00004229B:F08 7 23001 RTAOOOOO 185 AF.c.24.1 M00001578B:E04 8 6455 RTAOOOOO 192AF.g.23.1 M00004157CA09 9 13595 RTAOOOOO 189AF.f.8.1 M00003851B:D10 0 39442 RTAOOOOO 120A.O.21.1 M00001467A:E10 1 17036 RTA00000191AF.f.l3.1 M00004035D:B06 2 RTAOOOOO 183 AF.g.9.1 M00001513B:G03 3 7005 RTA00000181AF.k.24.1 M00001454B:C12 4 6268 RTAOOOOO 126A.0.23.1 M00001551A:B10 5 16130 RTAOOOOO 119A.C.13.1 M00001453A:E11 6 23201 RTAOOOOO 187AF.a.14.1 M00001657D:C03 7 5321 RTA00000183AF.k.8.1 M00001534A:F09 8 13157 RTAOOOOO 186AF.a.6.1 M00001614C:F10 9 2102 RTAOOOOO 193 AF.n.7.1 M00004377C:F05

80 1058 RTAOOOOO 126A.e.20.3 M00001548A:H09

81 40392 RTAOOOOO 180AF.J.8.1 M00001429D:D07 2 RTA00000183AF.e.23.1 M00001506DA09 3 11476 RTAOOOOO 187AF.p.19.1 M00003747D:C05 SEQ ID NO: Cluster ID Sequence Name Clone Name

84 3584 RTAOOOOO 177AF.h.20.1 M00001349B:B08

85 10470 RTAOOOOO 180AF.f.18.1 M00001424B:G09

86 39425 RTAOOOOO 133 A.f.1.1 M00001470A:C04

87 5175 RTAOOOOO 184 AF.f.3.1 M00001550A:G01

88 13576 RTAOOOOO 189AF.0.13.1 M00003885C:A02

89 7665 RTA00000134A.1.19.1 M00001535A:B01

90 16927 RTAOOOOO 177AF.h.9.3 M00001348B:B04

91 6660 RTAOOOOO 187AF.h.15.1 M00001679AA06

92 2433 RTA00000191AF.a.l5.2 M00003982C:C02

93 5097 RTAOOOOO 134A.k.1.1 M00001534A:D09

94 21847 RTAOOOOO 193 AF.j.9.1 M00004318C:D10

95 3277 RTA00000138A.1.5.1 M00001624A:B06

96 5708 RTAOOOOO 184AF.g.12.1 M00001552B:D04

97 945 RTAOOOOO 178AR.a.20.1 M00001362C:H1 1

98 16269 RTAOOOOO 178AF.p.1.1 M00001389A:C08 99 RTAOOOOO 183 AF.c.24.1 M00001504A:E01

100 16731 RTA00000181AF.a.20.1 M00001442C:D07

101 12439 RTAOOOOO 190AF.O.24.1 M00003975A:G1 1

102 3162 RTAOOOOO 177AF.J.12.3 M00001351BA08 103 RTAOOOOO 194AF.b.19.1 M00004505D:F08

104 RTAOOOOO 193AF.n.15.1 M00004384C:D02 105 RTAOOOOO 186AF.n.7.1 M00001651A:H01

106 10717 RTAOOOOO 181 AF.d.10.1 M00001447A:G03

107 4573 RTA00000189AF.J.12.1 M00003871C:E02 108 RTAOOOOO 186AF.h.14.1 M00001632D:H07

109 11443 RTAOOOOO 192AF.1.13.2 M00004185C:C03

110 5892 RTAOOOOO 184AF.d.1 1.1 M00001548A:E10

111 3162 RTA00000177AF.J.12.1 M00001351B:A08

112 10470 RTAOOOOO 185AF.k.6.1 M00001597D:C05

113 17055 RTAOOOOO 187AF.m.3.1 M00001682C:B12

114 2030 RTAOOOOO 193 AF.m.20.1 M00004372A:A03

115 6558 RTAOOOOO 184AF.m.21.1 M00001560D:F10

116 23255 RTAOOOOO 190AF.J .4.1 M00003922A:E06

117 9577 RTAOOOOO 179AF.0.17.1 M00001409C:D12 118 RTAOOOOO 180AF.a.11.1 M00001414CA07

119 8 RTA00000181AF.e.l7.1 M00001448D:C09

120 67907 RTAOOOOO 188AF.g.11.1 M00003774C:A03

121 12081 RTAOOOOO 133 A.d.14.2 M00001469A:C10

122 2448 RTAOOOOO 119A.J.21.1 M00001460A:F06

123 3389 RTAOOOOO 189AF.g.3.1 M00003857A:G10

124 39174 RTAOOOOO 124A.n.13.1 M00001541A:H03

125 24488 RTAOOOOO 190AF.n.16.1 M00003968B:F06

126 8210 RTAOOOOO 192AF.n.13.1 M00004197D:H01 SEQ ID NO: Cluster ID Sequence Name Clone Name

127 RTA00000135A.1.2.2 M00001545A:B02

128 40455 RTAOOOOO 190AF.m.10.2 M00003958C:G10

129 9577 RTAOOOOO 180AF.d.23.1 M00001421 C.F01

130 13183 RTAOOOOO 192AF.a.24.1 M000041 14C:F11

131 5214 RTAOOOOO 186AF.g.1 1.1 M00001630B:H09

132 67252 RTA00000187AF.O.6.1 M00001716D:H05

133 3108 RTA00000188AF.d.24.1 M00003763A:F06

134 2464 RTAOOOOO 178AF.n.18.1 M00001387A:C05

135 36313 RTA00000181AF.e.23.1 M00001448D:H01

136 23255 RTAOOOOO 177AF.e.14.3 M00001343D.H07

137 7985 RTAOOOOO 182AR.J.2.1 M00001481D:A05

138 8286 RTAOOOOO 183 AF.o.1.1 M00001540A:D06

139 22195 RTAOOOOO 180AF.g.7.1 M00001425B:H08

140 4573 RTAOOOOO 184 AF.h.9.1 M00001553B:F12

141 26875 RTAOOOOO 187AF.i.1.1 M00001679A.F10

142 7187 RTA00000177AF.i.8.2 M00001350A.H01

143 86859 RTA000001 18A.p.8.1 M00001452A:B12

144 4623 RTAOOOOO 185 AF.f.4.1 M00001586C:C05

145 RTA00000121A.C.10.1 M00001469A:A01

146 10185 RTA00000183AF.d.5.1 M00001504C:A07

147 RTAOOOOO 183 AF.p.4.1 M00001542B.B01

148 15069 RTA00000191AF.1.6.1 M00004081C:D10

149 39304 RTAOOOOO 1 18A.J.21.1 M00001450A:A02

150 8672 RTAOOOOO 190AF.f.11.1 M00003909D:C03

151 13576 RTAOOOOO 177AF.g.16.1 M00001347A:B10

152 6293 RTAOOOOO 185 AF.e.1 1.1 M00001583D:A10

153 16977 RTAOOOOO 192AF.g.3.1 M00004151D:B08

154 5345 RTA00000189AF.1.19.1 M00003879B:C11

155 4905 RTAOOOOO 193 AF.e.14.1 M00004269D:D06

156 17036 RTAOOOOO 191 AF.j.10.1 M00004072B:B05

157 5417 RTAOOOOO 191 AF.h.19.1 M00004059A:D06

158 7172 RTAOOOOO 178AF.f.9.1 M00001371C:E09

159 40044 RTAOOOOO 186AF.d.1.1 M00001621C:C08

160 4386 RTAOOOOO 184AF.J.4.1 M00001556B:C08

161 40044 RTAOOOOO 183 AF.g.22.1 M00001514C:D11

162 9685 RTAOOOOO 183 AF.c.11.1 M00001501D:C02

163 22155 RTAOOOOO 185 AF.n.9.1 M00001608B:E03

164 10515 RTAOOOOO 189AF.f.18.1 M00003853A:F12

165 6539 RTAOOOOO 185 AF.d.1 1.1 M00001579D:C03

166 15066 RTAOOOOO 180AF.e.24.1 M00001423B:E07

167 4261 RTAOOOOO 180AF.h.5.1 M00001426D:C08

168 13864 RTAOOOOO 125 A.m.9.1 M00001545A:D08

169 6539 RTAOOOOO 189AF.d.22.1 M00003844C:B1 1 SEQ ID NO: Cluster ID Sequence Name Clone Name

170 11465 RTAOOOOO 185AF.m.19.1 M00001607A:E1 1

171 3266 RTAOOOOO 184 AR.g.1.1 M00001551C:G09

172 102 RTA00000184AF.O.5.1 M00001563B:F06

173 16970 RTA00000181AR.i.l 8.2 M00001452C:B06

174 12971 RTAOOOOO 193 AF.a.20.1 M00004223D:E04

175 5007 RTAOOOOO 177AF.g.2.1 M00001346A:F09

176 3765 RTAOOOOO 135A.d.1.1 M00001541A:D02

177 11294 RTAOOOOO 184AF.J.6.1 M00001556B:G02

178 3681 RTA00000131A.g.l5.2 M00001449A:D12

179 9283 RTA00000181AR.m.21.2 M00001455D.F09

180 18699 RTAOOOOO 182AF.m.16.1 M00001490B:C04

181 86110 RTA00000181AF.f.l2.1 M00001449C:D06

182 39648 RTAOOOOO 178AR.1.8.2 M00001383A:C03

183 7337 RTAOOOOO 123A.b.17.1 M00001528A:C04

184 1334 RTAOOOOO 178AF.J.7.1 M00001379A.A05

185 17076 RTAOOOOO 188AF.d.21.1 M00003762C:B08

186 22794 RTA00000138A.b.5.1 M00001601A:D08

187 39171 RTAOOOOO 186AF.1.7.1 M00001644C:B07

188 8551 RTAOOOOO 179AF.p.21.1 M00001412B:B10

189 5857 RTAOOOOO 118A.g.14.1 M00001449A:A12

190 9443 RTAOOOOO 183 AF.c.1.1 M00001500C:E04

191 9457 RTAOOOOO 193AF.i.14.2 M00004307C:A06

192 7206 RTAOOOOO 182AF.0.15.1 M00001494D:F06

193 22979 RTAOOOOO 178AF. 22.1 M00001382C:A02

194 40455 RTAOOOOO 190AR.m.10.1 M00003958C:G10

195 7221 RTAOOOOO 191 AF.p.9.1 M00004105C:A04 196 RTA00000191AF.J.9.1 M00004072A:C03

197 7239 RTAOOOOO 126A.m.4.2 M00001550AA03

198 31587 RTAOOOOO 189AF.1.20.1 M00003879B:D10

199 16317 RTAOOOOO 190AF.e.6.1 M00003907D:H04

200 13576 RTAOOOOO 189AR.0.13.1 M00003885CA02

201 5779 RTA00000177AF.g.l4.3 M00001346D:G06

202 6124 RTA00000191AR.e.2.3 M00004028DA06

203 9952 RTAOOOOO 180AF.C.20.1 M00001418B:F03 204 RTAOOOOO 188AF.-.8.1 M00003784D:D12

205 5779 RTAOOOOO 177AF.g.14.1 M00001346D:G06

206 39490 RTAOOOOO 128A.b.4.1 M00001557A:F03

207 4416 RTAOOOOO 187AF.h.13.1 M00001678D:F12

208 4009 RTAOOOOO 179AF.e.20.1 M00001396A:C03

209 5336 RTAOOOOO 183 AF.b.13.1 M00001500A:C05

210 39186 RTA00000121A.p.l5.1 M00001512AA09

211 40122 RTAOOOOO 190AF.n.23.1 M00003970C:B09

212 12532 RTAOOOOO 190AF.g.2.1 M00003912B:D01 SEQ ID NO: Cluster ID Sequence Name Clone Name

213 8078 RTA00000177AR.1.13.1 M00001353A:G12

214 3900 RTAOOOOO 190AF.g.13.1 M00003914C:F05

215 7589 RTAOOOOO 120A.p.23.1 M00001468A:F05

216 8298 RTAOOOOO 127A.d.19.1 M00001553A.H06

217 4443 RTAOOOOO 177AF.b.20.4 M00001341A:E12

218 26295 RTAOOOOO 193 AF.i.24.2 M00004312A:G03

219 3389 RTAOOOOO 183AF.m.19.1 M00001537B:G07

220 7015 RTAOOOOO 187AF.f.18.1 M00001673C:H02

221 8526 RTAOOOOO l δOAF.d.1.1 M00001418D:B06

222 4665 RTA00000186AF.m.3.1 M00001648CA01

223 1399 RTAOOOOO 129A.0.10.1 M00001604A:B10

224 9244 RTAOOOOO 127A.1.3.1 M00001556A:C09 225 RTAOOOOO 179 AF.j.13.1 M00001400B:H06

226 82498 RTAOOOOO 1 18A.m.10.1 M00001450A:B12

227 35702 RTAOOOOO 187AR.C.15.2 M00001663A:E04

228 38759 RTAOOOOO 120A.m.12.3 M00001467A:B07

229 39648 RTAOOOOO 178AF.1.8.1 M00001383A:C03

230 19105 RTAOOOOO 133 A.e.15.1 M00001469A.H12

231 85064 RTA00000131A.m.23.1 M00001452A:F05

232 9285 RTA00000191AF.m.l 8.1 M00004086D:G06

233 9285 RTAOOOOO 190AF.d.7.1 M00003906C:E10

234 39391 RTAOOOOO 138A.C.3.1 M00001604A:F05 235 RTAOOOOO 178AF.d.20.1 M00001368D:E03

236 39498 RTAOOOOO 1 19A.J.20.1 M00001460A:F12

237 7798 RTAOOOOO 189AF. 12.1 M00003876D:E12

238 7798 RTAOOOOO 189AF.C.18.1 M00003839A:D08

239 19829 RTAOOOOO 125 A.h.24.4 M00001544A:G02 240 RTAOOOOO 188AF.d.1 1.1 M00003761DA09

241 4275 RTA00000120A.J.14.1 M00001466A:E07

242 22113 RTAOOOOO 125 A.c.7.1 M00001542AA09

243 40314 RTAOOOOO 186AF.C.15.1 M00001619C:F12

244 10944 RTAOOOOO 126A.h.17.2 M00001549A:D08

245 39809 RTAOOOOO 190AF.e.3.1 M00003907DA09

246 22085 RTA00000135A.e.5.2 M00001541A.F07

247 19255 RTAOOOOO 135 A.m.18.1 M00001545A:C03

248 14311 RTAOOOOO 192AF.o.2.1 M00004203B:C12

249 8479 RTAOOOOO 189AF.J.22.1 M00003875C:G07 250 RTAOOOOO 189AF.J.23.1 M00003875D:D1 1

251 4193 RTAOOOOO 184AF.e.13.1 M00001549B:F06

252 22814 RTAOOOOO 184AF.h.14.1 M00001553D:D10

253 39563 RTA00000179AF.k.20.1 M00001402A:E08

254 39420 RTAOOOOO 134A.0.23.1 M00001537A:F12

255 11589 RTAOOOOO 177AF.b.17.4 M00001340D.F10 SEQ ID NO: Cluster ID Sequence Name Clone Name

256 4937 RTA00000191AF.p.21.1 M00004108A:E06

257 39412 RTAOOOOO 133A. 17.1 M0000151 1A:H06

258 4837 RTAOOOOO 185 AR.k.3.2 M00001597C:H02

259 13046 RTAOOOOO 193AF.h.19.1 M00004296C:H07

260 4141 RTAOOOOO 177AF.p.20.3 M00001361A:A05

261 38085 RTAOOOOO 123 A.e.15.1 M00001531A:D01 262 RTAOOOOO 189 AF.p.8.1 M00003891C:H09

263 11451 RTAOOOOO 192AF.p.17.1 M00004214C:H05

264 14507 RTAOOOOO 189AR.1.23.2 M00003879D:A02

265 40054 RTAOOOOO 180AF.p.10.1 M00001439C:F08

266 39423 RTAOOOOO 134A.k.22.1 M00001535A:F10

267 39453 RTA00000135A.g.l l.l M00001542A:E06

268 10751 RTAOOOOO 187AF.k.7.1 M00001679D:D03

269 10751 RTAOOOOO 187AF.k.6.1 M00001679D:D03

270 78091 RTAOOOOO 187AFJ.6.1 M00001679C:F01

271 39539 RTAOOOOO 127A.i.21.1 M00001555A:B02 272 RTAOOOOO 182AF.1.15.1 M00001487B:H06

273 RTAOOOOO 194AF.d.13.1 M00004896A:C07 274 RTAOOOOO 128 A.c.20.1 M00001558A:H05

275 9283 RTA00000181AR.m.22.2 M00001455D:F09

276 39168 RTA00000121A.1.10.1 M00001507A:H05

277 39458 RTA00000126A.p.l5.2 M00001552A:D1 1

278 14391 RTAOOOOO 177AF.m.17.3 M00001355B:G10

279 39195 RTA00000137A.C.16.1 M00001555A:C01

280 7212 RTAOOOOO 193AF.b.14.1 M00004230B:C07

281 4015 RTA00000136A.e.l2.1 M00001549A:B02

282 12977 RTAOOOOO 189AF.J.19.1 M00003875B:F04 283 RTAOOOOO 178AF.m.13.1 M00001384BA11

284 14391 RTA00000191AF.1.7.1 M00004081C:D12

285 RTAOOOOO 194AF.C.23.1 M00004691D:A05

286 RTA00000181AF.b.7.1 M00001443B:F01

287 8358 RTAOOOOO 183AF.i.5.1 M00001528B:H04

288 1267 RTA00000125A.O.5.1 M00001546A:G11 289 RTAOOOOO 189AF.f.7.1 M00003851B:D08

290 16347 RTA00000184AF.e.l5.1 M00001549C:E06

291 7899 RTAOOOOO 193AF.a.17.1 M00004223B:D09

292 2379 RTAOOOOO 178AF.a.6.1 M00001361D:F08

293 39478 RTA00000133A.i.5.1 M00001471A:B01

294 39392 RTAOOOOO 134A.m.16.1 M00001536A:C08

295 5053 RTAOOOOO 184AF.0.12.1 M00001564A:B12

296 16999 RTAOOOOO 185 AF.k.9.1 M00001598A:G03

297 39180 RTA00000126A.n.8.2 M00001551A:F05

298 1037 RTA00000121A.f.8.1 M00001470A:B10 UJ UJ UJ UI UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UI UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ ui to 4^ 4*ι UJ UJ UJ UJ UJ UJ UJ UJ UJ UJ tO ISJ tO tO tO tO tO tO tO tO ι-i H- H- H- H- *- τ- ι-ι ι-. ^ © © © © © © © © © © VO

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SEQ ID NO: Cluster DD Sequence Name Clone Name

342 39486 RTAOOOOO 128A.m.22.2 M00001561A:C05

343 21877 RTAOOOOO 189AF.b.21.1 M00003833A:E05

344 6874 RTAOOOOO 192AF.a.14.1 M000041 1 1D.A08

345 6874 RTAOOOOO 189AF.e.9.1 M00003846B:D06

346 37285 RTA00000191AF.f.l l . l M00004035C:A07 347 RTAOOOOO 193 AF.j.20.1 M00004327B:H04

348 7674 RTA000001 18A.g.9.1 M00001416A:H01

349 2797 RTAOOOOO 180AF.i.19.1 M00001429A:H04 350 RTAOOOOO 184AF.g.22.1 M00001552D:A01

351 7802 RTAOOOOO 185 AF.n.5.1 M00001608A:B03

352 16921 RTAOOOOO 193AF.h.15.1 M00004295D:F12

353 11494 RTAOOOOO 192AF.J.6.1 M00004172C:D08

354 17062 RTAOOOOO 177AF.b.8.4 M00001340B:A06

355 16245 RTAOOOOO 177AF. 9.3 M00001352A:E02

356 83103 RTA00000119A.e.24.2 M00001454A:A09

357 4309 RTAOOOOO 186AF.e.22.1 M00001624C:F01

358 13072 RTA00000181AR.m.5.2 M00001455B:E12

359 4059 RTAOOOOO 177AF.n.18.3 M00001357D:D1 1

360 5178 RTAOOOOO 178AF.n.10.1 M00001386C:B12

361 1120 RTAOOOOO 1 18A.p.15.3 M00001452A:D08

362 6420 RTAOOOOO 183 AF.d.11.1 M00001504D:G06

363 13913 RTAOOOOO 186AF.e.6.1 M00001623D.F10 364 RTAOOOOO 192AF.C.2.1 M00004121B:G01

365 3956 RTAOOOOO 183 AF.g.3.1 M00001512D:G09

366 14364 RTA00000183AF.g.l2.1 M00001513C:E08

367 6880 RTA00000191AF.m.20.1 M00004087D:A01

368 84182 RTAOOOOO 180AF.h.19.1 M00001428A:H10

369 2790 RTAOOOOO 177AF.e.2.1 M00001343C:F10

370 4561 RTAOOOOO 184AF.L21.1 M00001555D:G10

371 8847 RTAOOOOO 180AF.b.16.1 M00001416B:H11

372 56020 RTA00000193AF.g.2.1 M00004285B:E08

373 1531 RTAOOOOO 119A.0.3.1 M00001461A:D06

374 6420 RTAOOOOO 177AFT.10.3 M00001345A:E01 375 RTA00000188AF.b.l2.1 M00003754C:E09

376 RTAOOOOO 180AF. 24.1 M00001432C:F06 377 RTA00000184AF.a.8.1 M00001544A:E06

378 2696 RTAOOOOO 134A.m.13.1 M00001536A.B07

379 260 RTA00000185AR.U2.2 M00001594B:H04

380 11350 RTAOOOOO 189AF.a.24.2 M00003826B:A06

381 2428 RTA00000123A.1.21.1 M00001533A:C1 1

382 4313 RTAOOOOO 122A.n.3.1 M00001517A:B07 383 RTAOOOOO 184AF.p.3.1 M00001566B:D1 1

384 697 RTAOOOOO 188AF.d.6.1 M00003759B:B09 SEQ ID NO: Cluster ID Sequence Name Clone Name

385 5619 RTA00000188AF.1.9.1 M00003796C:D05

386 4568 RTA00000122A.d.l 5.3 M00001513A:B06 387 RTAOOOOO 177AF.i.6.2 M00001350A:B08

388 5622 RTAOOOOO 178AF.a.1 1.1 M00001362B:D10

389 7514 RTAOOOOO 184 AF.k.21.1 M00001558B:H1 1

390 5619 RTAOOOOO 189AF.f.17.1 M00003853A:D04

391 7570 RTAOOOOO 187AF.g.24.1 M00001677DA07

392 23358 RTAOOOOO 190AF.O.21.1 M00003974D:H02

393 23210 RTAOOOOO 190AF.O.20.1 M00003974D:E07

394 5192 RTAOOOOO 184AF. 2.1 M00001557B:H10

395 13538 RTAOOOOO 180AF.a.24.1 M00001415A:H06 396 RTAOOOOO 189AF.h.17.1 M00003867A:D10

397 RTAOOOOO 192AF.0.11.1 M00004205D:F06 398 RTAOOOOO 184AF.1.11.1 M00001559B:F01

399 4718 RTAOOOOO 189AF.g.5.1 M00003857A:H03

400 14929 RTAOOOOO 177AF.m.1.2 M00001353D:D10

401 4908 RTAOOOOO 192AF.J.2.1 M00004171D:B03 402 RTAOOOOO 178AF.k.16.1 M00001381D:E06

403 RTAOOOOO 194AF.C.24.1 M00004692A:H08

404 17732 RTAOOOOO 178ARL2.2 M00001376B:G06

405 17062 80.Al.sp6: 130208.Seq M00001340B:A06

406 11589 80.Bl .sp6:130220.Seq M00001340D:F10

407 4443 80.Cl .sp6:130232.Seq M00001341A:E12

408 39805 80.Dl .sp6: 130244.Seq M00001342B:E06

409 2790 80.El .sp6:130256.Seq M00001343C:F10

410 23255 80.Fl .sp6:130268.Seq M00001343D:H07

411 6420 80.Gl .sp6: 130280.Seq M00001345A:E01

412 5007 80.Hl .sp6:130292.Seq M00001346A:F09

413 13576 80.D2.sp6: 130245.Seq M00001347A:B10

414 16927 80.E2.sp6: 130257.Seq M00001348B:B04

415 16985 80.F2.sp6:130269.Seq M00001348B:G06

416 3584 80.G2.sp6: 130281. Seq M00001349B:B08 417 80.H2.sp6:130293.Seq M00001350A:B08

418 7187 80A3.sp6:130210.Seq M00001350A:H01

419 16245 80.D3.sp6:130246.Seq M00001352A:E02

420 8078 80.E3.sp6:130258.Seq M00001353A:G12

421 14929 80.F3.sp6:130270.Seq M00001353D:D10

422 14391 80.G3.sp6:130282.Seq M00001355B:G10

423 4141 80.B4.sp6:130223.Seq M00001361A:A05

424 2379 80.C4.sp6:130235.Seq M00001361D:F08

425 5622 80.D4.sp6:130247.Seq M00001362B:D10

426 945 8O.E4.sp6:130259.Seq M00001362C:H1 1

427 40132 80.F4.sp6:130271.Seq M00001365C:C10 SEQ ID NO: Cluster DD Sequence Name Clone Name

428 80.G4.sp6:130283.Seq M00001368D:E03

429 6867 80.H4.sp6:130295.Seq M00001370A:C09

430 7172 80.A5.sp6: 130212.Seq M00001371C:E09

431 17732 80.B5.sp6:130224.Seq M00001376B:G06

432 39833 80.C5.sp6:130236.Seq M00001378B:B02

433 1334 80.D5.sp6:130248.Seq M00001379A:A05

434 39886 80.E5.sp6:130260.Seq M00001380D:B09 435 80.F5.sp6: 130272.Seq M00001381D.Ε06

436 22979 80.G5.sp6: 130284.Seq M00001382CA02

437 39648 80.H5.sp6: 130296.Seq M00001383A:C03 438 80.B6.sp6:130225.Seq M00001384B:A1 1

439 5178 80.C6.sp6:130237.Seq M00001386C:B12

440 2464 80.D6.sp6:130249.Seq M00001387A:C05

441 7587 80.E6.sp6:130261.Seq M00001387B:G03

442 5832 80.F6.sp6:130273.Seq M00001388D:G05

443 16269 80.G6.sp6:130285.Seq M00001389A:C08

444 6583 80.H6.sp6: 130297.Seq M00001394A:F01

445 4009 80.A7.sp6:130214.Seq M00001396A:C03 446 80.B7.sp6:130226.Seq M00001400B:H06

447 39563 80.C7.sp6:130238.Seq M00001402A:E08

448 5556 80.D7.sp6:130250.Seq M00001407B:D1 1

449 9577 80.E7.sp6: 130262.Seq M00001409C:D12

450 7005 80.F7.sp6: 130274.Seq M00001410A:D07

451 8551 80.G7.sp6:130286.Seq M00001412B:B10 452 80.H7.sp6:130298.Seq M00001414A:B01

453 80A8.sp6:130215.Seq M00001414C:A07

454 13538 80.B8.sp6: 130227.Seq M00001415A:H06

455 8847 80.C8.sp6: 130239.Seq M00001416B:H1 1

456 36393 80.D8.sp6: 13025 l.Seq M00001417A:E02

457 9952 80.E8.sp6:130263.Seq M00001418B:F03

458 9577 80.G8.sp6:130287.Seq M00001421C:F01

459 15066 80.H8.sp6:130299.Seq M00001423B:E07

460 10470 80.A9.sp6:130216.Seq M00001424B:G09

461 22195 80.B9.sp6:130228.Seq M00001425B:H08 462 80.C9.sp6:130240.Seq M00001426B.D12

463 4261 80.D9.sp6:130252.Seq M00001426D:C08

464 84182 80.E9.sp6:130264.Seq M00001428A:H10

465 40392 80.H9.sp6:130300.Seq M00001429D:D07

466 16731 80.C10.sp6:130241.Seq M00001442C:D07 467 80.D10.sp6:130253.Seq M00001443B:F01

468 13532 80.E10.sp6:130265.Seq M00001445A:F05

469 8 80.H10.sp6:130301.Seq M00001448D:C09

470 36313 80.Al l .sp6:130218.Seq M00001448D:H01 SEQ ID NO: Cluster ID Sequence Name Clone Name

471 5857 80.Bl l .sp6: 130230.Seq M00001449A:A12

472 41633 80.Cl l .sp6:130242.Seq M00001449A:B12

473 36535 80.Dl l .sp6:130254.Seq M00001449A:G10

474 86110 80.El l .sp6: 130266.Seq M00001449C:D06

475 32663 80.Fl l .sp6: 130278.Seq M00001450A:A1 1

476 27250 80.Gl l .sp6:130290.Seq M00001450A:D08

477 16970 80.Hl l .sp6: 130302.Seq M00001452C:B06

478 16130 80.A12.sp6: 130219.Seq M00001453A:E1 1

479 16653 80.B12.sp6: 13023 l .Seq M00001453C:F06

480 7005 80.C12.sp6:130243.Seq M00001454B:C12

481 13072 80.F12.sp6: 130279.Seq M00001455B:E12

482 9283 80.G12.sp6:130291.Seq M00001455D:F09

483 23255 100.Cl.sp6: 131446.Seq M00001343D:H07

484 13576 100.El.sp6: 131470.Seq M00001347A:B10

485 7187 100.C2.sp6: 131447.Seq M00001350A:H01

486 14391 100.E3.sp6: 131472.Seq M00001355B:G10

487 945 100.E4.sp6:131473.Seq M00001362C:H1 1

488 7172 100.A5.sp6: 131426.Seq M00001371C:E09

489 39648 100A6.sp6:131427.Seq M00001383A:C03

490 84182 100.G9.sp6: 131502.Seq M00001428A:H10

491 8 100.Bl l.sp6:131444.Seq M00001448D:C09

492 36535 100.Dl l .sp6:131468.Seq M00001449A:G10

493 82498 100.Fl l .sp6: 131492.Seq M00001450A:B12

494 16970 100.C12.sp6:131457.Seq M00001452C:B06

495 16130 100.D12.sp6:131469.Seq M00001453A:E1 1

496 7005 121.Dl.sp6: 131917.Seq M00001454B:C12 497 121.G6.sp6:131958.Seq M00001506D:A09

498 18957 121.F7.sp6:131947.Seq M00001528A:F09

499 40044 122.El .sp6: 132121.Seq M00001621C:C08

500 5214 122.C2.sp6:132098.Seq M00001630B:H09

501 6660 122.B5.sp6: 132089.Seq M00001679A:A06

502 13183 123.D5.sp6:132305.Seq M00004114C:F11

503 6455 123.E7.sp6: 132319.Seq M00004157C:A09

504 5319 123.F7.sp6:132331.Seq M00004169C:C12

505 11443 123.A8.sp6: 132272.Seq M00004185C:C03 506 123.C8.sp6:132296.Seq M00004191D:B1 1

507 8210 123.E8.sp6:132320.Seq M00004197D:H01

508 9457 123.Dl l.sp6:13231 l .Seq M00004307C:A06

509 6420 172.El.sp6: 133925.Seq M00001345A:E01

510 16245 172.D2.sp6: 133914.Seq M00001352A:E02

511 8078 172.C3.sp6: 133903.Seq M00001353A:G12

512 14929 172.D3.sp6:133915.Seq M00001353D:D10

513 14391 172.H3.sp6:133963.Seq M00001355B:G10 SEQ ID NO: Cluster ID Sequence Name Clone Name

514 6583 172.B8.sp6:133896.Seq M00001394A:F01

515 4009 172.D8.sp6:133920.Seq M00001396A:C03 516 172.B9.sp6: 133897.Seq M00001400B:H06

517 176.A3.sp6:134514.Seq M00001632D:H07

518 19267 176.G3.sp6:134586.Seq M00001645A:C12

519 78091 176.G5.sp6:134588.Seq M00001679C:F01

520 17055 176.D6.sp6:134553.Seq M00001682C:B12

521 6539 176.D9.sp6:134556.Seq M00003844C:B1 1 522 177.H4.sp6: 134791. Seq M00004121B:G01

523 5257 177.F5.sp6:134768.Seq M00004146C:C1 1

524 11494 177.E6.sp6:134757.Seq M00004172C:D08 525 177.G7.sp6:134782.Seq M00004205D:F06

526 11451 177.D8.sp6:134747.Seq M00004214C:H05

527 9283 173.D2.SP6: 134106.Seq M00001455D:F09

528 16283 173.F3.SP6:134131.Seq M00001467A:D08

529 10539 173.B5.SP6: 134085.Seq M00001499BA1 1

530 6420 173.F5.SP6:134133.Seq M00001504D:G06

531 3956 173.H5.SP6: 134157.Seq M00001512D:G09 532 173.G7.SP6:134147.Seq M00001544A:E06

533 1577 173.C9.SP6: 134101.Seq M00001556A:F1 1

534 9635 173.D9.SP6:1341 13.Seq M00001557A:F01

535 5192 173.E9.SP6:134125.Seq M00001557B:H10

536 6539 173.A12.SP6:134080.Seq M00001579D:C03

537 945 180.C2.sp6: 135940.Seq M00001362C:H1 1

538 7005 180.H5.sp6:136003.Seq M00001410A:D07

539 39304 180.G9.sp6:135995.Seq M00001450A:A02

540 27250 180.B10.sp6:135936.Seq M00001450A:D08

541 35555 184.A5.sp6:135530.Seq M00001528A:C04

542 19255 184.B10.sp6:135547.Seq M00001545A:C03

543 6268 184.C12.sp6:135561.Seq M00001551A:B10

544 3277 217.El .sp6:139406.Seq M00001624A:B06

545 39171 217.A12.sp6:139369.Seq M00001644C:B07

546 11460 219.F2.sp6:139035.Seq M00001676B:F05

547 10539 219.F6.sp6:139039.Seq M00001680D:F08

548 11476 219.H8.sp6:139065.Seq M00003747D:C05

549 4016 79.Al.sp6:130016.Seq M00001395A:C03

550 7674 79.Cl.sp6:130040.Seq M00001416A:H01

551 3681 79.El.sp6: 130064.Seq M00001449A:D12

552 39304 79.Fl .sp6:130076.Seq M00001450A:A02

553 82498 79.Gl .sp6: 130088.Seq M00001450A:B12

554 84328 79A2.sp6:130017.Seq M00001452A:B04

555 86859 79.B2.sp6:130029.Seq M00001452A:B12

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SEQ ID NO: Cluster DD Sequence Name Clone Name

643 4193 89.G9.sp6:130747.Seq M00001549B:F06

644 16347 89.H9.sp6:130759.Seq M00001549C:E06

645 7239 89.A10.sp6: 130676.Seq MOOOO1550A:A03

646 5175 89.B10.sp6:130688.Seq M00001550A:G01

647 22390 89.C10.sp6:130700.Seq M00001551A:G06

648 3266 89.D10.sp6:130712.Seq M00001551C:G09

649 5708 89.E10.sp6:130724.Seq M00001552B.D04 650 89.F10.sp6:130736.Seq M00001552D:A01

651 8298 89.G10.sp6:130748.Seq M00001553A:H06

652 4573 89.H10.sp6:130760.Seq M00001553B:F12

653 22814 89.Al l.sp6:130677.Seq M00001553D:D10

654 39539 89.Bl l.sp6:130689.Seq M00001555A:B02

655 39195 89.Cl l.sp6:130701.Seq M00001555A:C01

656 4561 89.Dl l.sp6:130713.Seq M00001555D:G10

657 9244 89.El l.sp6:130725.Seq M00001556A:C09

658 1577 89.Fl l.sp6:130737.Seq M00001556A:F11

659 4386 89.Hl l .sp6:130761.Seq M00001556B:C08

660 11294 89.A12.sp6:130678.Seq M00001556B.G02

661 5192 89.D12.sp6:130714.Seq M00001557B:H10

662 8761 89.E12.sp6:130726.Seq M00001557D:D09 663 89.F12.sp6:130738.Seq M00001558A:H05

664 7514 89.G12.sp6:130750.Seq M00001558B:H11 665 89.H12.sp6:130762.Seq M00001559B.F01

666 6558 90.Al.sp6:130859.Seq M00001560D:F10

667 102 90.Bl .sp6:130871.Seq M00001563B:F06 668 90.Dl.sp6:130895.Seq M00001566B:D11

669 5749 90.El .sp6:130907.Seq M00001571C:H06

670 6539 90.Gl.sp6:130931.Seq M00001579D:C03

671 6293 90.A2.sp6:130860.Seq M00001583D:A10 672 90.C2.sp6:130884.Seq M00001590B:F03

673 260 90.D2.sp6:130896.Seq M00001594B:H04

674 4837 90.E2.sp6:130908.Seq M00001597C:H02

675 10470 90.F2.sp6:130920.Seq M00001597D:C05

676 16999 90.G2.sp6:130932.Seq M00001598A:G03

677 22794 90.H2.sp6:130944.Seq M00001601A:D08

678 11465 90.A3.sp6:130861.Seq M00001607A:E11

679 7802 90.B3.sp6:l 30873. Seq M00001608A:B03

680 22155 90.C3.sp6:130885.Seq M00001608B:E03 681 90.D3.sp6:130897.Seq M00001608D:A11

682 13157 90.E3.sp6:130909.Seq M00001614C:F10

683 17004 90.F3.sp6:130921.Seq M00001617C:E02

684 40314 90.G3.sp6:130933.Seq M00001619C:F12

685 40044 90.H3.sp6:130945.Seq M00001621C:C08 SEQ ID NO: Cluster ID Sequence Name Clone Name

686 13913 90.A4.sp6: 130862.Seq M00001623D:F10

687 3277 90.B4.sp6: 130874.Seq M00001624A:B06

688 4309 90.C4.sp6: 130886.Seq M00001624C:F01

689 5214 90.D4.sp6:130898.Seq M00001630B:H09

690 90.E4.sp6: 130910.Seq M00001632D:H07

691 39171 90.F4.sp6:130922.Seq M00001644C:B07

692 19267 90.G4.sp6: 130934.Seq M00001645A:C12

693 4665 90.H4.sp6:130946.Seq M00001648C:A01

694 90A5.sp6:130863.Seq M00001651A:H01

695 23201 90.B5.sp6:130875.Seq M00001657D:C03

696 76760 90.C5.sp6: 130887.Seq M00001657D:F08

697 23218 90.D5.sp6:130899.Seq M00001662C:A09

698 35702 90.E5.sp6:13091 l .Seq M00001663A:E04

699 6468 90.F5.sp6:130923.Seq M00001669B:F02

700 14367 90.G5.sp6: 130935.Seq M00001670C:H02

701 7015 90.H5.sp6: 130947.Seq M00001673C:H02

702 8773 90.A6.sp6: 130864.Seq M00001675A:C09

703 11460 90.B6.sp6: 130876.Seq M00001676B:F05

704 7570 90.D6.sp6:130900.Seq M00001677D:A07

705 4416 90.E6.sp6:130912.Seq M00001678D:F12

706 6660 90.F6.sp6:130924.Seq M00001679A:A06

707 90.H6.sp6:130948.Seq M00001679A:F06

708 26875 90A7.sp6: 130865.Seq M00001679A:F10

709 6298 90.B7.sp6:130877.Seq M00001679B:F01

710 78091 90.C7.sp6:130889.Seq M00001679C:F01

711 10751 90.D7.sp6: 13090 l.Seq M00001679D:D03

712 10539 90.F7.sp6:130925.Seq M00001680D:F08

713 17055 90.G7.sp6:13093JSeq M00001682C:B12

714 5382 90.A8.sp6:130866.Seq M00001688C:F09

715 4393 90.B8.sp6:130878.Seq M00001693C:G01

716 67252 90.C8.sp6:130890.Seq M00001716D:H05

717 40108 90.D8.sp6:130902.Seq M00003741D:C09

718 11476 90.E8.sp6:130914.Seq M00003747D:C05

719 90.F8.sp6:130926.Seq M00003754C:E09

720 697 90.G8.sp6:130938.Seq M00003759B:B09

721 90.H8.sp6:130950.Seq M00003761D.A09

722 17076 90A9.sp6:130867.Seq M00003762C:B08

723 3108 90.B9.sp6: 130879.Seq M00003763A:F06

724 67907 90.C9.sp6: 130891. Seq M00003774C:A03

725 90.D9.sp6: 130903.Seq M00003784D:D12

726 11350 90.F9.sp6: 130927.Seq M00003826B:A06

727 7899 90.H9.sp6: 13095 l.Seq M00003837D:A01

728 7798 90.A10.sp6:130868.Seq M00003839A:D08 SEQ ID NO: Cluster ID Sequence Name Clone Name

729 6539 90.B10.sp6: 130880.Seq M00003844C:B1 1

730 6874 90.C10.sp6:130892.Seq M00003846B:D06 731 90.D10.sp6: 130904.Seq M00003851B:D08

732 13595 90.E10.sp6: 130916.Seq M00003851B:D10

733 5619 90.F10.sp6:130928.Seq M00003853A:D04

734 10515 90.G10.sp6: 130940.Seq M00003853A:F12

735 4622 90.H10.sp6: 130952.Seq M00003856B:C02

736 3389 90.Al l .sp6:130869.Seq M00003857A:G10

737 4718 90.Bl l .sp6:130881.Seq M00003857A:H03 738 90.Cl l .sp6:130893.Seq M00003867A:D10

739 12977 90.Fl l.sp6:130929.Seq M00003875B:F04

740 8479 90.Gl l .sp6:130941.Seq M00003875C:G07 741 90.Hl l .sp6: 130953.Seq M00003875D:D1 1

742 7798 90A12.sp6: 130870.Seq M00003876D:E12

743 5345 90.B12.sp6: 130882.Seq M00003879B:C1 1

744 31587 90.C12.sp6:130894.Seq M00003879B:D10

745 14507 90.D12.sp6: 130906.Seq M00003879D:A02

746 13576 90.F12.sp6: 130930.Seq M00003885C:A02 747 90.G12.sp6:130942.Seq M00003891C:H09

748 9285 90.H12.sp6:130954.Seq M00003906C:E10

749 39809 99.Al.sp6:131230.Seq M00003907D:A09

750 16317 99.Bl .sp6:131242.Seq M00003907D:H04

751 8672 99.Cl.sp6:131254.Seq M00003909D:C03

752 12532 99.Dl .sp6:131266.Seq M00003912B:D01

753 3900 99.El.sp6: 131278.Seq M00003914C:F05

754 23255 99.Fl .sp6: 131290.Seq M00003922A:E06

755 24488 99.C2.sp6:131255.Seq M00003968B:F06

756 40122 99.D2.sp6:131267.Seq M00003970C:B09

757 23210 99.E2.sp6:131279.Seq M00003974D:E07

758 23358 99.F2.sp6: 131291. Seq M00003974D:H02

759 3430 99A3.sp6:131232.Seq M00003981A:E10

760 2433 99.B3.sp6:131244.Seq M00003982C:C02

761 9105 99.C3.sp6:131256.Seq M00003983A:A05

762 6124 99.D3.sp6: 131268.Seq M00004028DA06

763 40073 99.E3.sp6:131280.Seq M00004028D:C05

764 37285 99.H3.sp6:131316.Seq M00004035CA07

765 17036 99.A4.sp6:131233.Seq M00004035D:B06

766 3706 99.C4.sp6:131257.Seq M00004068B:A01 767 99.D4.sp6:131269.Seq M00004072A:C03

768 15069 99.F4.sp6:131293.Seq M00004081C:D10

769 9285 99.H4.sp6:131317.Seq M00004086D:G06

770 6880 99A5.sp6:131234.Seq M00004087D:A01

771 5325 99.C5.sp6:131258.Seq M00004093D:B12 SEQ ID NO: Cluster ID Sequence Name Clone Name

772 7221 99.D5.sp6:131270.Seq M00004105C:A04

773 4937 99.E5.sp6: 131282. Seq M00004108A:E06

774 6874 99.F5.sp6: 131294.Seq M000041 11D:A08

775 13183 99.G5.sp6:131306.Seq M000041 14C:F1 1 776 99.H5.sp6:131318.Seq M00004121B.G01

777 13272 99.A6.sp6: 131235.Seq M00004138B:H02

778 5257 99.B6.sp6:131247.Seq M00004146C:C1 1

779 6455 99.D6.sp6: 131271. Seq M00004157C:A09

780 5319 99.E6.sp6:131283.Seq M00004169C:C12

781 4908 99.F6.sp6:131295.Seq M00004171D:B03

782 11494 99.G6.sp6:131307.Seq M00004172C:D08

783 11443 99.A7.sp6:131236.Seq M00004185C:C03 784 99.B7.sp6: 131248.Seq M00004191D:B1 1

785 8210 99.C7.sp6:131260.Seq M00004197D:H01

786 14311 99.D7.sp6: 131272.Seq M00004203B:C12 787 99.E7.sp6:131284.Seq M00004205D:F06

788 12971 99.B8.sp6:131249.Seq M00004223D:E04

789 6455 99.C8.sp6:131261.Seq M00004229B:F08

790 7212 99.D8.sp6:131273.Seq M00004230B:C07

791 4905 99.H8.sp6: 131321. Seq M00004269D:D06

792 16914 99A9.sp6:131238.Seq M00004275C:C11

793 16921 99.D9.sp6:131274.Seq M00004295D:F12

794 13046 99.E9.sp6:131286.Seq M00004296C:H07

795 9457 99.F9.sp6:131298.Seq M00004307C:A06

796 26295 99.G9.sp6:131310.Seq M00004312A:G03

797 21847 99.H9.sp6:131322.Seq M00004318C:D10 798 99.H10.sp6:131323.Seq M00004505D:F08

799 99.Bl l.sp6:131252.Seq M00004692A:H08 800 99.Dl l.sp6: 131276.Seq M00005180C:G03

801 39304 RTAOOOOO 118A.J.21.1. Seq_THCl 51859

802 2428 RTAOOOOO 123 A.1.21.1.Seq_THC205063

803 1058 RTA00000126A.e.20.3.Seq_THC217534

804 5097 RTAOOOOO 134A.k.1.1.Seq_THC215869

805 20212 RTA00000134A.1.22.1.Seq_THC128232

806 23255 RTAOOOOO 177AF.e.l 4.3. Seq_THC228776

807 2790 RTA00000177AF.e.2.1.Seq_THC229461

808 6420 RTAOOOOO 177AF.f.10.3.Seq_THC226443

809 4059 RTA00000177AF.n.l8.3.Seq_THC123051 810 RTAOOOOO 179AF.J.13. l .Seq_THC 105720

811 9952 RTAOOOOO 180AF.C.20.1.Seq_THC 162284

812 13238 RTA00000181AF.m.4.1.Seq_THC140691

813 9685 RTAOOOOO 183 AF.c.11.1.Seq_THC 109544 814 RTAOOOOO 183 AF.c.24.1.Seq_THC 125912 SEQ ID NO: Cluster IE > Sequence Name Clone Name

815 6420 RTAOOOOO 183 AF, .d.ll.l.Seq_THC226443

816 6974 RTAOOOOO 183 AF, .d.9.1.Seq_THC223129

817 40044 RTAOOOOO 183 AF .g.22.1.Seq_THC232899

818 RTAOOOOO 183 AF. .g.9.1.Seq_THC198280

819 5892 RTAOOOOO 184AF, .d.ll.l.Seq_THC161896

820 40044 RTAOOOOO 186AF. .d.l.l.Seq_THC232899

821 RTAOOOOO 186AF .h.14.l.Seq_THC112525

822 19267 RTAOOOOO 186AF, .1.12.1.Seq_THC178183

823 8773 RTAOOOOO 187AF. .f.24.1.Seq_THC220002

824 7570 RTAOOOOO 187AF. .g.24.1.Seq_THC168636

825 11476 RTAOOOOO 187AF, .p.l9.1.Seq_THC108482

826 RTA00000188AF, .d.ll.l.Seq_THC212094

827 17076 RTAOOOOO 188AF. .d.21.1.Seq_THC208760

828 697 RTAOOOOO 188AF, .d.6.1.Seq_THC178884

829 67907 RTAOOOOO 188AF. .g.ll.l.Seq_THC123222

830 5619 RTAOOOOO 188AF. .1.9. l .Seq THC 167845

831 4718 RTAOOOOO 189AF, .g.5.1.Seq_THC196102

832 39809 RTAOOOOO 190AF. .e.3.1.Seq_THC150217

833 23255 RTAOOOOO 190AF. j.4.1.Seq_THC228776

834 40122 RTAOOOOO 190AF. .n.23.1.Seq_THC 109227

835 23210 RTAOOOOO 190AF. .o.20.1.Seq_THC207240

836 23358 RTAOOOOO 190AF, .o.21.1.Seq_THC207240

837 5693 RTAOOOOO 190AF. .p.l7.2.Seq_THC173318

838 2433 RTA00000191AF, .a.l5.2.Seq_THC79498

839 5257 RTAOOOOO 192AF. .f.3.1.Seq_THC213833

840 16392 RTAOOOOO 192AF. .l.l .l .Seq_THC202071

841 RTAOOOOO 193 AF. .c.21.1.Seq_THC222602

842 26295 RTAOOOOO 193 AF. .i.24.2.Seq_THC 197345

843 RTAOOOOO 193 AF, .m.5.1.Seq_THC173318

844 RTAOOOOO 193 AF. .n.l5.1.Seq_THC215687

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 5 All Differential Data for Libs 1-4 and 8-9

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl Lib2 Lib3 Lib4 LibS Lib9

M00001340B:A06 17062 3 0 0 0 0 0

M00001340D.F 10 1 1589 2 2 1 3 3 8

M00001341A:E12 4443 10 6 2 6 3 1 1

M00001342B:E06 39805 2 0 0 0 1 0

M00001343C:F10 2790 7 15 13 14 6 0

M00001343D:H07 23255 3 0 1 1 0 0

M00001345A:E01 6420 8 0 2 0 1 0

M00001346A:F09 5007 4 8 3 6 2 6

M00001346D:E03 6806 5 2 1 2 0 3

M00001346D:G06 5779 5 4 3 4 0 0

M00001346D:G06 5779 5 4 3 4 0 0

M00001347A:B10 13576 5 0 0 0 12 1 1

M00001348B:B04 16927 4 0 0 2 0 0

M00001348B:G06 16985 4 0 0 0 0 0

M00001349B:B08 3584 5 1 1 5 0 0 2

M00001350A:H01 7187 5 3 1 0 1 0

M00001351BAO8 3162 10 14 1 6 6 5

M00001351B:A08 3162 10 14 1 6 6 5

M00001352A:E02 16245 4 0 0 0 0 0

M00001353A:G12 8078 4 3 1 0 1 0

M00001353D:D10 14929 4 0 0 1 23 16

M00001355B:G10 14391 3 1 0 0 0 0

M00001357D:D11 4059 8 6 8 16 0 1

M00001361AA05 4141 5 2 10 16 4 27

M00001361D:F08 2379 26 13 4 2 2 3

M00001362B:D10 5622 7 4 2 13 1 2

M00001362C:H11 945 9 21 2 1 0 0

M00001365C:C10 40132 2 0 0 0 3 0

M00001370A:C09 6867 7 3 0 0 0 0

M00001371C:E09 7172 3 5 1 2 0 1

M00001376B.G06 17732 1 3 5 0 1 4

M00001378B:B02 39833 2 0 0 0 0 0

M00001379AA05 1334 27 38 35 28 3 0

M00001380D:B09 39886 2 0 0 0 0 0

M00001382CA02 22979 2 1 0 0 0 0

M00001383A.C03 39648 2 0 0 0 0 0

M00001383A:C03 39648 2 0 0 0 0 0

M00001386C:B12 5178 5 5 4 2 5 2

M00001387A:C05 2464 5 19 25 16 1 0

M00001387B:G03 7587 6 2 1 0 0 0

M00001388D:G05 5832 10 3 0 1 5 0

M00001389A:C08 16269 3 0 0 0 1 1

M00001394A:F01 6583 2 7 3 2 0 0

M00001395A:C03 4016 5 14 0 6 0 0

M00001396A:C03 4009 6 4 13 5 4 10

M00001402A.E08 39563 2 0 0 0 0 0 Table 5 All Differential Data for Libs 1-4 and 8-9

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl Lib2 Lib3 Lib4 LibS Lib9

M00001407B.D11 5556 8 1 5 0 2 0

M00001409C:D12 9577 5 2 0 1 1 1 12

M00001410A:D07 7005 8 2 0 0 0 0

M00001412B:B10 8551 4 4 0 3 0 0

M00001415A:H06 13538 5 0 0 0 9 1

M00001416A.H01 7674 5 2 0 5 0 0

M00001416B:H1 1 8847 4 1 3 0 6 1

M00001417A:E02 36393 2 0 0 1 0 0

M00001418B:F03 9952 4 2 1 1 0 0

M00001418D:B06 8526 3 2 1 5 1 0

M00001421C.F01 9577 5 2 0 1 1 1 12

M00001423B:E07 15066 4 0 0 0 0 0

M00001424B:G09 10470 5 1 0 2 0 1

M00001425B:H08 22195 3 0 0 0 0 0

M00001426D:C08 4261 4 9 7 9 12 15

M00001428A:H10 84182 1 0 0 0 0 0

M00001429A:H04 2797 15 1 1 18 16 1 14

M00001429B 1 1 4635 7 9 2 0 0 0

M00001429D:D07 40392 2 0 1 8 12 16

M00001439C:F08 40054 1 0 0 0 0 0

M00001442C:D07 16731 3 1 0 0 0 0

M00001445A:F05 13532 3 2 1 0 1 2

M00001446A:F05 7801 5 2 4 6 1 0

M00001447A:G03 10717 7 2 0 5 8 0

M00001448D:C09 8 1850 2127 1703 3133 1355 122

M00001448D:H01 36313 2 0 0 0 1 30

M00001449A 12 5857 6 2 3 4 0 0

M00001449A:B12 41633 1 1 0 0 0 0

M00001449A:D12 3681 12 5 10 1 2 5

M00001449A:G10 36535 2 0 0 0 0 0

M00001449C:D06 861 10 1 0 0 0 0 0

M00001450AA02 39304 2 0 0 0 0 0

M00001450A:A11 32663 1 1 0 0 0 0

M00001450A:B12 82498 1 0 0 0 0 0

M00001450A:D08 27250 2 0 0 0 0 0

M00001452A:B04 84328 1 0 0 0 0 0

M00001452A:B 12 86859 1 0 0 0 0 0

M00001452A:D08 1120 44 41 5 11 5 0

M00001452A:F05 85064 1 0 0 0 0 0

M00001452C:B06 16970 4 0 0 0 3 4

M00001453A:E11 16130 3 1 0 0 0 1

M00001453C:F06 16653 3 1 0 0 0 0

M00001454AA09 83103 1 0 0 0 0 0

M00001454B:C12 7005 8 2 0 0 0 0

M00001454D:G03 689 58 95 17 36 66 95

M00001455A.E09 13238 4 1 0 0 0 0

M00001455B:E12 13072 4 1 0 0 0 0

M00001455D:F09 9283 4 1 0 1 0 1 Table 5 All Differential Data for Libs 1-4 and 8-9

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl Lib2 Lib3 Lib4 LibS Lib9

M00001455D:F09 9283 4 1 0 1 0 1

M00001460A:F06 2448 23 22 2 3 3 1

M00001460A:F12 39498 2 0 0 0 0 0

M00001461A.D06 1531 20 23 32 17 14 14

M00001463C:B1 1 19 1415 1203 1364 525 479 774

M00001465A:B1 1 10145 2 0 2 0 0 0

M00001466A:E07 4275 1 1 2 5 0 4 2

M00001467A:B07 38759 2 0 0 0 1 1

M00001467A:D04 39508 2 0 0 0 0 0

M00001467A:D08 16283 3 0 0 0 0 0

M00001467A:D08 16283 3 0 0 0 0 0

M00001467A:E10 39442 2 0 0 0 0 0

M00001468A:F05 7589 6 2 1 1 1 0

M00001469A:C10 12081 4 0 0 0 0 0

M00001469A:H12 19105 2 0 2 0 1 0

M00001470A:B10 1037 53 48 4 22 0 0

M00001470A:C04 39425 2 0 0 0 0 0

M00001471A:B01 39478 2 0 0 0 0 0

M00001481D:A05 7985 3 1 4 0 1 0

M00001490B:C04 18699 2 1 0 0 0 3

M00001494D:F06 7206 4 3 3 1 2 0

M00001497A:G02 2623 12 4 31 4 6 1

M00001499B:A1 1 10539 2 1 1 0 1 0

M00001500A:C05 5336 9 2 4 8 3 15

M00001500A:E11 2623 12 4 31 4 6 1

M00001500C:E04 9443 4 2 1 1 0 0

M00001501D:C02 9685 3 2 0 7 2 3

M00001504CA07 10185 5 1 0 0 2 4

M00001504C:H06 6974 7 3 0 1 0 0

M00001504D:G06 6420 8 0 2 0 1 0

M00001507A:H05 39168 2 0 0 0 0 0

M00001511A:H06 39412 2 0 0 0 0 0

M00001512AA09 39186 2 0 0 0 0 0

M00001512D:G09 3956 9 9 5 2 0 0

M00001513A:B06 4568 10 4 0 9 2 0

M00001513C.E08 14364 1 0 0 0 0 0

M00001514C:D11 40044 2 0 0 0 0 0

M00001517A:B07 4313 13 6 1 0 1 0

M00001518C:B11 8952 3 4 0 4 2 0

M00001528A:C04 7337 4 4 3 16 12 21

M00001528A:F09 18957 3 0 0 0 0 0

M00001528B:H04 8358 3 3 2 0 0 0

M00001531A:D01 38085 2 0 0 0 0 0

M00001532BA06 3990 6 12 4 1 3 1

M00001533A:C11 2428 14 14 13 9 2 19

M00001534A.C04 16921 4 0 0 1 2 1

M00001534A:D09 5097 6 5 1 1 3 2

M00001534A:F09 5321 11 7 1 5 10 26 ₅₅₇A₇₀₆₅2_1:

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4* 4* 4* σs σs Os σs to to t © o σs 4* to t CO 03 > > > α > > α CO > 03 > > o > > > > O 03 > > > > > > > CO > > > > α e ii b b CO b tή ^'-b tή 03 tή tή © o D b - o b b © b o © b X © b © b b © b © > o © b CO © © be b b b © © b b © o © © to t to © to © t σs 4* 5 t eos 5 © σs σs 00 t © eo ^1 to σs e-o to to to to 00 -0 * t to so co to to so as to to σs © to so to

4*. σs so ~4 s © t - Os σs co so © © to so σs t -o 4*. σs 4*. to so σs o co -p. to -o σs

4*. © co

© 4*. σs

© © 4* o © ~J 4* © © © 4* to O ^α © 4* © © co o — o O

to to t CO to UJ 4*. © © © tθ H- 4_- © CO

CΛ © © O CO Ul ι-. © tO UJ OO 4*ι CΛ t © © H^H t O O

HH --. © *-- to co to oo © o © cΛ Co co © ' .-^π ,© © to © © to CΛ ~J 4_. © H- © H- © © © ©

© © © CO © © _ © © © _ ©

SO © 0 © CΛ © tO © © © tO so © t0 © © © 0 © ©

Table 5 All Differential Data for Libs 1-4 and 8-9

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl Lib2 Lib3 Lib4 Lib8 Lib9

M00001557A:F01 9635 3 0 2 1 0 0

M00001557A:F03 39490 2 0 0 0 1 0

M00001557B.H10 5192 8 5 0 5 0 0

M00001557D:D09 8761 3 4 0 1 0 1

M00001558B:H1 1 7514 5 3 0 0 0 0

M00001560D:F10 6558 4 3 4 0 0 5

M00001561A:C05 39486 2 0 0 0 0 0

M00001563B:F06 102 289 233 278 1 16 123 184

M00001564A:B12 5053 1 1 4 2 2 1 1

M00001571C:H06 5749 4 1 9 0 0 0

M00001578B:E04 23001 2 1 0 2 0 0

M00001579D:C03 6539 8 3 0 0 0 1

M00001583D 10 6293 3 5 2 6 0 0

M00001586C:C05 4623 3 4 12 2 1 1

M00001587A:B 1 1 39380 2 0 0 0 0 0

M00001594B:H04 260 189 188 27 2 15 0

M00001597C:H02 4837 6 2 10 0 3 1

M00001597D:C05 10470 5 1 0 2 0 1

M00001598A:G03 16999 4 0 0 0 0 0

M00001601A:D08 22794 2 0 0 0 0 0

M00001604A.B10 1399 49 27 19 7 10 23

M00001604A:F05 39391 2 0 0 0 0 0

M00001607A:E1 1 1 1465 5 0 0 0 0 0

M00001608A:B03 7802 5 4 0 1 0 0

M00001608B:E03 22155 3 0 0 0 0 0

M00001614C:F10 13157 4 1 0 3 1 0

M00001617C:E02 17004 4 0 1 0 1 0

M00001619C:F12 40314 2 0 0 0 1 0

M00001621C:C08 40044 2 0 0 0 0 0

M00001623D:F10 13913 2 1 2 0 0 1

M00001624A:B06 3277 10 1 1 8 3 5 1

M00001624C:F01 4309 4 13 3 10 0 0

M00001630B:H09 5214 10 2 2 2 4 3

M00001644C:B07 39171 2 0 0 0 0 0

M00001645A:C12 19267 2 0 0 0 0 1

M00001648CA01 4665 5 9 0 0 0 0

M00001657D:C03 23201 3 0 0 0 3 0

M00001657D:F08 76760 1 0 2 2 0 5

M00001662CA09 23218 3 0 0 0 0 0

M00001663A:E04 35702 2 0 0 0 0 0

M00001669B.F02 6468 4 3 3 8 1 0

M00001670C:H02 14367 3 0 0 0 0 0

M00001673C:H02 7015 6 3 1 2 1 1

M00001675A:C09 8773 4 1 4 4 4 6

M00001676B:F05 114.60 4 2 0 0 0 0

M00001677C:E10 14627 1 2 1 0 1 0

M00001677DA07 7570 5 3 0 0 0 0

M00001678D:F12 4416 9 5 2 6 1 3 Table 5 All Differential Data for Libs 1 -4 and 8-9

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl Lib2 Lib3 Lib4 Lib8 Lib9

M00001679A:A06 6660 7 0 4 2 1 0

M00001679A:F10 26875 1 0 0 0 1 0

M00001679B:F01 6298 2 4 5 3 1 0

M00001679C:F01 78091 1 0 0 0 0 0

M00001679D.-D03 10751 3 2 0 1 0 1

M00001679D.D03 10751 3 2 0 1 0 1

M00001680D:F08 10539 2 1 1 0 1 0

M00001682C:B12 17055 4 0 0 0 0 0

M00001686A:E06 4622 7 6 4 2 3 0

M00001688C:F09 5382 6 2 6 2 0 3

M00001693C:G01 4393 10 6 2 4 1 1

M00001716D:H05 67252 1 0 0 1 0 0

M00003741D:C09 40108 2 0 0 0 0 0

M00003747D:C05 1 1476 6 0 0 0 0 0

M00003759B:B09 697 76 52 30 72 21 30

M00003762C:B08 17076 4 0 0 0 0 0

M00003763A:F06 3108 14 1 1 7 5 0 1

M00003774CA03 67907 1 0 0 0 0 0

M00003796C:D05 5619 3 5 3 3 0 4

M00003826B:A06 11350 3 3 0 0 1 0

M00003833A.E05 21877 2 1 0 0 0 1

M00003837D:A01 7899 5 4 0 2 1 0

M00003839A:D08 7798 5 2 2 0 0 1

M00003844C:B1 1 6539 8 3 0 0 0 1

M00003846B:D06 6874 6 3 0 0 0 0

M00003851B.D10 13595 4 0 1 0 0 1

M00003853A:D04 5619 3 5 3 3 0 4

M00003853A:F12 10515 5 1 0 1 1 2

M00003856B:C02 4622 7 6 4 2 3 0

M00003857A:G10 3389 4 1 1 13 2 0 0

M00003857A.H03 4718 4 5 5 2 4 6

M00003871C:E02 4573 5 7 2 5 0 1

M00003875B:F04 12977 5 0 0 0 0 0

M00003875B:F04 12977 5 0 0 0 0 0

M00003875C:G07 8479 4 3 1 1 2 4

M00003876D:E12 7798 5 2 2 0 0 1

M00003879B:C1 1 5345 7 1 7 4 6 27

M00003879B:D10 31587 1 1 0 0 1 0

M00003879D:A02 14507 3 1 0 0 3 1

M00003885C:A02 13576 5 0 0 0 12 11

M00003885CA02 13576 5 0 0 0 12 1 1

M00003906C.E10 9285 4 3 0 0 1 2

M00003907D:A09 39809 1 0 0 0 2 1

M00003907D:H04 16317 3 0 0 0 0 0

M00003909D:C03 8672 4 4 0 0 0 0

M00003912B:D01 12532 4 1 0 1 0 1

M00003914C:F05 3900 9 6 8 1 7 13

M00003922A:E06 23255 3 0 1 1 0 0 Table 5 All Differential Data for Libs 1 -4 and 8-9

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl Lib2 Lib3 Lib4 Lib8 Lib9

M00003958A:H02 18957 3 0 0 0 0 0

M00003958A:H02 18957 3 0 0 0 0 0

M00003958C:G10 40455 2 0 0 0 0 0

M00003958C:G10 40455 2 0 0 0 0 0

M00003968B:F06 24488 2 0 1 4 0 0

M00003970C:B09 40122 2 0 0 0 0 0

M00003974D:E07 23210 3 0 0 0 0 0

M00003974D:H02 23358 3 0 0 0 1 0

M00003975A:G1 1 12439 4 0 0 0 0 0

M00003978B:G05 5693 7 4 1 3 1 1

M00003981A.E10 3430 9 10 7 3 0 0

M00003982C:C02 2433 10 13 21 18 8 8

M00003983AA05 9105 5 1 1 1 0 0

M00004028DA06 6124 4 8 1 9 1 0

M00004028D:C05 40073 2 0 1 0 0 1

M00004031A.A12 9061 5 2 0 0 0 0

M00004031AA12 9061 5 2 0 0 0 0

M00004035CA07 37285 2 0 0 1 0 1

M00004035D:B06 17036 4 0 0 0 0 0

M00004059A:D06 5417 10 4 0 9 2 0

M00004068BA01 3706 7 14 4 22 1 0

M00004072B:B05 17036 4 0 0 0 0 0

M00004081C:D10 15069 3 0 0 1 0 0

M00004081C:D12 14391 3 1 0 0 0 0

M00004086D:G06 9285 4 3 0 0 1 2

M00004087D:A01 6880 2 6 1 1 0 0

M00004093D:B12 5325 5 5 2 0 2 1

M00004093D:B12 5325 5 5 2 0 2 1

M00004105CA04 7221 5 2 2 2 0 0

M00004108A:E06 4937 4 9 3 1 3 1

M0000411 1D:A08 6874 6 3 0 0 0 0

M000041 14C:F1 1 13183 2 3 0 7 0 1

M00004138B:H02 13272 3 2 0 3 0 0

M00004146C:C11 5257 2 8 5 5 5 25

M00004151D:B08 16977 4 0 0 0 0 0

M00004157CA09 6455 3 1 6 0 0 0

M00004169C:C12 5319 6 2 8 2 2 3

M00004171D:B03 4908 6 7 2 2 2 0

M00004172C:D08 1 1494 4 0 0 0 0 0

M00004183C:D07 16392 3 0 0 0 0 0

M00004185C:C03 11443 5 1 0 0 0 0

M00004197D:H01 8210 2 6 0 0 0 0

M00004203B:C12 1431 1 4 0 0 0 1 2

M00004212B:C07 2379 26 13 4 2 2 3

M00004214C:H05 1 1451 3 2 1 2 1 1

M00004223A:G10 16918 4 0 0 0 0 0

M00004223B:D09 7899 5 4 0 2 1 0

M00004223D:E04 12971 4 0 0 0 1 0 Table 5 All Differential Data for Libs 1-4 and 8-9

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones

ID Libl Lib2 Lib3 Lib4 Lib8 Lib9

M00004229B:F08 6455 3 1 6 0 0 0

M00004230B:C07 7212 3 5 2 1 3 0

M00004269D:D06 4905 7 6 3 1 3 1

M00004275C.C1 1 16914 3 0 0 1 0 0

M00004283BA04 14286 3 1 0 1 1 1

M00004285B:E08 56020 1 0 0 0 0 0

M00004295D:F12 16921 4 0 0 1 2 1

M00004296C:H07 13046 4 1 0 1 0 0

M00004307CA06 9457 2 0 5 0 0

M00004312A:G03 26295 2 0 0 0 0 0

M00004318C.D10 21847 2 1 0 0 0 0

M00004372AA03 2030 13 10 32 4 0 0

M00004377C:F05 2102 12 20 23 21 6 5

Table 6 All Differential Data for Libs 15-20

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl5 Libl6b Libl 7 Libl8 Libl9 Lib20

M00001340BA06 17062 0 0 0 0 0 0

M00001340D:F10 1 1589 0 0 0 0 0 0

M00001341A.E12 4443 0 0 0 1 0 0

M00001342B.E06 39805 0 0 0 0 0 0

M00001343C:F10 2790 0 0 0 0 0 0

M00001343D:H07 23255 0 0 0 0 0 0

M00001345A:E01 6420 0 0 0 0 0 0

M00001346A:F09 5007 0 0 0 0 0 0

M00001346D:E03 6806 0 0 0 0 0 0

M00001346D:G06 5779 0 0 0 0 0 0

M00001346D:G06 5779 0 0 0 0 0 0

M00001347A:B10 13576 0 0 0 0 0 0

M00001348B:B04 16927 0 0 0 0 0 0

M00001348B:G06 16985 0 0 0 0 0 0

M00001349B:B08 3584 0 0 0 0 0 0

M00001350A:H01 7187 0 0 0 0 0 0

M00001351B:A08 3162 0 1 0 0 1 0

M00001351B:A08 3162 0 1 0 0 1 0

M00001352A:E02 16245 0 0 0 0 0 0

M00001353A:G12 8078 0 0 0 0 0 0

M00001353D:D10 14929 0 3 1 0 5 0

M00001355B:G10 14391 0 0 0 0 0 0

M00001357D:D1 1 4059 0 0 0 0 0 0

M00001361AA05 4141 0 0 0 0 0 0

M00001361D:F08 2379 0 0 0 0 0 0

M00001362B:D10 5622 0 0 0 0 0 0

M00001362C:H11 945 0 0 0 0 0 1

M00001365C:C10 40132 0 0 0 0 0 0

M00001370A:C09 6867 0 0 0 0 0 0

M00001371C:E09 7172 0 0 0 0 0 0

M00001376B:G06 17732 0 0 0 0 0 1

M00001378B:B02 39833 0 0 0 0 0 0

M00001379AA05 1334 0 0 0 0 0 1

M00001380D:B09 39886 0 0 0 0 0 0

M00001382CA02 22979 0 0 0 0 0 0

M00001383A:C03 39648 0 0 0 0 0 0

M00001383A:C03 39648 0 0 0 0 0 0

M00001386C.B 12 5178 0 0 0 0 0 0

M00001387A:C05 2464 0 0 0 0 0 0

M00001387B:G03 7587 0 0 0 0 0 0

M00001388D:G05 5832 0 0 0 0 0 0

M00001389A:C08 16269 0 1 0 0 0 0

M00001394A:F01 6583 1 4 1 0 0 0

M00001395A:C03 4016 0 0 0 0 0 0

M00001396A:C03 4009 0 0 0 0 0 0

M00001402A:E08 39563 0 0 0 0 0 0

M00001407B:D1 1 5556 0 0 0 0 0 0

M00001409C:D12 9577 0 0 0 0 0 0 Table 6 All Differential Data for Libs 15-20

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl5 Liblόb Libl7 Libl8 Libl9 Lib20

M00001410A:D07 7005 0 0 0 0 0 0

M00001412B:B10 8551 0 0 0 0 0 0

M00001415A:H06 13538 0 0 0 0 0 0

M00001416A:H01 7674 0 0 0 0 0 0

M00001416B:H1 1 8847 0 0 0 0 0 0

M00001417A:E02 36393 0 0 0 0 0 0

M00001418B:F03 9952 0 0 0 0 0 0

M00001418D:B06 8526 0 0 0 0 0 0

M00001421 C.F01 9577 0 0 0 0 0 0

M00001423B:E07 15066 0 0 0 0 0 0

M00001424B:G09 10470 0 0 0 0 0 0

M00001425B:H08 22195 0 0 0 0 0 0

M00001426D:C08 4261 0 0 1 0 0 1

M00001428A:H10 84182 0 0 0 0 0 0

M00001429A:H04 2797 0 0 0 0 0 0

M00001429B 1 1 4635 0 0 0 0 0 0

M00001429D:D07 40392 0 0 0 0 0 0

M00001439C:F08 40054 0 0 0 0 0 0

M00001442C:D07 16731 0 0 0 0 0 0

M00001445A:F05 13532 0 0 0 0 0 0

M00001446A:F05 7801 0 0 0 0 0 0

M00001447A:G03 10717 0 0 0 0 0 0

M00001448D:C09 8 1 6 6 1 14 1

M00001448D.H01 36313 0 3 0 0 3 0

M00001449AA12 5857 0 0 0 0 0 0

M00001449A:B 12 41633 0 0 0 0 0 0

M00001449A:D12 3681 0 0 0 0 0 0

M00001449A:G10 36535 0 0 0 0 0 0

M00001449C:D06 861 10 0 0 0 0 0 0

M00001450AA02 39304 0 0 0 0 0 0

M00001450A 11 32663 0 0 0 0 0 0

M00001450A:B12 82498 0 0 0 0 0 0

M00001450A:D08 27250 0 0 0 0 0 0

M00001452A.B04 84328 0 0 0 0 0 0

M00001452A:B12 86859 0 0 0 0 0 0

M00001452A:D08 1120 0 0 0 0 0 0

M00001452A:F05 85064 0 0 0 0 0 0

M00001452C:B06 16970 0 0 2 0 1 0

M00001453A:E11 16130 0 0 0 0 0 0

M00001453C:F06 16653 0 0 0 0 0 0

M00001454AA09 83103 0 0 0 0 0 0

M00001454B:C12 7005 0 0 0 0 0 0

M00001454D:G03 689 0 2 2 0 4 2

M00001455A:E09 13238 0 0 0 0 0 0

M00001455B:E12 13072 0 0 0 0 0 0

M00001455D:F09 9283 0 0 0 0 0 0

M00001455D:F09 9283 0 0 0 0 0 0

M00001460A:F06 2448 0 0 0 0 0 0

M00001460A:F12 39498 0 0 0 0 0 0 Table 6 All Differential Data for Libs 15-20

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl5 Libl6b Libl7 Libl8 Libl9 Lib20

M00001461A:D06 1531 0 0 0 0 0 0

M00001463C.B11 19 2 13 13 0 69 10

M00001465A.B 1 1 10145 0 0 0 0 0 0

M00001466A:E07 4275 0 0 0 0 0 0

M00001467A.B07 38759 0 0 0 0 0 0

M00001467A:D04 39508 0 0 0 0 0 0

M00001467A:D08 16283 0 0 0 0 0 0

M00001467A:D08 16283 0 0 0 0 0 0

M00001467A.E10 39442 0 0 0 0 0 0

M00001468A:F05 7589 0 0 0 0 0 0

M00001469A:C10 12081 0 0 0 0 0 0

M00001469A:H12 19105 0 0 0 0 0 0

M00001470A:B10 1037 0 0 0 0 0 0

M00001470A:C04 39425 0 0 0 0 0 0

M00001471A:B01 39478 0 0 0 0 0 0

M00001481D:A05 7985 0 0 0 0 0 0

M00001490B:C04 18699 0 0 0 0 0 0

M00001494D:F06 7206 0 0 0 0 0 0

M00001497A:G02 2623 0 0 0 0 0 0

M00001499B:A 1 1 10539 0 0 0 0 0 0

M00001500A:C05 5336 0 0 0 0 0 0

M00001500A:E1 1 2623 0 0 0 0 0 0

M00001500C:E04 9443 0 0 0 0 0 0

M00001501D:C02 9685 0 0 0 0 0 0

M00001504CA07 10185 0 0 0 0 0 0

M00001504C:H06 6974 0 0 0 0 0 0

M00001504D:G06 6420 0 0 0 0 0 0

M00001507A:H05 39168 0 0 0 0 0 0

M0000151 1A:H06 39412 0 0 0 0 0 0

M00001512AA09 39186 0 0 0 0 0 0

M00001512D:G09 3956 0 0 1 0 0 0

M00001513A:B06 4568 0 0 0 0 0 0

M00001513C:E08 14364 0 0 0 0 0 0

M00001514C.D1 1 40044 0 1 0 0 0 0

M00001517A:B07 4313 0 0 0 0 0 0

M00001518C:B11 8952 0 0 0 0 0 0

M00001528A:C04 7337 0 0 0 0 0 0

M00001528A:F09 18957 0 0 0 0 0 0

M00001528B.H04 8358 0 0 0 0 0 0

MOO0O1531A:DOl 38085 0 0 0 0 0 0

M00001532BA06 3990 1 1 0 0 0 0

M00001533A:C11 2428 0 0 1 0 0 0

M00001534A:C04 16921 0 0 0 0 0 0

M00001534A:D09 5097 0 0 0 0 0 0

M00001534A:F09 5321 0 1 0 0 2 0

M00001534C:A01 41 19 0 0 0 0 0 0

M00001535A:B01 7665 0 0 0 0 0 0

M00001535A:C06 20212 0 0 0 0 0 0

M00001535A:F10 39423 0 0 0 0 0 0

© © 0 © © 0 0 © tO CΛ © 0 © 0 © © © © — © © © © © © 0 © 0 © © 0 © © © 0 0 0 0 0 © © © © © 0 © © © co © Os en o ^σ3^' o

000 © © © 0 © O CΛ O © © © © © 0 © 0 © 0 — © © © o o © o o © — O O O O O O O o © © o © © © © o ©σ c" ©^B s

© © © © © o o o © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © o © © o o © © oσ ε" ©^B s

O

F 5^*

© © © 0 © © © © © C O © © © © © © © © © C © © 0 0 © © © 0 © © © © --J O © 0 © 0 © 0 © © 0 © © © © ^ s ___ ^B to

SB _ 3

© 0 © © 0 © © 0 © — © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © © o o © ©

Table 6 All Differential Data for Libs 15-20

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl5 Liblόb Libl7 Libl8 Libl9 Lib20

M00001560D.F10 6558 0 0 0 0 0 0

M00001561A:C05 39486 0 0 0 0 0 0

M00001563B:F06 102 22 38 65 7 43 10

M00001564A:B12 5053 0 0 1 0 0 0

M00001571C:H06 5749 0 0 0 0 0 0

M00001578B:E04 23001 0 0 0 0 0 0

M00001579D:C03 6539 0 0 0 0 0 0

M00001583DA10 6293 0 0 0 0 0 0

M00001586C:C05 4623 0 0 0 0 1 0

M00001587A:B1 1 39380 0 0 0 0 0 0

M00001594B:H04 260 0 0 0 0 1 0

M00001597C:H02 4837 0 0 0 0 0 0

M00001597D:C05 10470 0 0 0 0 0 0

M00001598A:G03 16999 1 1 1 0 0 0

M00001601A:D08 22794 0 0 0 0 0 0

M00001604A:B10 1399 0 0 0 0 0 0

M00001604A:F05 39391 0 0 0 0 0 0

M00001607A:E1 1 1 1465 0 0 0 0 0 0

M00001608A:B03 7802 0 0 0 0 0 0

M00001608B:E03 22155 0 0 0 0 0 0

M00001614C:F10 13157 0 0 0 0 0 0

M00001617C:E02 17004 0 0 0 0 1 0

M00001619C:F12 40314 0 0 0 0 0 0

M00001621C.C08 40044 0 1 0 0 0 0

M00001623D:F10 13913 0 0 0 0 0 0

M00001624A:B06 3277 0 0 0 0 0 0

M00001624C:F01 4309 0 0 0 0 0 0

M00001630B:H09 5214 1 0 0 1 1 0

M00001644C.B07 39171 0 0 0 0 0 0

M00001645A:C12 19267 0 0 0 0 1 0

M00001648CA01 4665 0 0 0 0 0 0

M00001657D:C03 23201 0 0 0 0 0 0

M00001657D:F08 76760 0 0 0 0 0 0

M00001662CA09 23218 0 0 0 0 0 0

M00001663A.E04 35702 0 0 0 0 0 0

M00001669B:F02 6468 0 0 0 0 0 0

M00001670C:H02 14367 0 0 0 0 0 0

M00001673C:H02 7015 0 0 0 0 0 0

M00001675A:C09 8773 0 0 0 0 0 0

M00001676B:F05 1 1460 0 0 0 0 0 0

M00001677C:E10 14627 0 1 0 0 0 0

M00001677DA07 7570 0 0 0 0 0 0

M00001678D:F12 4416 0 0 0 0 0 0

M00001679A:A06 6660 0 0 0 0 0 0

M00001679A:F10 26875 0 0 0 0 0 0

M00001679B:F01 6298 0 0 0 0 0 0

M00001679C:F01 78091 0 0 0 0 0 0

M00001679D:D03 10751 0 0 0 0 0 0

M00001679D:D03 10751 0 0 0 0 0 0 Table 6 All Differential Data for Libs 15-20

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl5 Liblόb Libl7 Libl8 Libl9 Lib20

M00001680D:F08 10539 0 0 0 0 0 0

M00001682C:B 12 17055 0 0 0 0 0 0

M00001686A.E06 4622 0 0 0 0 0 0

M00001688C:F09 5382 0 0 0 0 0 0

M00001693C:G01 4393 0 0 0 0 0 0

M00001716D:H05 67252 0 0 0 0 0 0

M00003741D:C09 40108 0 0 0 0 0 0

M00003747D:C05 1 1476 0 0 0 0 0 0

M00003759B:B09 697 0 0 0 0 1 0

M00003762C:B08 17076 0 0 0 0 0 0

M00003763A:F06 3108 0 0 0 0 0 0

M00003774C:A03 67907 0 0 0 0 0 0

M00003796C:D05 5619 0 0 0 0 0 0

M00003826B:A06 1 1350 0 0 0 0 0 0

M00003833A:E05 21877 0 0 0 0 0 0

M00003837D:A01 7899 0 0 0 0 0 0

M00003839A:D08 7798 0 0 0 0 0 0

M00003844C:B 1 1 6539 0 0 0 0 0 0

M00003846B:D06 6874 0 0 1 0 0 0

M00003851B:D10 13595 0 0 0 0 0 0

M00003853A:D04 5619 0 0 0 0 0 0

M00003853A:F12 10515 0 0 0 0 0 0

M00003856B:C02 4622 0 0 0 0 0 0

M00003857A:G10 3389 0 0 0 0 0 0

M00003857A:H03 4718 0 0 0 0 0 0

M00003871C:E02 4573 0 0 0 0 0 0

M00003875B:F04 12977 0 0 0 0 0 0

M00003875B:F04 12977 0 0 0 0 0 0

M00003875C:G07 8479 0 0 0 0 0 1

M00003876D:E12 7798 0 0 0 0 0 0

M00003879B:C11 5345 0 0 0 2 0 1

M00003879B:D10 31587 0 0 0 0 0 0

M00003879DA02 14507 0 0 0 0 0 0

M00003885C:A02 13576 0 0 0 0 0 0

M00003885C:A02 13576 0 0 0 0 0 0

M00003906C:E10 9285 0 0 0 0 0 0

M00003907DA09 39809 0 0 0 0 0 0

M00003907D:H04 16317 0 0 0 0 0 0

M00003909D:C03 8672 . 0 0 0 0 0 0

M00003912B:D01 12532 0 0 0 0 0 0

M00003914C:F05 3900 0 0 0 0 1 0

M00003922A:E06 23255 0 0 0 0 0 0

M00003958A:H02 18957 0 0 0 0 0 0

M00003958A:H02 18957 0 0 0 0 0 0

M00003958C:G10 40455 0 0 0 0 0 0

M00003958C:G10 40455 0 0 0 0 0 0

M00003968B:F06 24488 0 0 0 0 0 0

M00003970C:B09 40122 0 0 0 0 0 0

M00003974D:E07 23210 0 0 0 0 0 0 Table 6 All Differential Data for Libs 15-20

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl5 Liblόb Libl7 Libl8 Libl9 Lib20

M00003974D:H02 23358 0 0 0 0 0 0

M00003975A:G1 1 12439 0 0 0 0 0 0

M00003978B:G05 5693 0 0 0 0 0 0

M00003981A.E10 3430 0 0 0 0 1 0

M00003982C:C02 2433 0 0 0 0 0 0

M00003983A:A05 9105 0 0 0 0 0 0

M00004028DA06 6124 0 0 0 0 0 0

M00004028D:C05 40073 0 0 0 0 0 0

M00004031A:A12 9061 0 0 0 0 0 0

M00004031A 12 9061 0 0 0 0 0 0

M00004035C:A07 37285 0 0 0 0 0 0

M00004035D:B06 17036 0 0 0 0 0 0

M00004059A:D06 5417 0 0 0 0 0 0

M00004068BA01 3706 0 0 0 0 0 0

M00004072B:B05 17036 0 0 0 0 0 0

M00004081C:D10 15069 0 0 0 0 0 0

M00004081C:D12 14391 0 0 0 0 0 0

M00004086D:G06 9285 0 0 0 0 0 0

M00004087DA01 6880 0 0 0 0 0 0

M00004093D:B12 5325 1 1 0 1 0 1

M00004093D:B12 5325 1 1 0 1 0 1

M00004105CA04 7221 0 0 0 0 0 0

M00004108A:E06 4937 0 0 0 0 0 0

M0000411 1DA08 6874 0 0 1 0 0 0

M00004114C:F1 1 13183 0 0 0 0 0 0

M00004138B:H02 13272 0 0 0 0 0 0

M00004146C:C11 5257 0 1 0 0 0 0

M00004151D:B08 16977 0 0 0 0 0 0

M00004157CA09 6455 0 0 0 0 0 0

M00004169C.C12 5319 0 0 0 0 0 0

M00004171D:B03 4908 0 0 0 0 0 0

M00004172C:D08 1 1494 0 0 0 0 0 0

M00004183C:D07 16392 0 0 0 0 0 0

M00004185C:C03 11443 0 0 0 0 0 0

M00004197D:H01 8210 0 0 0 0 0 0

M00004203B:C12 1431 1 0 0 0 0 0 0

M00004212B:C07 2379 0 0 0 0 0 0

M00004214C:H05 11451 0 0 0 0 0 0

M00004223A.G10 16918 0 0 0 0 0 0

M00004223B:D09 7899 0 0 0 0 0 0

M00004223D:E04 12971 0 0 0 0 0 0

M00004229B:F08 6455 0 0 0 0 0 0

M00004230B:C07 7212 0 0 0 0 0 0

M00004269D:D06 4905 0 0 0 0 0 0

M00004275C:C11 16914 0 0 0 0 0 0

M00004283BA04 14286 0 0 0 0 0 0

M00004285B:E08 56020 0 0 0 0 0 0

M00004295D:F12 16921 0 0 0 0 0 0

M00004296C:H07 13046 0 0 0 0 0 0 rable 6 All Differential Data for Libs 15-20

Clone Name Cluster Clones in Clones in Clones in Clones in Clones in Clones in

ID Libl5 Liblόb Libl 7 Libl8 Libl9 Lib20

M00004307CA06 9457 0 0 0 0 0 0

M00004312A:G03 26295 0 0 0 0 0 0

M00004318C.D10 21847 0 0 0 0 0 0

M00004372AA03 2030 0 0 0 0 0 0

M00004377C:F05 2102 0 0 0 0 0 0

Table 7 All Differential Data for Libs 12-14

Clone Name Cluster ID Clones in Clones in Clones in

Libl2 Libl3 Libl4

M00001340B:A06 17062 0 0 0

M00001340D:F10 1 1589 0 0 0

M00001341A.E12 4443 4 2 0

M00001342B:E06 39805 0 0 0

M00001343C:F10 2790 0 0 0

M00001343D:H07 23255 0 0 0

M00001345A:E01 6420 0 0 0

M00001346A:F09 5007 0 0 0

M00001346D:E03 6806 0 1 1

M00001346D:G06 5779 0 0 0

M00001346D:G06 5779 0 0 0

M00001347A:B10 13576 0 0 0

M00001348B:B04 16927 0 0 0

M00001348B:G06 16985 0 0 0

M00001349B:B08 3584 0 0 0

M00001350A:H01 7187 0 0 0

M00001351B:A08 3162 0 0 1

M00001351B.A08 3162 0 0 1

M00001352A:E02 16245 0 0 0

M00001353A:G12 8078 0 0 0

M00001353D:D10 14929 0 1 0

M00001355B:G10 14391 0 0 0

M00001357D:D1 1 4059 0 0 0

M00001361A:A05 4141 1 2 1

M00001361D:F08 2379 0 0 0

M00001362B:D10 5622 0 2 1

M00001362C:H11 945 0 0 0

M00001365C.C10 40132 0 0 0

M00001370A:C09 6867 0 0 0

M00001371C:E09 7172 0 0 1

M00001376B:G06 17732 2 0 0

M00001378B:B02 39833 0 0 0

M00001379A:A05 1334 0 0 0

M00001380D:B09 39886 0 0 0

M00001382C:A02 22979 1 0 0

M00001383A:C03 39648 0 0 0

M00001383A:C03 39648 0 0 0

M00001386C:B12 5178 0 0 0

M00001387A:C05 2464 0 0 0

M00001387B:G03 7587 0 0 0

M00001388D:G05 5832 0 0 0

M00001389A:C08 16269 2 0 0

M00001394A:F01 6583 0 0 0

M00001395A:C03 4016 0 0 0

M00001396A:C03 4009 2 0 0

M00001402A:E08 39563 0 0 0

M00001407B:D11 5556 0 0 0 Table 7 All Differential Data for Libs 12-14

Clone Name Cluster ID Clones in Clones in Clones in

Libl2 Libl3 Libl4

M00001409C:D12 9577 0 0 0

M00001410A.D07 7005 0 0 0

M00001412B.B10 8551 0 0 0

M00001415A.H06 13538 0 0 0

M00001416A.H01 7674 0 0 0

M00001416B:H11 8847 1 0 0

M00001417A:E02 36393 0 0 0

M00001418B:F03 9952 0 0 0

M00001418D:B06 8526 0 0 0

M00001421C:F01 9577 0 0 0

M00001423B:E07 15066 0 0 0

M00001424B:G09 10470 0 0 0

M00001425B:H08 22195 0 0 0

M00001426D:C08 4261 0 0 0

M00001428A:H10 84182 0 0 0

M00001429A:H04 2797 0 0 0

M00001429B:A11 4635 0 0 0

M00001429D:D07 40392 0 0 0

M00001439C:F08 40054 0 0 0

M00001442C:D07 16731 0 0 0

M00001445A:F05 13532 0 0 0

M00001446A:F05 7801 0 1 0

M00001447A:G03 10717 0 0 0

M00001448D:C09 8 7 6 9

M00001448D:H01 36313 1 0 0

M00001449A:A12 5857 0 0 0

M00001449A:B12 41633 0 0 0

M00001449A:D12 3681 1 0 0

M00001449A:G10 36535 0 0 0

M00001449C:D06 86110 0 0 0

M00001450A:A02 39304 0 1 0

M00001450A:A11 32663 0 0 0

M00001450A:B12 82498 0 0 0

M00001450A:D08 27250 0 0 0

M00001452A:B04 84328 0 0 0

M00001452A:B12 86859 0 0 0

M00001452A:D08 1120 0 0 0

M00001452A:F05 85064 0 0 0

M00001452C:B06 16970 1 0 0

M00001453A:E11 16130 0 0 0

M00001453C:F06 16653 0 0 0

M00001454A:A09 83103 0 0 0

M00001454B:C12 7005 0 0 0

M00001454D:G03 689 0 0 1

M00001455A:E09 13238 0 0 0

M00001455B:E12 13072 0 0 0

M00001455D:F09 9283 0 0 0

M00001455D:F09 9283 0 0 0 Table 7 All Differential Data for Libs 12-14

Clone Name Cluster ID Clones in Clones in Clones in

Libl2 Libl3 Libl4

M00001460A:F06 2448 0 0 0

M00001460A:F12 39498 0 0 0

M00001461A:D06 1531 0 0 1

M00001463C:B1 1 19 17 32 31

M00001465A:B11 10145 0 0 0

M00001466A:E07 4275 0 0 0

M00001467A:B07 38759 0 0 0

M00001467A:D04 39508 0 0 0

M00001467A:D08 16283 0 0 0

M00001467A:D08 16283 0 0 0

M00001467A:E10 39442 0 0 0

M00001468A:F05 7589 0 0 0

M00001469A:C10 12081 0 0 0

M00001469A:H12 19105 0 0 0

M00001470A:B10 1037 0 0 0

M00001470A:C04 39425 0 0 0

M00001471A:B01 39478 0 0 0

M00001481D:A05 7985 0 0 0

M00001490B:C04 18699 0 0 0

M00001494D:F06 7206 0 0 0

M00001497A:G02 2623 1 0 0

M00001499B:A1 1 10539 0 1 0

M00001500A:C05 5336 0 0 0

M00001500A:E11 2623 1 0 0

M00001500C:E04 9443 0 0 0

M00001501D:C02 9685 0 0 0

M00001504C:A07 10185 0 0 0

M00001504C:H06 6974 0 0 0

M00001504D:G06 6420 0 0 0

M00001507A:H05 39168 0 0 0

M00001511A:H06 39412 0 0 0

M00001512A:A09 39186 0 0 0

M00001512D:G09 3956 0 0 0

M00001513A.B06 4568 0 0 0

M00001513C:E08 14364 0 0 0

M00001514C:D11 40044 0 0 0

M00001517A:B07 4313 0 0 0

M00001518C:B11 8952 0 0 0

M00001528A:C04 7337 1 2 2

M00001528A:F09 18957 0 0 0

M00001528B:H04 8358 0 0 0

M00001531A:D01 38085 0 0 0

M00001532B:A06 3990 0 0 0

M00001533A.C11 2428 0 0 0

M00001534A:C04 16921 0 0 0

M00001534A:D09 5097 0 0 0

M00001534A:F09 5321 4 7 6

M00001534C:A01 4119 0 0 0 Table 7 All Differential Data for Libs 12-14

Clone Name Cluster ID Clones in Clones in Clones in

Libl2 Libl3 Libl4

MO0001535A:B01 7665 0 2 4

M00001535A:C06 20212 0 0 0

M00001535A:F10 39423 0 0 0

M00001536A:B07 2696 0 0 0

M00001536A:C08 39392 0 0 0

M00001537A:F12 39420 0 0 0

M00001537B:G07 3389 0 0 0

M00001540A:D06 8286 0 0 0

M00001541A.D02 3765 0 0 0

M00001541A:F07 22085 0 0 0

M00001541A:H03 39174 0 0 0

M00001542AA09 22113 0 0 0

M00001542A:E06 39453 0 0 0

M00001544A:E03 12170 0 0 0

M00001544A:G02 19829 0 0 0

M00001544B:B07 6974 0 0 0

M00001545A:C03 19255 0 0 0

M00001545A:D08 13864 0 0 0

M00001546A.G11 1267 0 0 0

M00001548A:E10 5892 0 1 0

M00001548A:H09 1058 1 3 0

M00001549A:B02 4015 0 1 0

M00001549A:D08 10944 1 0 0

M00001549B:F06 4193 0 0 0

M00001549C:E06 16347 0 0 0

M00001550A:A03 7239 0 1 0

M00001550A:G01 5175 1 0 0

M00001551A:B10 6268 0 0 1

M00001551A:F05 39180 0 0 0

M00001551A:G06 22390 0 0 1

M00001551C:G09 3266 0 0 0

M00001552A:B12 307 6 11 4

M00001552A:D1 1 39458 0 0 0

M00001552B:D04 5708 0 0 0

M00001553A:H06 8298 0 0 0

M00001553B:F12 4573 0 0 0

M00001553D:D10 22814 0 0 0

M00001555A:B02 39539 0 0 0

M00001555A:C01 39195 0 0 0

M00001555D:G10 4561 0 0 0

M00001556A:C09 9244 0 1 0

M00001556A:F11 1577 0 0 2

M00001556A:H01 15855 1 1 0

M00001556B.C08 4386 3 0 1

M00001556B:G02 11294 0 0 0

M00001557A:D02 7065 0 0 0

M00001557A:D02 7065 0 0 0

M00001557A:F01 9635 0 0 0 Table 7 All Differential Data for Libs 12-14

Clone Name Cluster ID Clones in Clones in Clones in

Libl2 Libl3 Libl4

M00001557A:F03 39490 0 0 0

M00001557B.H10 5192 0 0 0

M00001557D:D09 8761 0 0 0

M00001558B.H1 1 7514 0 0 0

M00001560D:F10 6558 0 0 0

M00001561A:C05 39486 0 0 0

M00001563B:F06 102 2 1 2

M00001564A:B12 5053 0 0 0

M00001571C:H06 5749 0 0 0

M00001578B:E04 23001 0 0 0

M00001579D:C03 6539 0 0 0

M00001583D:A10 6293 0 0 0

M00001586C:C05 4623 0 0 0

M00001587A:B1 1 39380 0 0 0

M00001594B:H04 260 1 0 0

M00001597C:H02 4837 1 0 0

M00001597D:C05 10470 0 0 0

M00001598A:G03 16999 4 2 6

M00001601A.D08 22794 0 0 0

M00001604A:B10 1399 6 3 3

M00001604A:F05 39391 0 0 0

M00001607A:E11 11465 0 0 0

M00001608A:B03 7802 0 0 0

M00001608B:E03 22155 0 0 0

M00001614C:F10 13157 0 0 0

M00001617C:E02 17004 0 0 0

M00001619C:F12 40314 0 0 0

M00001621C:C08 40044 0 0 0

M00001623D.F10 13913 0 0 0

M00001624A:B06 3277 0 0 0

M00001624C:F01 4309 0 0 0

M00001630B:H09 5214 0 1 2

M00001644C:B07 39171 0 0 0

M00001645A:C12 19267 0 0 0

M00001648C:A01 4665 0 0 0

M00001657D:C03 23201 0 0 0

M00001657D:F08 76760 0 0 0

M00001662C:A09 23218 0 0 0

M00001663A:E04 35702 0 0 0

M00001669B:F02 6468 0 0 0

M00001670C:H02 14367 0 0 0

M00001673C:H02 7015 0 0 0

M00001675A:C09 8773 0 0 0

M00001676B:F05 11460 2 0 0

M00001677C:E10 14627 0 0 0

M00001677D:A07 7570 0 0 0

M00001678D:F12 4416 1 2 0

M00001679A:A06 6660 0 0 0

— t oo CO σs so CO UJ 4*1 ►— CΛ sl t 4* *1 UJ 4*. CΛ σs σs sl sl t σs O —

00 CO so t 5 o cc Λ — s] OS _^-. CΛ CΛ CΛ CΛ CO sl to sl UJ OS σs 00 CΛ ^" 00 ^•-J sl o oo 4n so o so C sl 00 sl CO SO SO ∞ O P so 4*.

CΛ CΛ © sl © P sl sl sl

SO σs σs sl sl CΛ O0 CO 00 so so 4*ι SO 00 SO sl © SO sl 00 σs OS O o

P P P P P P P P P P P P 4» P — P P P P P P P P P P P P P P P © © © © © ©

O O — © © © © © © © P © 00 © © © 0 © © © © © — © © © © o o © © © © © ©

© © © © © © O O P P P P UJ © © © © © © © © — © © © © © © © © © © © © © ©

Table 7 All Differential Data for Libs 12-14

Clone Name Cluster ID Clones in Clones in Clones in

Libl2 Libl3 Libl4

M00003958A:H02 18957 0 0 0

M00003958C.G10 40455 0 0 0

M00003958C:G10 40455 0 0 0

M00003968B:F06 24488 0 0 0

M00003970C:B09 40122 0 0 0

M00003974D:E07 23210 0 0 0

M00003974D:H02 23358 0 0 0

M00003975A:G1 1 12439 0 0 0

M00003978B:G05 5693 0 0 0

M00003981A:E10 3430 0 0 0

M00003982C:C02 2433 2 4 0

M00003983A:A05 9105 0 0 0

M00004028D:A06 6124 0 0 0

M00004028D:C05 40073 0 1 0

M00004031A:A12 9061 0 0 0

M00004031A:A12 9061 0 0 0

M00004035C:A07 37285 0 0 0

M00004035D:B06 17036 0 0 0

M00004059A:D06 5417 0 0 0

M00004068B:A01 3706 0 0 0

M00004072B:B05 17036 0 0 0

M00004081C:D10 15069 0 0 0

M00004081C:D12 14391 0 0 0

M00004086D:G06 9285 0 0 0

M00004087D:A01 6880 0 0 0

M00004093D:B12 5325 0 0 0

M00004093D:B12 5325 0 0 0

M00004105C:A04 7221 0 0 0

M00004108A:E06 4937 0 0 0

M00004111D:A08 6874 0 0 0

M00004114C:F11 13183 0 0 0

M00004138B:H02 13272 0 0 0

M00004146C:C1 1 5257 0 0 1

M00004151D:B08 16977 0 0 0

M00004157C:A09 6455 0 0 0

M00004169C:C12 5319 0 0 0

M00004171D:B03 4908 0 0 0

M00004172C:D08 11494 0 0 0

M00004183C:D07 16392 0 0 0

M00004185C:C03 11443 2 0 0

M00004197D:H01 8210 0 0 0

M00004203B:C12 14311 0 0 0

M00004212B:C07 2379 0 0 0

M00004214C:H05 1 1451 0 0 0

M00004223A:G10 16918 0 0 0

M00004223B:D09 7899 0 0 0

M00004223D:E04 12971 0 0 0

M00004229B:F08 6455 0 0 0 Table 7 All Differential Data for Libs 12-14

Clone Name Cluster ID Clones in Clones in Clones in

Libl2 Libl3 Libl4

M00004230B:C07 7212 0 0 1

M00004269D:D06 4905 0 0 0

M00004275C:C1 1 16914 0 0 0

M00004283BA04 14286 0 0 0

M00004285B:E08 56020 0 0 0

M00004295D:F12 16921 0 0 0

M00004296C:H07 13046 0 0 0

M00004307C:A06 9457 1 0 0

M00004312A:G03 26295 0 0 0

M00004318C:D10 21847 0 0 0

M00004372A:A03 2030 0 0 0

M00004377C:F05 2102 0 0 0

Claims

We Claim:

1. A library of polynucleotides, the library comprising the sequence information of at least one of SEQ ID NOS: 1-844.

2. The library of claim 1, wherein the library is provided on a nucleic acid array.

3. The library of claim 1, wherein the library is provided in a computer-readable format.

4. The library of claim 1, wherein the library comprises a differentially expressed polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOS:9, 39, 42, 52, 62, 74, 119, 172, 317, and 379.

5. The library of claim 1, wherein the library comprises a polynucleotide differentially expressed in a human breast cancer cell, where the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS: 4, 9, 39, 42, 52, 62, 65, 66, 68, 74, 81, 114, 123, 144, 130, 157, 162, 172, 178, 183, 202, 214, 219, 223, 258, 298, 317, 338, 379, 384, 386, and 388.

6. The library of claim 1, wherein the library comprises a polynucleotide differentially expressed in a human colon cancer cell, where the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS: 1, 39, 52, 97, 119, 134, 172, 176, 241, 288, 317, 357, 362, and 374.

7. The library of claim 1, wherein the library comprises a polynucleotide differentially expressed in a human lung cancer cell, where the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS: 9, 34, 42, 62, 74, 106, 119, 135, 154, 160, 260, 308, 323, 349, 361, 369, 371, 379, 395, 381, and 400.

8. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to an identifying sequence of SEQ ID NOS: 1-844 or a degenerate variant thereof.

9. An isolated polynucleotide according to claim 8, wherein the polynucleotide comprises a seqeuence encoding a polypeptide of a protein family selected from the group consisting of: 4 transmembrane segments integral membrane proteins, 7 transmembrane receptors, ATPases associated with various cellular activities (AAA), eukaryotic aspartyl proteases, GATA family of transcription factors, G-protein alpha subunit, phorbol esters/diacylglycerol binding proteins, protein kinase, protein phosphatase 2C, protein tyrosine phosphatase, trypsin, wnt family of developmental signaling proteins, and WW/rsp5/WWP domain containing proteins.

10. The polynucleotide of claim 9, wherein the polynucleotide comprises a sequence of one of SEQ ID NOS: 24, 41, 101, 157, 291, 305, 315, 341, 63, 1 16, 134, 136, 151, 384, 404, 308, 213, 367, 188, 251, 202, 315, 367, 397, 256, 382, 169, 23, 291, 324, 330, 341, 353, 188, 379 , and 395.

11. The polynucleotide of claim 8, wherein the polynucleotide comprises a seqeuence encoding a polypeptide having a functional domain selected from the group consisting of: Ank repeat, basic region plus leucine zipper transcription factors, bromodomain, EF-hand, SH3 domain, WD domain/G-beta repeats, zinc finger (C2H2 type), zinc finger (CCHC class), and zinc-binding metalloprotease domain.

12. The polynucleotide of claim 11, wherein the polynucleotide comprises a sequence of one of SEQ ID NOS: 116, 251, 374, 97, 136, 242, 379, 306, 386, 18, 335, 61, 306, 386, 322, 306, and 395.

13. A recombinant host cell containing the polynucleotide of claim 8.

14. An isolated polypeptide encoded by the polynucleotide of claim 8.

15. An antibody that specifically binds a polypeptide of claim 14.

16. A vector comprising the polynucleotide of claim 8.

17. A polynucleotide comprising the nucleotide sequence of an insert contained in a clone deposited as ATCC accession number xx, xx, xx, xx, xx, xx, xx, xx, or xx.

18. A method of detecting differentially expressed genes correlated with a cancerous state of a mammalian cell, the method comprising the step of: detecting at least one differentially expressed gene product in a test sample derived from a cell suspected of being cancerous, where the gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS:4, 9, 39, 42, 52, 62, 65, 66, 68, 74, 81, 114, 123, 144, 130, 157, 162, 172, 178, 183, 202, 214, 219, 223, 258, 298, 317, 338, 379, 384, 386, 388, 1, 39, 52, 97, 119, 134, 172, 176, 241, 288, 317, 357, 362, 374, 9, 34, 42, 62, 74, 106, 119, 135, 154, 160, 260, 308, 323, 349, 361, 369, 371, 379, 395, 381, and 400; wherein detection of the differentially expressed gene product is correlated with a cancerous state of the cell from which the test sample was derived.

19. The method of claim 18, wherein said detecting step is by hybridization of the test sample to a reference array, wherein the reference array comprises an identifying sequence of at least one of SEQ ID NOS: 1-844.

20. The method of claim 18, wherein the cell is a breast tissue derived cell, and the differentially expressed gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS: 4, 9, 39, 42, 52, 62, 65, 66, 68, 74, 81, 114, 123, 144, 130, 157, 162, 172, 178, 183, 202, 214, 219, 223, 258, 298, 317, 338, 379, 384, 386, and 388.

21. The method of claim 18, wherein the cell is a colon tissue derived cell, and the differentially expressed gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS: 1, 39, 52, 97, 119, 134, 172, 176, 241, 288, 317, 357, 362, and 374.

22. The method of claim 18, wherein the cell is a lung tissue derived cell, and the differentially expressed gene product is encoded by a gene corresponding to a sequence of at least one of SEQ ID NOS: 9, 34, 42, 62, 74, 106, 119, 135, 154, 160, 260, 308, 323, 349, 361, 369, 371, 379, 395, 381, and 400.