JP2003518920A - New human genes and gene expression products - Google Patents

Abstract

  (57) [Summary] The present invention provides a novel polynucleotide. The present invention relates to novel members of the protein family and polynucleotides that are differentially expressed in cancer cells as compared to normal cells, and differentially expressed in metastatic cancer cells as compared to normal or non-metastatic cancer cells. There is further provided a polynucleotide expressed in The present invention also provides a method of detecting a differentially expressed gene that correlates with a cancerous state of a mammalian cell, the method comprising at least one test sample from a cell suspected of being cancerous. Detecting the two differentially expressed gene products.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to novel polynucleotides of human origin and their encoded gene products.

BACKGROUND OF THE INVENTION The identification of novel polynucleotides, particularly those that encode expressed gene products, is an advance in drug discovery, diagnostic techniques, and understanding of the progression and nature of complex diseases such as cancer. Is important in. Identification of genes expressed in different cell types isolated from different sources such as disease state or stage, stage of development, exposure to various environmental factors, tissue of origin, species from which the tissue is isolated, etc. , Is the key to identifying the genetic factors responsible for the phenotype associated with these various differences.

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

SUMMARY OF THE INVENTION The present invention relates to novel human polynucleotides and their variants, polypeptides encoding them and their variants, genes corresponding to these polynucleotides, and these genes. Related to proteins expressed by. The present invention also relates to diagnostic and therapeutic agents (including probes, antisense nucleotides, and antibodies) containing such novel human polynucleotides, their corresponding genes, or gene products. The polynucleotide of the present invention is
It corresponds to a polynucleotide containing sequence information of at least one of SEQ ID NOs: 1-3351.

Various aspects and embodiments of the invention will be readily apparent to those of skill in the art upon reading the description provided herein.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides polynucleotides containing the disclosed nucleotide sequences, full-length cDNA sequences, mRNA genomic sequences, and genes corresponding to these sequences and degenerate variants thereof. , And polypeptides and polypeptide variants encoded by the polynucleotides of the invention.

Polypeptide variants differ from wild-type in that they have one or more amino acid substitutions that either enhance, add, or decrease the biological activity of the wild-type protein. .

Six of the polypeptides disclosed herein encode novel members of the MKK kinase family; this coding region is found within the nucleotide region in brackets: SEQ ID NO: 29 ( Nucleotides 295-421); SEQ ID NO: 31 (298-397); SEQ ID NO: 196 (37-322); SEQ ID NO: 3175
(Nucleotides 14-164); SEQ ID NO: 3190 (229-390); and SEQ ID NO: 3281 (15-182). Twenty-four of the polypeptides encode novel members of the family of transcription factor proteins with basic regions and leucine zippers: SEQ ID NO: 410 (42-191); SEQ ID NO: 552 (11).
6-288); SEQ ID NO: 768 (116-288); SEQ ID NO: 822 (108-
262); SEQ ID NO: 836 (158-353); SEQ ID NO: 1288 (73-23)
4); SEQ ID NO: 1365 (69-257); SEQ ID NO: 1540 (289-471)
); SEQ ID NO: 1549 (200-391); SEQ ID NO: 1556 (163-354)
); SEQ ID NO: 1557 (207-398); SEQ ID NO: 1563 (107-298)
); SEQ ID NO: 1622 (180-365); SEQ ID NO: 1630 (100-291)
); SEQ ID NO: 1704 (184-372); SEQ ID NO: 1808 (36-161)
SEQ ID NO: 1454 (49-209); SEQ ID NO: 2363 (48-211); SEQ ID NO: 2424 (43-194); SEQ ID NO: 3147 (190-369); SEQ ID NO: 3152 (129-320); SEQ ID NO: 3158 ( 167-334); and SEQ ID NO: 3208 (34-256).

SEQ ID NO: 186 (175-395); 2591 (60-165); 3307 (
43-321); and 3339 (94-342) encode a polypeptide having an SH2 domain, and SEQ ID NOs: 234 (23-121), 1832 ().
18-173), and 1835 (57-206) encode polypeptides having the SH3 domain. Nine polypeptides encode a novel member of a family of proteins with an Ank repeat region: SEQ ID NO: 187 (358).
-432); SEQ ID NO: 1268 (238-315); SEQ ID NO: 1804 (301
-378); SEQ ID NO: 1819 (278-355); SEQ ID NO: 1839 (224)
-307); SEQ ID NO: 1830 (184-267); SEQ ID NO: 2562 (18-
101); SEQ ID NO: 3015 (131-214); and SEQ ID NO: 3267 (9
7-180).

The following eleven polynucleotides encode a polypeptide having C2H2-type zinc fingers: SEQ ID NOs: 308 (110-172); 807 (339).
-392); 1324 (294-356); 1503 (154-216); 15
27 (156-212); 1674 (196-258); 1779 (64-12).
6); 1801 (295-351); 3081 (190-252); 3193 (
293-355); and 3306 (161-223). Eight polynucleotides encode a polypeptide of the ATPase family: SEQ ID NO: 431
(71-428); 639 (157-561); 2135 (2-401); 26.
84 (9-461); 2859 (100-320); 3178 (45-386).
3197 (281-343) and 3266 (8-139). A polypeptide having a type III fibronectin domain has the sequence of SEQ ID NO: 746 (209-427).
) And 1192 (186-416). The polypeptide having the EF hand domain is SEQ ID NO: 820 (341-406); 1755 (2
81-367) and 3285 (16-102). The six polypeptides of the protein kinase family are SEQ ID NO: 1157 (41-4
44); 1478 (54-437), 1496 (241-520); 2286 (
12-182); 2969 (5-387); and 3190 (118-390).
Coded by

The LIM domain-containing polypeptide is SEQ ID NO: 1269 (79-240); 1
309 (248-404); 1360 (222-377); and 1386 (2.
43-398). Two polypeptides of the family with the C2 domain (protein kinase C-like) are SEQ ID NO: 1325 (1-234
) And 2282 (183-353). WD domain, G
The polypeptide having the -β repeat motif is SEQ ID NO: 1336 (66-164).
1380 (42-140); 1711 (263-361); 1762 (236.
-334); 1909 (160-258); 2218 (127-225); 30.
47 (191-292); 3108 (275-367) and 3292 (208).
-300).

SEQ ID NO: 1410 (222-350) encodes a member of the trypsin family. SEQ ID NOs: 1417 (8-354); 2281 (20-387) and 2310 (20-371) encode members of the protein tyrosine phosphatase family. SEQ ID NOs: 1464 (4-180) and 1514 (2
-252) encodes a member of a family with an RNA recognition motif (also known as RRM, RBD, or RNP domain). SEQ ID NO: 14
96 (241-520) and 3297 (7-153) encode helicases with a conserved C-terminal domain. SEQ ID NO: 1538 (9-635) is
Encodes a member of the wnt family of developmental signaling proteins.

The three polynucleotides encode a polypeptide having a homeobox domain: SEQ ID NOs: 1676 (9-86); 1820 (123-299); and 1821 (127-303). The novel thioredoxin is SEQ ID NO: 1677.
(316-369). Two novel members of the ras family are SEQ ID NOs: 1688 (109-410) and 3258 (138-39).
4) coded by. A novel polypeptide having a phosphatidylinositol-specific phospholipase CY domain is SEQ ID NO: 1707 (92-43).
9) coded by. The novel serine carboxypeptidase is encoded by SEQ ID NO: 1744 (238-433). N in Ets domain
A novel polypeptide having terminal homology is SEQ ID NO: 1811 (184-315).
) Coded by. A novel polypeptide having a bromo domain is encoded by SEQ ID NO: 1814 (127-294). A novel polypeptide having a double-stranded RNA binding motif is encoded by SEQ ID NO: 1818 (9-146). A novel polypeptide having the G protein α subunit is encoded by SEQ ID NO: 1846 (12-398).

SEQ ID NOs: 1911 (35-151) and 1980 (60-197) are C3
It encodes a polypeptide having an HC4 type zinc finger domain (RING finger). SEQ ID NO: 2065 (253-306) encodes a polypeptide having a CCHC zinc finger domain. SEQ ID NO: 2216 (90-
179) encodes a polypeptide having a WW / rsp5 / WWP domain. SEQ ID NO: 2428 (25-350) encodes a polypeptide member of the bispecific phosphatase family having a catalytic domain.

SEQ ID NOs: 2577 (0-311); 3183 (14-215); and 319.
5 (0-215) encodes a member of the four-transmembrane segment integral membrane protein family. SEQ ID NOs: 2826 (116-400) and 2871 (1
98-392) encode polypeptides of the DEAD and DEAH box helicase families. SEQ ID NO: 2944 (18-281) encodes a polypeptide having a calpain large subunit (domain III).

SEQ ID NO: 3274 (11-187) encodes a eukaryotic transcription factor having a forkhead domain. SEQ ID NO: 3345 (65-271) encodes a polypeptide having a PDZ domain, and SEQ ID NO: 3351 (124-27).
0) encodes a polypeptide in the family of phorbol ester / glycerol binding proteins.

Polynucleotide compositions encompassed by the present invention, methods for obtaining cDNA or genomic DNA encoding full-length gene products, expression of these polynucleotides and genes, identification of structural motifs of polynucleotides and genes,
Identification of the function of the gene product encoded by the gene corresponding to the polynucleotide of the invention, use of the provided polynucleotide as a probe and in mapping and in tissue profiling, the corresponding polypeptide for raising antibodies And the use of other gene products, and the use of polynucleotides and their encoded gene products for therapeutic and diagnostic purposes are described below.

(Polynucleotide Composition) The scope of the present invention relating to a polynucleotide composition includes SEQ ID NOs: 1 to 335.
A polynucleotide having the sequence set forth in any one of 1; the biological material or other biological material described herein by hybridization under stringent conditions, particularly conditions of high stringency Polynucleotides obtained from biological sources (especially human sources); Genes corresponding to the provided polynucleotides; Provided polynucleotides and variants of their corresponding genes, especially organisms of the encoded gene products Biological activity (eg, biological activity attributable to a gene product corresponding to a provided polynucleotide as a result of assignment of the gene product to a protein family and / or identification of functional domains present in the gene product). Examples of the variants to be retained include, but are not necessarily limited to. Other nucleic acid compositions contemplated by and within the scope of the present invention will be readily apparent to those of ordinary skill in the art given the disclosure herein. As used herein with respect to the nucleic acids of the composition, “polynucleotide” and “nucleic acid” are not intended to be limited with respect to the length or structure of the nucleic acids, unless otherwise indicated.

The invention features polynucleotides that are expressed in human tissue, specifically human colon, breast, and / or lung tissue. The novel nucleic acid composition of the present invention comprises the sequence shown in any one of SEQ ID NOs: 1 to 3531 or its identified sequence. An "identifying sequence" is a contiguous sequence of residues of at least about 10 nt to about 20 nt long, usually at least about 50 nt to about 100 nt long that uniquely identifies a polynucleotide sequence, eg, any sequence above about 20 nt. It exhibits less than 90% sequence identity, usually about 80% to less than about 85%, to contiguous nucleotide sequences. Accordingly, the novel nucleic acid compositions of the present invention include a full-length cDNA or mR containing an identifying sequence of contiguous nucleotides from any one of SEQ ID NOs: 1-3351.
Including NA.

The polynucleotide of the present invention also includes polynucleotides having sequence similarity or sequence identity. Nucleic acids with sequence similarity are labeled under conditions of low stringency, eg, 50 ° C. and 10 × SSC (0.9 M saline / 0.09 M).
Sodium citrate) and 1
Retains binding when subjected to washing at 55 ° C in SSC. Sequence identity may be determined under stringent conditions, such as above 50 ° C. and 0.1 × SSC (9 mM
It can be determined by hybridization with saline / 0.9 mM sodium citrate). Hybridization methods and conditions are well known in the art, see, eg, US Pat. No. 5,707,829. A nucleic acid that is substantially identical to a provided polynucleotide sequence, such as an allelic variant, a genetically altered version of a gene, etc., under stringent hybridization conditions, is provided with the provided polynucleotide sequence (sequence Numbers 1-3
351). By using probes, in particular labeled probes of DNA sequences, homologous or related genes can be isolated. The source of homologous genes can be any species, eg primates, especially humans: rodents (eg rats and mice); dogs, cats, cows, sheep, horses, yeasts, nematodes and the like.

Preferably, hybridization is performed using at least 15 contiguous nucleotides (nt) of at least one of SEQ ID NOs: 1-3351. That is, if at least 15 consecutive nts of one of the disclosed SEQ ID NOs are used as probes, the probes will preferentially hybridize to nucleic acids containing complementary sequences and will be unique to the selected probe. Enables identification and recovery of nucleic acids that hybridize to Probes from more than one SEQ ID NO: can hybridize to the same nucleic acid if the cDNA from which they correspond corresponds to one mRNA. Probes over 15 nt may be used, for example from about 18 nt to about 100 nt, indicating that 15 nt is sufficient sequence for unique identification.

The polynucleotides of the present invention also include naturally occurring variants of nucleotide sequences (eg, degenerate variants, allelic variants, etc.). Variants of the polynucleotides of the invention are identified by hybridization of the nucleotide sequences disclosed herein with putative variants, preferably under stringent conditions. For example, by using appropriate wash conditions, a variant of a polynucleotide of the invention will have an allelic variant of at most about 25-30% base pair (based on the selected polynucleotide probe).
bp) can be identified if it shows inadequacy. Generally, allelic variants are 15-
Including 25% bp mismatch and only 5-15%, or 2-5%, or 1
~ 2% bp mismatch, as well as single bp mismatch.

The present invention also includes homologs corresponding to the polynucleotides of SEQ ID NOs: 1-3351, wherein the source of homologous genes is any mammalian species, eg primates, especially humans; rodents. (Eg, rat); dog, cat, cow, sheep, horse,
It can be yeast, nematodes and the like. Between mammalian species (eg human and mouse), homologs generally have substantial sequence similarity, eg, at least 75 nucleotide sequences between nucleotide sequences.
%, Usually at least 90%, more usually at least 95% sequence identity.
Sequence similarity is calculated based on the reference sequence, which is a conserved motif,
It may be a subset of longer sequences, such as coding regions, flanking regions and the like. A reference sequence will usually be at least about 18 contiguous nt long, more usually at least about 30 nt long, and can be extended to the complete sequence being compared. Algorithms for sequence analysis are known in the art (eg Altsc.
hul et al. Mol. Biol. (1990) 215: 403-10 BLAST).

Generally, variants of the invention will be at least about 65%, preferably at least about 7%.
5%, more preferably at least about 85% sequence identity, and at least about 90%, 91%, 92%, 93%, 94%, 95%, or 96%,
Most preferably it can exceed 97%, 98% or 99%. For purposes of this invention, the preferred method of calculating percent identity is Smith-W using
The aterman algorithm. The overall DNA sequence identity was determined by the following search parameters: gap open penalty.
ty), 12; and gap extension penalties.
MPSRCH program (Oxford Molecular) using affine gap search with 1).
Must be greater than 65% as determined by the Smith-Waterman homology search algorithm as performed in.

The nucleic acids of the invention are useful in cDNA or genomic DNA, and fragments thereof, particularly those encoding biologically active gene products and / or in the methods disclosed herein (eg, diagnostics). , As a unique identifier of the differentially expressed gene of interest, etc.). As used herein, the term "cDNA"
Is intended to include all nucleic acids that share the arrangement of sequence elements found in the native mature mRNA species, where the sequence elements are exons and 3'non-coding and 5'non-coding regions. Usually mRNA species have contiguous exons, with intron intervention and, if present, nuclear RNA.
It is removed by splicing to produce a continuous open reading frame encoding the polypeptide of the invention.

The genomic sequence of interest includes the nucleic acids present between the start and stop codons and includes all introns normally present in the native chromosome, as defined in the listed sequences. It is further 3 ′ found in mature mRNA
It may include untranslated regions and 5'untranslated regions. It may further include specific transcriptional and translational regulatory sequences (eg, promoters, enhancers, etc.), of approximately 1 kb, but possibly a longer flanking genome at either the 5'and 3'ends of the transcribed region. Contains DNA. Genomic DNA can be isolated as fragments of 100 kbp or less; and is substantially free of flanking chromosomal sequences. Genomic DNA flanking the coding region in either 3'and 5'or internal regulatory sequences, such as are sometimes found in introns, are present for proper tissue, stage-specific, or disease state-specific expression. Contains the required sequences.

The nucleic acid composition of the present invention may encode all or a part of the polypeptide of the present invention. Double-stranded or single-stranded fragments can be obtained from DNA sequences by chemical synthesis of oligonucleotides according to conventional methods, such as by restriction enzyme digestion, PCR amplification and the like. The isolated polynucleotides and polynucleotide fragments of the invention are at least about 10, about 15, about 20, selected from the polynucleotide sequences as set forth in SEQ ID NOs: 1-3351.
It comprises about 35, about 50, about 100, about 150 to about 200, about 250 to about 300, or about 350 consecutive nts. These fragments also have a length intermediate to those specifically mentioned (eg 35, 36, 37, 38, 39, etc .; 150,
151, 152, 153, 154, etc.). For the most part, fragments will be at least about 15 nt, usually at least 18 nt or 25 nt.
nt and up to at least about 50 consecutive nt lengths. In a preferred embodiment, the polynucleotide molecule comprises at least 12 nt of contiguous sequence selected from the group consisting of the polynucleotides set forth in SEQ ID NOs: 1-3351.

Probes specific for the polynucleotides of the present invention can be made using the polynucleotide sequences disclosed in SEQ ID NOs: 1-3351. The probe is preferably at least about 12,15,16,18,20,22,24, or 25 nt fragment of the corresponding contiguous sequence of SEQ ID NOs: 1-3351, and 2,1,0.5. , 0.1, or 0.05 kb in length. Probes can be chemically synthesized or can be made from longer polynucleotides using restriction enzymes. The probe can be labeled with, for example, a radioactive tag, a biotinylated tag, or a fluorescent tag. Preferably, the probes are SEQ ID NOs: 1-33
Designed based on the identified sequence of one of the 51 polynucleotides. More preferably, the probe is designed based on a contiguous sequence of one of the polynucleotides of the invention that remains unmasked after application of a masking program (eg, XBLAST) to mask low complexity to the sequence. (Ie, unmasked regions are selected, as indicated by the polynucleotides outside the poly-n stretch of masked sequence generated by the masking program).

The polynucleotides of the present invention are isolated and obtained in sufficient purity, generally other than intact chromosomes. Usually, the polynucleotide is DNA
Or RNA, obtained substantially free of other naturally occurring nucleic acid sequences, and generally of at least about 50%, usually at least about 90% pure,
And is typically "recombinant," for example flanked by one or more nucleotides normally not associated with the naturally occurring chromosome.

The polynucleotide of the present invention can be provided as a linear molecule or in a circular molecule, and can be provided in an autonomously replicating molecule (vector) or in a molecule without a replication sequence. . Expression of polynucleotides may be regulated by themselves or by other regulatory sequences known in the art. The polynucleotides of the invention can be incorporated into various techniques available in the art (eg, transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acid, liposome-mediated DNA transfer, DNA coating). Intracellular delivery of latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate mediated transfection, etc.) can be used to introduce into appropriate host cells.

The nucleic acid composition of the invention may be used as a probe for the detection of mRNA of the invention in a biological sample (eg an extract of human cells), eg to produce a polypeptide, a polynucleotide. To make additional copies of the ribozyme or to make antisense oligonucleotides, and single-stranded DNA
It can be used as a probe or as a triplex forming oligonucleotide. The probes described herein include, for example, SEQ ID NOs: 1 to 1 in a sample.
It can be used to determine the presence or absence of a polynucleotide sequence as shown in 3351 or variants thereof. These and other uses are described in more detail below.

(Use of Polynucleotide to Obtain Full-Length cDNA, Gene, and Promoter Region) A full-length cDNA molecule containing the disclosed polynucleotide can be obtained as follows. One of SEQ ID NOs: 1-3351, or at least 12, 1
Polynucleotides having those moieties containing 5, 18, or 20 nt may be probed as described in US Pat. No. 5,654,173,
Used as a hybridization probe to detect hybridizing members of a cDNA library using cloning methods and clone selection techniques. Libraries of cDNA are made from selected tissues, such as normal or tumor tissues, or from, eg, drug-treated mammalian tissues. Preferably, the tissue is the same tissue from which the polynucleotide of the invention was isolated. This is because both the polynucleotides and cDNAs described herein represent expressed genes. Most preferably cD
NA libraries are made from the biological materials described herein in the Examples. The selection of cell types for library construction can be done after the organism of the protein encoded by the gene corresponding to the polynucleotide of the invention is known. This indicates which tissues and cell types are likely to express the relevant gene, and thus represents a suitable source for mRNA to make cDNA. As described in the examples, the cDNA of the present invention is isolated from a particular cell or tissue type, and such cells and tissues are preferred for obtaining the relevant nucleic acid.

Techniques for generating and probing nucleic acid sequence libraries are described in, for example, Sa
Mbrook et al., Molecular Cloning: A Laborato.
ry Manual, 2nd Edition, (1989) Cold Spring Harb
or Press, Cold Spring Harbor, NY. cDNA can be prepared by using primers based on the sequences from SEQ ID NOs: 1-3351. In one embodiment, a cDNA library can be made exclusively of polyadenylated mRNA. Therefore, poly-T primers can be used to prepare cDNA from mRNA.

Members of the library are obtained which are longer than the polynucleotide provided and which preferably contain the complete coding sequence of the native message. To confirm that the entire cDNA has been obtained, RNA protection experiments are performed as follows. Hybridization of full-length cDNA to mRNA was performed using RNase
Protects RNA from degradation. If the cDNA is not full length, the part of the mRNA that does not hybridize is subjected to RNase degradation. This is assayed by changes in electrophoretic mobility in polyacrylamide gels or by detection of released monoribonucleotides, as is known in the art. Sa
Mbrook et al., Molecular Cloning: A Laborato.
ry Manual, 2nd Edition, (1989) Cold Spring Harb
or Press, Cold Spring Harbor, NY. Partial c
To obtain additional sequences 5'to the ends of the DNA, 5'RACE (PC
R Protocols: A Guide to Methods and A
applications, (1990) Academic Press, Inc.
． ) Can be done.

Genomic DNA is isolated using the provided polynucleotides in a manner similar to the isolation of full length cDNA. Briefly, the provided polynucleotide,
Alternatively, that portion is used as a probe for a library of genomic DNA. Preferably, the library is, but need not be, derived from the cell type used to make the polynucleotide of the invention. Most preferably, the genomic DNA is obtained from the biological material described herein in the examples. Such libraries are described in Sambrook et al., 9.4-9.
As described in detail at 30, it may be in a vector suitable to carry a large segment of the genome, such as P1 or YAC. In addition, genomic sequences can be isolated from human BAC libraries, which can be isolated, for example, from Research.
ch Genetics, Inc. Huntsville, Alabama, U
Commercially available from SA. To obtain additional 5'or 3'sequence, Sa
Chromosome walking is performed as described in mbrook et al., which isolates contiguous and overlapping fragments of genomic DNA. They are,
Restriction digestion enzymes and DNA ligase are used to map and splice together as is known in the art.

Using the polynucleotide sequences of the present invention, the corresponding full-length gene is a cDNA
It can be isolated using both classical and PCR methods for constructing and probing libraries. Using either method, Northern blots are preferably performed on a number of cell types to determine which cell line expresses the gene of interest at the highest level. Classical methods of constructing cDNA libraries are taught in Sambrook et al. (supra). Using these methods, cDNA can be generated from mRNA and inserted into a viral or expression vector. Typically, an mRNA containing a poly (A) tail
The library can be generated using poly (T) primers. Similarly, cDN
A libraries can be generated using the sequences of the invention as primers.

The PCR method is used to amplify a member of the cDNA library containing the desired insert. In this case, the desired insert comprises the full-length cDNA-derived sequence corresponding to the polynucleotide of the invention. Such PCR methods include gene trap and RACE methods, such as those described in Gruber et al., WO 95/04745 and Gruber et al., US Pat. No. 5,500,356. The kit is, for example, Life Technology
es, Gaithersburg, Maryland, USA are commercially available for performing gene trap experiments. In a preferred embodiment of RACE, the common primer is designed to anneal to an optional adapter sequence linked to the ends of the cDNA (Apte and Siebert, Biotechni).
ques (1993) 15: 890-893: Edwards et al., Nuc. Ac
ids Res. (1991) 19: 5227-5232). When a single gene-specific RACE primer is paired with a common primer, preferential amplification of the sequence between this single gene-specific primer and this common primer occurs. Commercially available cDNA pools modified for use in RACE are available.

The promoter region of the gene is generally located 5'to the start site for RNA polymerase II. Several hundred promoter regions contain "TATA" boxes (eg, TATTA or TATAA sequences) that are susceptible to mutation. The promoter region uses a primer from the coding region of the gene
'Can be obtained by performing RACE. Alternatively, the cDNA can be used as a probe for genomic sequences, and the region 5'to the coding region is identified by "walking up." If the gene is highly or differentially expressed, promoters from this gene may be useful in regulatory constructs for heterologous genes.

Once the full-length cDNA or gene is obtained, the DNA encoding the variant
Can be prepared by site-directed mutagenesis (Sambrook et al., 15.3).
~ 15.63). The choice of codons or nucleotides to be replaced can be based on the disclosure herein regarding any changes in amino acids to achieve altered protein structure and / or function.

As an alternative method of obtaining DNA or RNA from biological material, nucleic acids containing nucleotides having the sequence of one or more polynucleotides of the present invention can be synthesized. Accordingly, the present invention is suitable for 15 nt (corresponding to at least 15 consecutive nt of one of SEQ ID NOS: 1-3351) to one or more biological manipulations (including replication and expression) of nucleic acid molecules. It includes nucleic acid molecules of lengths up to the maximum length. The present invention provides (a) the complete gene size and SEQ ID NOs: 1-335.
A nucleic acid comprising at least one of 1; (b) a nucleic acid of (a) also comprising at least one additional polynucleotide or gene operably linked to allow expression of the fusion protein; (c) A nucleic acid vector containing (a) or (b); (d) a plasmid containing (a) or (b); and (e) (a
) Or (b) is included in the recombinant virus particle, but is not limited thereto. Once the polynucleotide disclosed herein is provided, (a)-(
The construction and preparation of e) is well within the skill of the art.

The sequence of a nucleic acid containing at least 15 consecutive nts of at least one of SEQ ID NOs: 1-3351, preferably the entire sequence of at least any one of SEQ ID NOs: 1-3351 is restricted. , And A, T, G, and / or
Or C (for DNA), and A, U, G, and / or C (R
NA) or any of their modified bases, including inosine and pseudouracil. The choice of sequence depends on the desired function and can be dictated by the desired coding region, the desired intron-like region, and the desired regulatory region. If the entire sequence of any one of SEQ ID NOs: 1-3351 is within the nucleic acid, the resulting nucleic acid is referred to herein as a polynucleotide comprising the sequence of any one of SEQ ID NOs: 1-3351. Be seen.

(Expression of Full-length cDNA or Polypeptide Encoded by Full-length Gene) A provided polynucleotide (eg, a polynucleotide having a sequence of one of SEQ ID NOs: 1-3351), a corresponding cDNA, or a full-length cDNA The gene is
Used to express a partial or complete gene product. Sequence number 1-3
A construct of a polynucleotide having the sequence of 351 can be produced synthetically. Alternatively, single-step assembly of genes and entire plasmids from multiple members of oligodeoxyribonucleotides is described, for example, in Stemmer et al., Gene.
(Amsterdam) (1995) 164 (1): 49-53. In this method, assembly PCR (oligodeoxyribonucleotide (
(Synthesis of long DNA sequences derived from multiple members of oligo). This method is based on DNA shuffling (Stemmer, Nature (1994).
370: 389-391) and do not rely on DNA ligase, but instead during the assembly process, rely on DNA polymerase to assemble even longer DNA fragments.

Suitable polynucleotide constructs are described, for example, in Sambrook et al., Molcu.
lar Cloning: A Laboratory Manual, 2nd Edition,
(1989) Cold Spring Harbor Press, Cold
Using standard recombinant DNA techniques described in Spring Harbor, NY, and in the United States Dept. of HHS, Nati
onal Institute of Health (NIH) Guideli
Purified under current rules as described in nes for Recombinant DNA Research. The gene product encoded by the polynucleotide of the present invention is expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Vectors, host cells, and methods for obtaining expression in these are well known in the art. Suitable vectors and host cells are described in US Pat. No. 5,654,173.

A polynucleotide molecule comprising a polynucleotide sequence provided herein,
Generally, it is propagated by placing the molecule in a vector. Viral and non-viral vectors (including plasmids) are used. The choice of plasmid depends on the type of cell in which it is desired to grow and the purpose of growth. Certain vectors are useful for amplifying and producing 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 whole animals or humans. Selection of the appropriate vector is well within the skill of the art. Many such vectors are commercially available. Methods for preparing vectors containing the desired sequence are well known in the art.

The polynucleotides set forth in SEQ ID NOs: 1-3351, or their corresponding full-length polynucleotides, are linked to regulatory sequences as appropriate to obtain the desired expression characteristics. These can include promoters (attached at either the 5'end of the sense strand or the 3'end of the antisense strand), enhancers, terminators, operators, repressors, and inducers. The promoter 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 ligated to the desired nucleotide sequence using the techniques described above for ligation into vectors. Any technique known in the art can be used.

When any suitable host cell or organism is used to replicate and / or express a polynucleotide or nucleic acid of the invention, the resulting replicated nucleic acid, RNA, expressed protein or Polypeptides are within the scope of this invention as the product of their host cells or organisms. The product is recovered by any suitable means known in the art.

Once the gene corresponding to the selected polynucleotide is identified, its expression can be regulated in cells in which the gene is native. For example, a cell's endogenous genes can be regulated by exogenous regulatory sequences as disclosed in US Pat. No. 5,641,670.

Identification of Functional and Structural Motif of Novel Genes Translation of the nucleotide sequence of a provided polynucleotide, cDNA, or complete gene can be aligned with individual known sequences. Similarity to individual sequences can be used to determine the activity of the polypeptide encoded by the polynucleotide of the invention. Also, a sequence exhibiting similarity to more than one individual sequence may exhibit activity characteristic of either or both of the individual sequences.

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

[0050] Typically, the selected polynucleotides are translated in all 6 frames to determine the best alignment with the individual sequences. The sequences disclosed herein in the sequence listing are in the 5'to 3'direction and translation in 3 frames may be sufficient. These amino acid sequences are commonly referred to as query sequences,
This is aligned with the individual sequence. The database with individual sequences is "Comp
uter Methods for Macromolecular Seque
ence Analysis ”Methods in Enzymology
(1996) 266, Doolittle, Academic Press, I.
nc. a division of Harcourt Brace & Co
． , San Diego, California, USA. Databases include Genbank, EMBL, and DNA Database.
of Japan (DDBJ).

The query sequence and individual sequences can be aligned using the above methods and computer programs, and BLAST: a (http //www.ncbi available in nlm.nhi.gov/BLAST of the World Wide Web.) Including. Another alignment algorithm is Fasta (Genetics Computi).
Available in ng Group (GCG) package, Madison, Wisc
onsin, USA, Oxford Molecular Group, Inc.
． Is a wholly owned subsidiary). Other techniques for alignment are Doolittle,
As described above. Preferably, alignment programs that allow gaps in the sequences are utilized to align the sequences. Smith-Waterman is
It is one type of algorithm that allows for gaps in sequence alignments. Met
h. Mol. Biol. (1997) 70: 173-187. Also, a GAP program using the Needleman and Wunsch alignment method
It can be used to align sequences. An alternative search strategy is MASPA
MPSRCH software running on an R computer is used. MPSRC
Hmit Smit to score sequences on a massively parallel computer.
Use the h-Waterman algorithm. This approach improves the ability to identify sequences that are faintly related matches and is particularly tolerant of small gaps and nucleotide sequence errors. The amino acid sequence encoded by the provided polynucleotide can be used to search both protein and DNA databases.

High Similarity Generally, as a result of alignments considered to be high similarities, the percentage of alignment region length is typically at least about 55% of the total length of the query sequence.
More typically at least about 58%; even more typically at least about 60% of the total residue length of the query sequence. Typically, the percent of alignment region length can be as high as about 62%; more usually as high as about 64%; and even more usually as high as about 66%. Further, for high similarities, the alignment regions typically have at least about 75% sequence identity; more typically at least about 78%; even more typically at least about 80% sequence identity. Shows sex. Usually, percent sequence identity can be as high as about 82%; more usually as high as about 84%; much more usually as high as about 86%.

The p-value is used in conjunction with these methods. If a high degree of similarity is found,
If the p-value is less than about 10 ^-2 ; more usually less than about 10 ^-3 ; even more usually less than about 10 ^-4 , the query sequence has a high degree of similarity to the profile sequence. Is considered to have. More typically, the p-values are not more than about 10 ⁻⁵ ; more typically about 10 ⁻¹⁰ or less; even more typically Is about 10 ⁻¹⁵ or less.

Similarity Determined by Sequence Identity Only Sequence identity can be used alone to determine the similarity of a query sequence to an individual sequence and can be indicative of sequence activity. Such alignments preferably allow gaps to align the sequences. Typically, the query sequence has at least about 15% sequence identity over the query sequence; more typically at least about 20%; even more typically at least about 25%; Has at least about 5
0% is associated with the profile sequence. Sequence identity, alone, is a measure of similarity when the query sequence is usually at least 80 residues long; more usually 90 residues; even more usually at least 95 amino acid residues long. Most useful. More typically, the query sequence is preferably 100 residues long;
Similarity can be determined based solely on sequence identity when more preferably 120 residues long; even more preferably 150 amino acid residues long.

Profiles and Alignment with Multiple Aligned Sequences Translations of the provided polynucleotides can be aligned with amino acid profiles that define either protein families or common motifs. In addition, the translation of the provided polynucleotide,
It can be aligned to a multiple sequence alignment (MSA) that includes polypeptide sequences of protein family or motif members. Similarity or identity to the profile sequence or MSA can be used to determine the activity of the gene product (eg, polypeptide) encoded by the provided polynucleotide or the corresponding cDNA or gene. For example, a sequence showing identity or similarity to a chemokine profile or MSA may exhibit chemokine activity.

Profiles can be manually designed by creating (1) MSA, which is an alignment of amino acid sequences of members of the family, and (2) creating a statistical representation of the alignment. Such methods are described, for example, in Birney et al., Nuc.
l. Acid Res. (1996) 24 (14): 2730-2739. MSAs for several protein families and motifs are publicly available. MSA is reviewed by Sonnhammer et al., Proteins (
1997) 28: 405-420. A brief description of these MSAs can be found in Pascarella et al., Prot. Eng. (1996) 9 (3): 24
9-251. The technology for profile construction from MSA is
Sonnhammer et al. (Supra); Birney et al. (Supra); and Comp.
uter Methods for Macromolecular Seque
ence Analysis ", Methods in Enzymology
(1996) 266, Doolittle, Academic Press, I.
nc. , San Diego, California, USA.

Similarities between query sequences and protein families or motifs are (
a) comparing the query sequence to the profile and / or (b)
It can be determined by aligning the query sequence with family or motif members. Typically, programs such as Searchwise allow query sequences to be aligned in multiples (also known as profiles (Birne.
y et al. (supra)))). Other techniques for comparing sequences and profiles are described in Sonnhammer et al. (Supra) and Doolittle (supra).

Next, Feng et al. Mol. Evol. (1987) 25: 351 and Higgins et al., CABIOS (1989) 5: 151 can be used to align a query sequence with a family or motif member (also known as MSA). Sequence alignments can be generated using any of a variety of software tools. Examples include PileUp, which creates multiple sequence alignments and is described by Feng et al. Mol. E
vol. (1987) 25: 351. Another method (GAP) is Ne
edleman et al. Mol. Biol. (1970) 48: 443. GAP is best suited for global alignment of sequences. The third method (BastFit) is described by Smith et al., Adv. Appl. Math (1981
) It works by using the 2: 482 local homology algorithm and inserting gaps to maximize the number of matches. Generally, the following factors are used to determine whether there are similarities between the query sequence and the profile or MSA: (1) Conserved residues found in the query sequence. Number, (2) percent 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 are:
When translating the nucleotide sequences to produce the best alignment, a large number of frameshifts may be produced. The fewer frameshifts needed to create an alignment, the stronger the similarity or identity between the query and the profile or MSA. For example, the weak similarity obtained without frameshifting may be a better indicator of the activity or structure of the query sequence than the strong similarity obtained with two frameshifts. Preferably, no more than 3 frameshifts are found in the alignment; more preferably no more than 2 frameshifts; even more preferably no more than 1 frameshift; even more preferably no frameshifts, Found in the alignment of the query with the profile or MSA.

Conserved residues are amino acids found at specific positions in all or some of the family or motif members. Alternatively, a position is considered conserved if only a particular class of amino acids is found at that particular position in all or some of the members of the family. For example, the N-terminal position can include a positively charged amino acid such as lysine, arginine, or histidine.

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

Three unrelated amino acids; more usually, two unrelated amino acids are found at a particular position in some or all of the members:
The residues are considered to be conserved. Unrelated amino acids are at least about 40% of all members of a class; more typically at least about 50%;
Even more typically, these residues are conserved if found in a particular position in at least about 60% of the members. Usually, a class or single amino acid is at least about 70% of members of a family or motif; more usually at least about 80%; even more usually at least about 90%; even more usually Residues are conserved if found in at least about 95%.

If the query sequence comprises at least about 25% of conserved residues of the profile or MSA; more usually at least about 30%; even more usually at least about 40%, then the query sequence is: It has similarities to the profile or MSA. Typically, 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%, The query sequence has a stronger similarity to the profile sequence or MSA.

Identification of Secreted and Membrane Bound Polypeptides Both the secreted and membrane bound polypeptides of the invention are of particular interest. For example, levels of secreted polypeptides can be assayed in convenient body fluids, such as blood, plasma, serum, and other body fluids such as urine, prostatic fluid and semen. Membrane-bound polypeptides are useful for constructing vaccine antigens or for inducing an immune response. Such antigens include all or part of the extracellular region of the membrane bound polypeptide. Since both secreted and membrane-bound polypeptides contain contiguous fragments of hydrophobic amino acids, hydrophobicity prediction algorithms can be used to identify such polypeptides.

Signal sequences are usually encoded by both secreted and membrane-bound polypeptide genes and direct the polypeptide to the surface of the cell. Signal sequences usually include stretches of hydrophobic residues. Such a signal sequence can be folded into a helix structure. Membrane-bound polypeptides typically include at least one transmembrane region with a stretch of hydrophobic amino acids that can cross the membrane. Some transmembrane regions also show helix structures. Hydrophobic fragments within a polypeptide can be identified by using computer algorithms. Such algorithms are described in Hopp and Woods, Pr.
oc. Natl. Acad. Sci. USA (1981) 78: 3824-38.
28; Kyte and Doolittle, J. et al. Mol. Biol. (1982
) 157: 105-132; and RAOAR algorithm, Degli E.
sposti et al., Eur. J. Biochem. (1990) 190: 207-
219 is included.

Another method of identifying secreted and membrane bound polypeptides is to translate the polynucleotides of the invention in all 6 frames, and at least 8
To determine if there are consecutive hydrophobic amino acids. A translated polypeptide having at least 8; more typically 10; and even more typically 12 contiguous hydrophobic amino acids is considered to be either a putative secreted polypeptide or a membrane-bound polypeptide. Hydrophobic amino acids include alanine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine.

Identification of Function of Expression Product of Full Length Gene Ribozymes, antisense constructs, and dominant negative mutants are used to determine the function of expression products of genes corresponding to the polynucleotides provided herein. Can be used for. The phosphoramidite method of oligonucleotide synthesis can be used to construct antisense molecules and ribozymes. Beau
Cage et al., Tet. Lett. (1981) 22: 1859 and U.S. Pat. No. 4,668,777. Automated synthesis devices are available to make oligonucleotides using this chemistry. Examples of such devices include Applied Biosystems, a division o.
f Perkin-Elmer Corp. , Foster City, Cal
Biosearch 8600, Type 392 and 39 from Ifornia, USA
Type 4; and Perceptive Biosystems, Framing
ham, Massachusetts, and Expedite of USA. Synthetic RNA, phosphate analog oligonucleotides, and chemically derivatized oligonucleotides can also be produced and covalently linked to other molecules.
RNA oligonucleotides can be synthesized, for example, using RNA phosphoramidites. This method is based on an automated synthesizer (eg, Applied Biosys).
tems, types 392 and 394, Foster City, California
nia, USA).

Oligonucleotides of up to 200 nucleotides, more typically 100 nucleotides, more typically up to 50 nucleotides; even more typically 30-40 nucleotides can be synthesized. These synthetic fragments can be annealed and ligated together to build larger fragments. For example,
See Sambrook et al., Supra. Trans-cleaving catalytic RNA (ribozyme) is an RNA molecule that possesses endoribonuclease activity. Ribozymes are designed specifically 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 disconnection event is m
It destabilizes RNA and prevents protein expression. Importantly, ribozymes can be used to inhibit the expression of genes of unknown function for the purpose of determining the function of genes of unknown function in in vitro or in vivo situations by detecting phenotypic effects.

Antisense nucleic acids are designed to bind specifically to RNA, thereby involving RNA-, which involves the inhibition of DNA replication, reverse transcription, or messenger RNA translation.
This results in the formation of DNA hybrids or RNA-RNA hybrids. Antisense polynucleotides based on the selected polynucleotide sequence may interfere with expression of the corresponding gene. Antisense polynucleotides are typically produced intracellularly by expression from an antisense construct containing the antisense strand as the transcribed strand. Antisense polynucleotides based on the disclosed polynucleotides include an mRN containing a sequence complementary to the antisense polynucleotide.
Binds to A and / or interferes with translation of its mRNA. The expression product of control cells and the expression product of cells treated with the antisense construct are compared to detect the protein product of the gene corresponding to the polynucleotide on which the antisense construct is based. The protein is isolated and identified using conventional biochemical methods.

Given the extensive background literature and clinical experience in antisense therapy,
One of ordinary skill in the art can use the selected polynucleotides of the invention as additional potential therapeutic agents. The choice of polynucleotides can be narrowed by first testing them for binding to "hot spot" regions of the cancerous cell's genome. If the polynucleotide is identified as binding to a "hot spot",
Testing the polynucleotides as antisense compounds in corresponding cancer cells is justified.

Dominant negative mutations for the corresponding proteins active as homomultimers are also readily generated. Variant polypeptides interact with wild-type polypeptides (made from other alleles) and form non-functional multimers. Thus, the mutation is in the substrate binding domain, catalytic domain, or cell localization domain. Preferably, the variant polypeptide is overproduced. A point mutation having such an effect is created. Furthermore, fusion of different length polypeptides to the ends of the protein can produce dominant negative mutants.
General strategies are available for making dominant negative mutants (eg Herskowitz, Nature (1987) 329:
219). Such techniques are loss of fu.
nucleation) mutations, which are useful for determining protein function.

Polypeptides and Variants thereof The polypeptides of the invention are not identical in sequence to the disclosed polynucleotides and to the disclosed polynucleotides due to the degeneracy of the genetic code. It includes a polypeptide encoded by a nucleic acid. Therefore, the present invention includes, within the scope of the present invention, a polypeptide encoded by the polynucleotide having any one of the sequences of SEQ ID NOs: 1-3351 or a variant thereof.

In general, as used herein, the term “polypeptide” refers to the full-length polypeptide encoded by the indicated polynucleotide, the polypeptide encoded by the gene indicated by the indicated polynucleotide. Refers to both peptides, and parts or fragments thereof. "Polypeptide" also includes variants of naturally-occurring proteins, wherein such variants are homologous or substantially similar to naturally-occurring proteins and are naturally-occurring. Can be of the same or different species as the protein (eg, human, mouse, or some other species that naturally expresses the indicated polypeptide, usually a mammalian species). In general, a variant polypeptide comprises at least about 80%, usually at least about 90%, and usually a differentially expressed polypeptide of the invention, as measured by BLAST using the parameters above. More usually have sequences that have at least about 98% sequence identity. Variant polypeptides may be naturally glycosylated or non-naturally glycosylated (ie, a polypeptide is a glycosylation pattern different from that found in the corresponding naturally occurring protein). Have).

The present invention also includes homologs (or fragments thereof) of the disclosed polypeptides, wherein the homologs are isolated from other species (ie, other animal or plant species), Such homologues are usually of mammalian species (eg, mouse,
Rodents such as rats: domestic animals (eg horses, cows, dogs, cats); and humans. By "homologue" is meant a polymorph having an amino acid sequence identity of at least about 35%, usually at least about 40%, and more usually at least about 60% to a particular differentially expressed protein as identified above. Peptides are contemplated, where sequence identity is determined using the BLAST algorithm with the above parameters.

Generally, the polypeptides of the invention are provided in non-naturally occurring environments,
For example, it is separated from their naturally occurring environment. In certain embodiments, a protein of the invention is present in a composition enriched for that protein relative to a control. As such, it provides a purified polypeptide,
Purified, as used herein, means that the protein is present in a composition that is substantially free of non-differentially expressed polypeptide, where substantially no protein is 90% of the composition. It is meant that less than%, usually less than 60%, and more usually less than 50% is composed of non-differentially expressed polypeptides.

Also within the scope of the invention are variants; variants of polypeptides include variants, fragments and fusions. Variants include amino acid substitutions, additions,
Or it may include a deletion. Amino acid substitutions may be conservative amino acid substitutions, or to eliminate non-essential amino acids (eg, to change glycosylation, phosphorylation, or acetylation sites), or one that is not essential for function. It may be a substitution to minimize misfolding due to the substitution or deletion of the above cysteine residues. Conservative amino acid substitutions result in overall charge, hydrophobicity / hydrophilicity,
And / or is a substitution that preserves the steric bulk of the amino acid being replaced. Variants are designed to retain the biological activity of a particular region of the protein (eg, the functional domain and / or the region associated with the consensus sequence if the polypeptide is a member of the protein family). Can be done. The choice of amino acid changes for the generation of variants is based on the accessibility of amino acids (internal vs. external) (eg Go et al., Int. J. Peptide Protein Res. (198).
0) 15: 211), the thermal stability of the modified polypeptides (eg Q
uerol et al., Prot. Eng. (1996) 9: 265), the desired glycosylation site (eg, Olsen and Thomsen, J. Gen.
． Microbiol. (1991) 137: 579), the desired disulfide bridge (eg, Clarke et al., Biochemistry (199).
3) 32: 4322; and Wakarchuk et al., Protein Eng.
(1994) 7: 1379), the desired metal binding site (eg, To
ma et al., Biochemistry (1991) 30:97, and Haeze.
rbrouck et al., Protein Eng. (1993) 6: 643), as well as desired substitutions within the proline loop (eg, Masul et al., Appl. En.
v. Microbiol. (1994) 60: 3579). Cysteine-depleted muteins can be produced as described in US Pat. No. 4,959,314.

Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. Fragments of interest are typically at least about 10 amino acids in length to at least about 15 amino acids in length, usually at least about 50 amino acids in length, and can be 300 amino acids in length or longer, but usually not more than about 1000 amino acids in length. , Wherein the fragment has a stretch of amino acids that is identical to a polypeptide encoded by a polynucleotide having the sequence of any of SEQ ID NOs: 1-3351, or homologs 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 variant.

Computer-Related Embodiments Generally, a library of polynucleotides is a collection of sequence information, which information is in biochemical form (eg, as a collection of polynucleotide molecules), or Provided either in electronic form (eg, as a collection of polypeptide nucleotide sequences stored in computer readable form, as in a computer system, and / or as part of a computer program). . Polynucleotide sequence information can be used, for example, as a resource for gene discovery, as an indication of the sequences expressed in the selected cell type (eg, a marker of the cell type), and / or for a given disease or disease state. As a marker, it can be used in a variety of ways. In general, a disease marker is all levels affected by a disease, either at elevated or decreased levels relative to normal cells (eg, cells of the same or similar type that are not substantially affected by the disease). A representation of the gene product present in the cell. For example, the polynucleotide sequence in the library can be a polynucleotide that represents the mRNA, polypeptide, or other gene product encoded by the polynucleotide, and is associated with normal (ie, substantially disease-free) breast cells. In comparison, they are either overexpressed or downregulated in ductal cells affected by the cancer.

The nucleotide sequence information for the library may be embodied in any suitable form, such as electronic or biochemical form. For example, libraries of sequence information embodied in electronic form include, for example, i) between cancer cells and normal cells; ii) between cancer cells and dysplastic cells; iii) cancer cells and cancer. Between cells affected by a disease or condition other than; iv) between metastatic cancer cells and normal and / or non-metastatic cancer cells; v) malignant cancer cells and non-malignant cancer cells cell(
Or normal cells), and / or vi) dysplastic cells relative to normal cells, are representative of genes that are differentially expressed (eg, overexpressed or repressively expressed). Includes accessible computer data files (or collections of nucleic acid molecules in biochemical form) containing nucleotide sequences.
Other combinations and comparisons of cells affected by various diseases or stages of disease will be readily apparent to those of skill in the art. A biochemical embodiment of the library comprises a collection of nucleic acids having the sequences of the genes in the library, wherein the nucleic acids are
As described in more detail below, it may correspond to the entire gene in the library or a fragment thereof.

Polynucleotide libraries of the invention generally include sequence information for multiple polynucleotide sequences, wherein at least one polynucleotide has any of SEQ ID NOs: 1-3351. By plural is meant at least 2, usually at least 3, and may include all of SEQ ID NOs: 1-3351. The length and number of polynucleotides in the library will vary with the nature of the library, eg, if the library is an oligonucleotide array, a cDNA array, a computer database of sequence information, and the like.

If the library is an electronic library, nucleic acid sequence information can be present in a variety of media. "Media" refers to an article of manufacture other than an isolated nucleic acid molecule that contains the sequence information of the present invention. Such an article of manufacture provides a genomic sequence or a subset thereof in a form that can be tested by means that are not directly applicable to the sequence as it is present in a nucleic acid. For example, a nucleotide sequence of the invention, eg, SEQ ID NOs: 1-3
The nucleic acid sequence of any of the 351 polynucleotides can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include: magnetic storage media (eg floppy disks, hard disk storage media, and magnetic tape); optical storage media (eg CD-ROM); electronic storage media (eg RA).
M and ROM); and hybrids of these categories (eg, magnetic / optical storage media), but are not limited thereto. One of ordinary skill in the art can readily understand what any computer-readable medium now known can be used to make an article of manufacture containing the sequence information records of the invention. "Recorded" refers to the process of storing information on a computer-readable medium, using any method known in the art. Any convenient data storage structure may be selected based on the means used to access the stored information. A wide variety of data processor programs and formats can be used for storage (eg, word processing text files, database formats, etc.). In addition to sequence information, electronic versions of the libraries of the invention may contain other computer readable information and / or other types of computer readable files (eg, searchable files, executable files, etc., such as , Including, but not limited to, search program software, etc.).

By providing the nucleotide sequences in computer readable form, the information can be accessed for a variety of purposes. Computer software for accessing sequence information is publicly available. For example, BLAS
T (Altschul et al., Supra), and BLA in the Sybase system
The ZE (Brutlag et al., Comp. Chem. (1993) 17: 203) search algorithm uses open reading frames (ORFs) from other organisms.
Can be used to identify ORFs within the genome that contain homologs for.

As used herein, “computer-based system” refers to the hardware, software, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), input means, output means, and data storage means. One of ordinary skill in the art can readily appreciate that any one of the currently available computer-based systems is suitable for use in the present invention. The data storage means may include any product containing a record of the sequence information of the invention as described above, or memory access means capable of accessing such product.

A “search tool” is a means for comparing the expression level of a target sequence or target structural motif, or a polynucleotide in a sample, with stored sequence information,
Refers to one or more programs executed in a computer-based system. Search means can be used to identify genomic fragments or regions that match a particular target sequence or target motif. A variety of known algorithms
Publicly known and commercially available (eg, MacPattern (EM
BL), BLASTN, and BLASTX (NCBI)). "Target sequence" is
Any polynucleotide or amino acid sequence of 6 or more contiguous nucleotides or two or more amino acid sequences, preferably about 10 to 100 amino acids or about 30 to 300 nucleotides. obtain. A variety of comparison means can be used to achieve comparison of sequence information from a sample with a data storage means (eg, to analyze target sequences, target motifs, or relative expression levels). Those skilled in the art will appreciate that any one of the publicly available homology search programs can be used as a search tool for the computer-based system of the present invention to achieve a comparison of target sequences and target motifs. Can be easily recognized. Computer programs for analyzing expression levels in samples and controls are also known in the art.

“Target structure motif” or “target motif” refers to any reasonably selected sequence or combination of sequences, where a sequence is a three-dimensional higher order formed upon folding of the target motif. Selection is based on structure or on the consensus sequence of regulatory or active sites. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include hairpin structures, promoter sequences, and other expression elements such as binding sites for transcription factors,
It is not limited to these.

Various structural formats for the input and output means may be used to input and output information in the computer-based system of the present invention. One format for the output means ranks the relative expression levels of different polynucleotides. Such a presentation provides one of skill in the art with a relative expression level rating for determining gene expression profiles.

As discussed above, “libraries” of the invention also include SEQ ID NOs: 1-3.
A biochemical library of 351 polynucleotides (eg, a collection of nucleic acids representing the provided polynucleotides). Biochemical libraries can take a variety of forms, such as solutions of cDNA, patterns of probe nucleic acids that are stably associated with the surface of a solid support (ie, arrays), and the like. Of particular interest are nucleic acid arrays in which one or more of SEQ ID NOS: 1-3351 is displayed on the array. By array is meant an article carrying at least one substrate with at least two different nucleic acid targets on one of its surfaces, where the number of different nucleic acids can be quite high, typically at least 10 nucleotides, usually at least It can be 20 nucleotides, and often at least 25 nucleotides. A variety of different array formats have been developed and are known to those of skill in the art. The arrays of the invention find use in a variety of applications, including gene expression analysis, drug screening, mutation analysis, etc., as disclosed in the exemplary patent documents listed above.

In addition to the nucleic acid libraries described above, a library of similar polypeptides is also provided, wherein the polypeptides of the library are at least one of the polypeptides encoded by SEQ ID NOs: 1-3351. Represents a part.

Use of Polynucleotide Probes in Mapping and Tissue Profiling Polynucleotide probes generally comprise at least 12 contiguous nucleotides of a polynucleotide as set forth in the Sequence Listing for a variety of purposes (eg, polynucleotides). Chromosomal mapping and detection of transcription levels). Further disclosure of preferred regions of the disclosed polynucleotide sequences can be found in the examples. Probes that specifically hybridize to the polynucleotides disclosed herein will have a detection signal that is at least 5, 10, or 20 times higher than background hybridization provided with other unrelated sequences. Should be provided.

Detection of expression levels. Nucleotide probes are used to detect expression of the gene corresponding to the provided polynucleotides. In Northern blots, mRNA is electrophoretically separated and contacted with the probe. The probe is detected as hybridizing to a particular size mRNA species. The amount of hybridization is quantified, for example, to determine the relative amount of expression under particular conditions. The probe is used for in situ hybridization to cells to detect expression. The probe can also be used in vivo for diagnostic detection of hybridizing sequences. The probe is typically
It is labeled with a radioactive isotope. Other types of detectable labels can be used (eg,
Chromophores, fluorophores, and enzymes). Other examples of nucleotide hybridization assays are described in WO92 / 02526 and US Pat. No. 5,124,246.

Alternatively, the polymerase chain reaction (PCR) is another means for detecting small amounts of target nucleic acid (eg, Mullis et al., Meth. Enzymol.
1987) 155: 335; U.S. Pat. No. 4,683,195; and U.S. Pat. No. 4,683,202). Two primer polynucleotides that hybridize to the target nucleic acid are used to prime the reaction. The primer may be in the polynucleotide of the sequence listing or at the 3'side and 5'to it.
It may consist of a side array. Alternatively, if the primers are 3'and 5'to these polynucleotides, then they need not hybridize to the polynucleotides or complements. After amplification of the target with a thermostable polymerase, the amplified target nucleic acid can be detected by methods known in the art (eg, Southern blot). mRNA or cDNA can also be obtained from Sambrook
K. et al., “Molecular Cloning: A Laboratory M”.
"" (New York Cold Spring Harbor L)
laboratory, 1989) and can be detected by traditional blotting techniques (eg, Southern blots, Northern blots, etc.) (eg, without PCR amplification). In general, mRNA, or cDNA made from mRNA using polymerase enzyme, can be purified and separated using gel electrophoresis and transferred to a solid support such as nitrocellulose. The solid support is exposed to a labeled probe, washed to remove any unhybridized probe, and the duplex containing the labeled probe is detected.

Mapping. The polynucleotides of the present invention can be used to identify the chromosome in which the corresponding gene resides. Such mapping may be useful in identifying the function of the gene associated with the polynucleotide due to its proximity to other genes with known function. Function can also be assigned to the gene associated with the polynucleotide if the particular syndrome or disease maps on the same chromosome. For example, the use of polynucleotide probes in the identification and quantification of nucleic acid sequence abnormalities is described in US Pat. No. 5,783,387.
An exemplary mapping method is fluorescence in situ hybridization (FISH).
), Which allows global genomic assessment of changes in relative copy number of DNA sequences, facilitating comparative genomic hybridization (eg, Valdes et al., Methods in Molecular Biolog).
y (1997) 68: 1). Polynucleotides can also be mapped to specific chromosomes using, for example, radial hybrids or chromosome-specific hybrid panels. Leach et al., Advances in Genet
ics (1995) 33: 63-99; Walter et al., Nature Gen.
etics (1994) 7:22; Walter and Goodfellow,
See Trends in Genetics (1992) 9: 352.
A panel on Radiative Hybrid Mapping is available at Research Gene.
tics, Inc. , Huntsville, Alabama, USA. The statistical program RHMAP can be used to build a map based on data from radiohybridization, along with a measure of the relative likelihood of one order relative to another. RHMAP is http: // www
． sph. umich. edu / group / statgen / softwar
It is available via the World Wide Web at e. In addition, commercially available programs are available for identifying regions of the chromosome that are commonly associated with diseases such as cancer.

Tissue typing or tissue profiling. Expression of the specific mRNA corresponding to the provided polynucleotide may be altered in different cell types and may be tissue specific. This change in mRNA levels in different cell types can be exploited using nucleic acid probe assays to determine tissue type. For example, PCR, branched DNA probe assays, or blotting techniques that utilize nucleic acid probes that are substantially identical or complementary to the polynucleotides listed in the Sequence Listing determine the presence or absence of the corresponding cDNA or mRNA. You can

Tissue typing can be used to identify a developing organ or tissue source of metastatic lesions by identifying the expression of specific markers for that organ or tissue. The origin of the lesion is identified if the polynucleotide is only expressed in a particular tissue type and a metastatic lesion is found to express the polynucleotide. Expression of a particular polynucleotide can be assayed by detection of either the corresponding mRNA or protein product.

Use of polymorphisms. The polynucleotides of the invention can be used in forensic analysis, genetic analysis, mapping, and diagnostic applications, where the corresponding region of the gene is polymorphic in the human population. Any means for detecting polymorphisms in a gene can be used, including electrophoresis of protein polymorphism variants, differential sensitivity to restriction enzyme cleavage, and hybridization to allele-specific probes. Examples include, but are not limited to:

Antibody Production The polynucleotides of the invention, and the corresponding mRNA, cDNA, or complete gene expression products, are prepared and used to raise antibodies for experimental, diagnostic and therapeutic purposes. obtain. For polynucleotides to which the corresponding gene has not been assigned, this provides an additional way to identify that corresponding gene. The polynucleotide or related cDNA is expressed and antibodies are prepared as described above. These antibodies are specific for the epitope on the polypeptide encoded by the polynucleotide and precipitate the corresponding native protein in cell or tissue preparations or in cell-free extracts of in vitro expression systems. Or can bind to the protein.

Methods for producing polyclonal and monoclonal antibodies that specifically bind a selected antigen are well known in the art. These antibodies specifically bind to epitopes present on the polypeptides encoded by the polynucleotides disclosed in the Sequence Listing. Typically, at least 6, 8, 10, or 12 consecutive amino acids are required to form the epitope.
Epitopes containing non-contiguous amino acids may require longer polypeptides (eg at least 15, 25 or 50 amino acids). Antibodies that specifically bind to the human polypeptides encoded by these provided polynucleotides, when used in Western blots or other immunochemical assays, will yield more than the detection signals provided with other proteins. At least 5 times, 10
It provides a 2-fold or 20-fold higher detection signal. Preferably, an antibody specific for the polypeptide of the invention does not bind to other proteins at detectable levels in immunochemical assays and is capable of immunoprecipitating that particular polypeptide from solution.

The present invention also contemplates naturally occurring antibodies specific for the polypeptides of the present invention. For example, serum antibodies to the polypeptides of the invention in the human population can be prepared by methods well known in the art (eg, passing the antiserum through a column to which the corresponding selected polypeptide or fusion protein is bound). Can be purified). The bound antibody can then be eluted from the column using, for example, a buffer having a high salt concentration.

In addition to the antibodies discussed above, the present invention also includes genetically engineered antibodies, antibody derivatives (eg, single chain antibodies, antibody fragments (eg, Fab, etc.)) according to methods well known in the art. Intended.

Other embodiments of the invention include humanized monoclonal antibodies capable of binding the polypeptides of the invention. The phrase "humanized antibody" refers to an antibody derived from a non-human antibody (typically a mouse monoclonal antibody). Alternatively, the humanized antibody is a parental (
It may be derived from a chimeric antibody that retains or substantially retains the antigen binding properties of the (non-human) antibody, but exhibits reduced immunogenicity when administered to a human as compared to its parent antibody. As used herein, the phrase “chimeric antibody” refers to an antibody that comprises sequences derived from two different antibodies (eg, US Pat. No. 4,816,567).
No.), the two antibodies are typically produced from different species. More typically, chimeric antibodies include human antibody fragments and mouse antibody fragments, generally human constant regions and mouse variable regions.

Since humanized antibodies are much less immunogenic in humans than the parental mouse monoclonal antibodies, they can be used in the treatment of humans with little risk of anaphylaxis. Accordingly, these antibodies may be used in therapeutic applications, including in vivo administration to humans (eg, use as radiation sensitizers for the treatment of neoplastic diseases, or methods for reducing side effects (eg, in cancer therapy). Use in).

Humanized antibodies can be completed by a variety of methods, including, for example: (1) human framework and constant region non-human complementarity determining regions (CDRs). Grafting (a process referred to in the art as "humanization"), or (2) replacement of an entire non-human variable domain (but this entire non-human variable domain is replaced on the human-like surface by substitution of surface residues). Cover (Cloak
ing))) (a process referred to in the art as "veneering"). In the present invention, humanized antibodies include both "humanized" or "veneered" antibodies. These methods are described, for example, in Jones
Et al., Nature 321: 522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci. , U. S. A. , 81: 6851-
6855 (1984); Morrison and Oi, Adv. Immunol
． , 44: 65-92 (1988); Verhoeyer et al., Science.
239: 1534-1536 (1988); Padlan, Molec. Imm
un. 28: 489-498 (1991); Padlan, Molec. Imm
unol. 31 (3): 169-217 (1994); and Kettleb.
orough, C.I. A. Et al., Protein Eng. 4 (7): 773-83.
(1991), each of which is incorporated herein by reference.

The phrase “complementarity determining region” refers to the native F of a native immunoglobulin binding site.
It refers to an amino acid sequence that defines both the binding affinity and specificity of the v region. For example,
Chothia et al. Mol. Biol. 196: 901-917 (1987)
); Kabat et al., U.S.P. S. Dept. of Health and Huma
n Services NIH Publication No. 91-324
2 (1991). The phrase "constant region" refers to the portion of the antibody molecule that confers effector functions. In the present invention, mouse constant regions are replaced by human constant regions. The constant region of the humanized antibody of interest is derived from human immunoglobulin. The heavy chain constant region can be selected from any of the five isotypes: α, δ, ε, γ or μ.

One method of humanizing an antibody is to align non-human heavy and light chain sequences with human heavy and light chain sequences, select a non-human framework, and perform such an alignment. Substituting the non-human framework with a human framework based on the following, performing molecular modeling to predict the conformation of the humanized sequence, and comparing with the conformation of the parent antibody.
Following this process, until the predicted conformation of the humanized sequence model closely approximates that of the non-human CDRs of the parent non-human antibody,
Repeat mutations of residues in the CDR regions that interfere with the structure of the CDRs. Such humanized antibodies can be further derivatized, eg, to facilitate uptake and clearance via the Ashwell receptor. For example, US Pat. No. 5,53
See 0,101 and 5,585,089, which patents are incorporated herein by reference.

Humanized antibodies can also be produced using transgenic animals that have been engineered to contain human immunoglobulin loci. For example, WO98 / 248
93 discloses a transgenic animal having a human Ig locus, wherein
These animals do not produce functional endogenous immunoglobulins due to inactivation of the endogenous heavy and light chain loci. WO 91/10741 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response against an immunogen, wherein these antibodies have primate constant and / or variable regions, And the endogenous immunoglobulin-encoding loci have been replaced or inactivated. WO96 / 30498 describes a Cre / Lox system for modifying immunoglobulin loci in mammals (eg, substituting all or part of a constant or variable region to form a modified antibody molecule). Disclose the use. WO94 /
02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus. US Pat. No. 5,939,598 discloses methods for producing transgenic mice that lack endogenous heavy chains and express exogenous immunoglobulin loci containing one or more heterologous constant regions.

Using the transgenic animals described above, an immune response to the selected antigenic molecule is generated and antibody producing cells are removed from the animal,
It can be used to generate hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, etc. are known in the art, eg W
It is used to immunize transgenic mice as described in O96 / 33735. This publication discloses monoclonal antibodies against various antigenic molecules including IL-6, IL-8, TNF, human CD4, L-selectin, gp39 and tetanus toxin. These monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effects of the corresponding protein. WO96 / 33735 is a monoclonal antibody against IL-8 (IL
(Derived from immune cells of transgenic mice immunized with −8) blocked the IL-8-induced function of neutrophils. Human monoclonal antibodies with specificity for this antigen used to immunize transgenic animals are also disclosed in WO 96/34096.

Polynucleotides or Arrays for Diagnostic Polynucleotide arrays consist of spotting a polynucleotide probe on a substrate (eg glass, nitrocellulose, etc.) in a two-dimensional matrix or array to which the probe is attached. Can be made by. The probe can be attached to the substrate either by covalent bonding or by non-specific interactions (eg, hydrophobic interactions). A sample of polynucleotides can be detectably labeled (eg, using a radioactive or fluorescent label) and then hybridized to a probe. Double-stranded polynucleotides, including the labeled sample polynucleotide bound to the probe polynucleotide, 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 799,897; WO 97/292.
12; WO97 / 27317; EP785,280; WO97 / 02357: US Pat. No. 5,593,839; US Pat. No. 5,578,832; EP728.
, 520; US Pat. No. 5,599,695; EP 721 016; US Pat. No. 5,556,752; WO 95/22058; and US Pat.
No. 734. Arrays can be used, for example, to test the differential expression of genes and to determine gene function. For example, the array can be used to detect the differential expression of polynucleotides between test cells and control cells (eg, between cancer cells and normal cells). For example, high expression of a particular message in cancer cells, which is not observed in the corresponding normal cells, may indicate a cancer-specific gene product. Exemplary uses of arrays are further described, for example, in Papalarado et al., Sem. Radiation Onco
l. (1998) 8: 217; and Ramsay, Nature Biote.
chnol. (1998) 16:40.

Differential Expression in Diagnostic Polynucleotides of the invention can also be used to detect differences in expression levels between two cells, for example as a method to identify abnormal or diseased tissue in humans. Can be used for. For polynucleotides that correspond to protein family profiles, tissue selection can be selected according to putative biochemical functions. Generally, the expression of the gene corresponding to a specific polynucleotide is compared between a human, first suspected diseased tissue, and a second normal tissue. Tissues suspected of being abnormal or diseased can be from different human tissue types, but preferably it is from the same tissue type; for example, intestinal polyps or other abnormal growths are normal. Should be compared to different intestinal tissues. Normal tissue is
The same tissue as the test sample, or any normal tissue of the patient, particularly tissue expressing a polynucleotide-related gene of interest (e.g., brain, thymus, testis, heart, prostate, placenta, spleen, small intestine, skeletal muscle, Mucosal lining of the pancreas and colon).
For example, a difference between a polynucleotide-related gene, mRNA, or protein in two tissues that is compared in molecular weight, amino acid sequence or nucleotide sequence, or relative amount is that the gene in human tissue suspected of being affected, Alternatively, it indicates a change in the gene that regulates this gene. Examples of the detection of differential expression or its use in diagnosing cancer are described in US Pat. Nos. 5,688,641 and 5,677,125.

Genetic predisposition to disease in humans can also be detected by comparing expression levels of mRNA or protein corresponding to the polynucleotides of the invention in fetal tissue to relevant levels in normal fetal tissue. Fetal tissues used for this purpose include, but are not limited to, amniotic fluid, chorionic villi, blood, and blastomere of in vitro fertilized embryos. Comparable normal polynucleotide-related genes can be obtained from any tissue. mRNA or protein is obtained from normal human tissue in which the polynucleotide-related gene is expressed. Differences, such as changes in the nucleotide sequence of the same product of a fetal polynucleotide-related gene or mRNA or in its size, or changes in the molecular weight, amino acid sequence, or relative amount of fetal proteins, can be due to Mutations may be exhibited that indicate a genetic predisposition to the disease. Generally, the diagnostic, prognostic, and other methods of the invention based on differential expression are suspected of having, or suffering from, a disease (eg, breast cancer, lung cancer, colon cancer, and / or metastatic forms thereof). Detecting the level or amount of gene products (particularly differentially expressed gene products) in test samples obtained from susceptible patients, as well as normal cells (eg, cells substantially free of cancer) and / or
Or comparing the levels found in other control cells with the levels detected (eg, to distinguish cancer cells from cells afflicted with dysplasia). In addition, the severity of the disease is detected in samples exhibiting detected levels of differentially expressed gene products and levels of differential gene products associated with diseases of varying degrees of severity. Can be evaluated by comparing It should be noted that the use of the term "diagnosis" herein is not necessarily meant to exclude "prognostic" or "prognosis", but rather for convenience.

The term “differentially expressed gene” generally refers to, for example, a gene product (eg,
An open reading frame encoding a polypeptide and / or introns of such genes and adjacent 5'and 3'non-coding nucleotide sequences (up to about 20 kb beyond the coding region) involved in the regulation of expression. Is likely to include more than one in either direction). The gene may be introduced into a suitable vector for extrachromosomal maintenance or for integration into the host genome. Generally, at least about 25%, usually at least about 50% to 75%, more usually at least about 90%.
Differences in expression levels associated with these reductions in expression levels are differentially expressed genes of interest (ie, genes that are under-expressed or down-regulated in test samples compared to control samples). Indicates. Further, the difference in expression levels associated with increased expression by at least about 25%, usually at least about 50% to 75%, more usually at least about 90% compared to control samples is at least about 1 and 1/2. Fold, usually at least 2 fold to about 10 fold, and can be about 100 fold to about 1,000 fold increase, which is differentially expressed gene of interest (ie, overexpressed). Or upregulated genes).

As used herein, a “differentially expressed polynucleotide” is
By a nucleic acid molecule (RNA or DNA) comprising a sequence representing a differentially expressed gene, for example, a differentially expressed polynucleotide is the detection of a differentially expressed polynucleotide in a sample by It includes a sequence that uniquely identifies the differentially expressed gene (eg, an open reading frame encoding a gene product), as correlated with the presence of the differentially expressed gene in the sample. "
"Differentially expressed polynucleotides" also include fragments of the disclosed polynucleotides (eg, fragments that retain biological activity), as well as homologous, substantially similar or substantially homologous to the disclosed polynucleotides. To be homologous (eg, having about 90% sequence identity).

As used herein, “diagnosis” generally refers to the determination of a patient's susceptibility to a disease or disorder, a determination as to whether the subject is currently suffering from the disease or disorder, And a decision regarding the prognosis of a subject suffering from a disease or disorder (
For example, identification of the pre-metastatic or metastatic cancer state, the stage of the cancer, or the responsiveness of the cancer to treatment). The invention is particularly directed to breast cancer (eg, carcinoma in situ (eg, 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 (eg, small cell carcinoma, non-small cell carcinoma, mesothelioma,
And other forms and / or stages of lung cancer), as well as the diagnosis of subjects in the context of colon cancer (eg, adenomatous polyps, colorectal cancer, and other forms and / or stages of colon cancer).

As used throughout this specification, “sample” or “biological sample” generally refers to a sample of biological fluid or tissue (especially a sample obtained from tissue, particularly a disease for which a diagnostic application is designed. (For example, a sample derived from a cell of a type related to ductal carcinoma). “Sample” is also meant to include the derivatives and fractions (eg, cell lysates) of such a sample. If the sample is solid tissue, cells of this tissue can be dissociated or tissue sections can be analyzed.

The methods of the invention useful in diagnosis or prognosis are typically performed by comparing the amount of a differentially expressed gene product of choice in a sample of interest to that of a control to obtain the gene product. Comprising the step of determining any relative difference in the expression of, which difference may be qualitatively and / or quantitatively measured. The quantification is
For example, it can be achieved by comparing the level of expression product detected in the sample with the amount of product present in the calibration curve. Comparisons are made visually; by using techniques such as densitometry with or without computerized assistance; preparing a representative library of cDNA clones of mRNA isolated from test samples. , Sequence the clones in the library to determine the number of cDNA clones corresponding to the same gene product, and compare the number of clones of the same gene product to the number of clones of the same gene product in the control sample. By analyzing the numbers; or using the array to detect the relative level of hybridization to a selected sequence or set of sequences and comparing this hybridization pattern to that of a control. Can be done by Differences in expression are then correlated with the presence or absence of aberrant expression patterns. A variety of different methods for determining the amount of nucleic acid in a sample are known to those of skill in the art (eg,
See WO 97/27317). In general, the diagnostic assays of the invention involve detection of the gene product of a polynucleotide sequence (eg, mRNA or polypeptide) corresponding to the sequences of SEQ ID NOs: 1-3351. The patient from whom the sample is obtained may be clearly healthy, may be predisposed to disease (as determined, for example, by family history or exposure to certain environmental factors), or changes in the gene product of the invention. Expression may have been previously identified as having a related condition.

Diagnosis is a gene encoded by at least one, preferably at least two or more, at least three or more, or at least four or more polynucleotides having the sequences set forth in SEQ ID NOS: 1-3351. Can be determined based on the detected expression level of the gene product of the product, and SEQ ID NOs: 1-3351
And / or the detection of the expression of a gene corresponding to an additional diagnostic marker and / or an additional sequence that may function as a reference sequence. When the diagnostic method is designed to detect the presence of cancer or the susceptibility of a patient to cancer, the assay is preferably encoded by the gene corresponding to the polynucleotide differentially expressed in the cancer. Includes detection of gene products. Examples of such differentially expressed polynucleotides are described in the Examples below. Given the information provided on the polynucleotides provided and their relative expression levels provided herein, assays using the detection of such polynucleotides and their expression levels in diagnosis and prognosis would be , Will be readily apparent to one of ordinary skill in the art.

Any of a variety of detectable labels can be used in connection with various embodiments of the diagnostic methods of the present invention. Suitable detectable labels include fluorescent dyes (eg fluorescein isothiocyanate (FITC), rhodamine, Texas red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM).
) 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxyfluorescein, 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 label (eg ³² P, ³⁵ S
, ³ H) and the like. The detectable label can include a two-step system (eg, biotin-avidin, hapten-anti-hapten antibody, etc.).

Reagents specific to the polynucleotides and polypeptides of the invention (eg, antibodies and nucleotide probes) can be supplied in kits for detecting the presence of expression products in a biological sample. The kit can also include a buffer or labeling component, and instructions for using the reagents to detect and quantify the expression product in the biological sample. Exemplary embodiments of the diagnostic method of the present invention are described in more detail below.

Polypeptide Detection in Diagnostic In one embodiment, test samples are assayed for the level of differentially expressed polypeptide. Diagnosis can be accomplished using any of a number of methods for determining the absence or presence, or altered amount, of a differentially expressed polypeptide in a test sample.
For example, detection can utilize staining of cells or histological sections with labeled antibodies, which is performed according to conventional methods. Cells can be permeabilized to stain cytoplasmic molecules. In general, an antibody that specifically binds the differentially expressed polypeptide of the invention is added to the sample and incubated for a period sufficient to allow binding to the epitope, usually at least about 10 minutes. It The antibody may be detectably labeled for direct detection (eg, using a radioisotope, an enzyme, a fluorophore, a chemiluminescer, etc.) or a second step for detecting binding. It can be used with antibodies or reagents (eg biotin and horseradish peroxidase conjugated avidin, secondary antibodies conjugated to fluorescent compounds (eg 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 and the like.
Any suitable alternative method of qualitative or quantitative detection of the level or amount of differentially expressed polypeptide (eg, ELISA, Western blot, immunoprecipitation,
Radioimmunoassays, etc.) can be used.

(Detection of mRNA) The diagnostic method of the present invention may also, or alternatively, be an mRNA encoded by a gene corresponding to the differentially expressed polynucleotide of the present invention.
The detection of NA may be included. Any suitable qualitative or quantitative method known in the art for detecting specific mRNA can be used. mRNA is
For example, it can be detected by 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 detect differences in the size or amount of mRNA transcripts between two samples. The mRNA expression level in a sample can be determined by making a library of expressed sequence tags (ESTs) from the sample, where the EST library is representative of the sequences present in the sample (Adams et al. (1991). Science 25
2: 1651). A listing of the relative representations of ESTs in the library can be used to approximate the relative representations of gene transcripts in the starting sample. The results of the EST analysis of the test sample are then compared to the EST analysis of the reference sample to correspond to the selected polynucleotide, particularly one or more differentially expressed genes described herein. The relative expression level of the polynucleotide can be determined. Alternatively, gene expression in the test sample can be determined by serial analysis of gene expression (SAGE
) Methodology (eg, Velculescu et al., Science (1995) 27.
0: 484) or differential display (DD) methodology (eg,
See US Pat. No. 5,776,683; and US Pat. No. 5,807,680).

Alternatively, gene expression can be analyzed using hybridization analysis. Oligonucleotides or cDNAs are DNA or R of specific sequence composition.
In order to selectively identify or capture NA, and the amount of RNA or cDNA that hybridizes to a known qualitatively or quantitatively determined capture sequence, a specific message within a pool of cellular messages in a sample It can be used to provide information about relative displays. Hybridization analysis uses, for example, array-based techniques with high density formats (including filters, microscope slides, or microchips) or solution-based techniques with spectroscopic analysis (eg, mass spectrometry). Can be designed to allow simultaneous screening of relative expression of hundreds to thousands of genes. One exemplary use of arrays in the diagnostic methods of the invention is described in more detail below.

Use of Single Genes in Diagnostic Applications The diagnostic methods of the invention can focus on the expression of a single differentially expressed gene. For example, a diagnostic method can include detecting a differentially expressed gene associated with a disease, or a polymorphism in such a gene (eg, a polymorphism in the coding or control regions). Disease-related polymorphisms may include mutations such as gene deletions or truncations, altered expression levels of the encoded protein and / or affecting the activity of the encoded protein.

Many methods are available for analyzing nucleic acids for the presence of particular sequences (eg, disease-associated polymorphisms). When large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into an appropriate vector and grown in sufficient quantity for analysis. Cells expressing the differentially expressed gene can be used as a source of mRNA, which can be directly assayed 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 a sufficient amount for analysis, and a detectable label is added in the amplification reaction to facilitate detection. Can be included (eg, using detectably labeled primers or detectably labeled oligonucleotides). Alternatively, various methods that utilize oligonucleotide ligation as a means of detecting polymorphisms are also known in the art. See, for example, Riley et al., Nucl. Acids Re
s. (1990) 18: 2887; and Delahunty et al., Am. J. H
um. Genet. (1996) 58: 1239.

Amplified or cloned sample nucleic acid can be analyzed by one of many methods known in the art. Nucleic acids can be sequenced by the dideoxy method or other methods, and the sequence of bases can be compared to a selected sequence (eg, wild type sequence). Hybridization with a polymorphic or variant sequence can also be used to determine its presence in a sample (eg, by Southern blot, dot blot, etc.). US Patent No. 5,445,9
No. 34 or WO 95/35505, hybridization patterns of polymorphic or variant sequences and control sequences to an array of oligonucleotide probes immobilized on a solid support are also associated with disease. It can be used as a means to identify polymorphic or variant sequences. Single-stranded conformational polymorphism (SSCP) analysis in gel matrix, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis showed D as a change in electrophoretic mobility.
Used to detect conformational changes created by NA sequence variations. Alternatively, if the polymorphism creates or destroys a recognition site for a restriction endonuclease, the sample is digested with that endonuclease and the product is size fractionated to determine if the fragment was digested. . Fractionation is performed by gel electrophoresis or capillary electrophoresis (especially acrylamide gel or agarose gel).

Screening for mutations in a gene may be based on the functional or antigenic properties of the protein. Protein shortening assays are useful in detecting deletions that can affect the biological activity of proteins. A variety of immunoassays designed to detect polymorphisms in proteins can be used in the screen. Functional protein assays have proven to be effective screening tools when many different genetic mutations lead to specific disease phenotypes. The activity of the encoded protein can be determined by comparison with the wild type protein.

Pattern Matching in Diagnostic Using Arrays In another embodiment, the diagnostic and / or prognostic method of the present invention involves the generation of a test expression pattern (TEP) of genes in a test sample. Includes detection of expression for a selected set. T
The EP is compared to a reference expression pattern (REP) generated by detecting the expression of a selected set of genes in a reference sample (eg, positive control sample or negative control sample). The selected set of genes comprises at least one of the genes of the invention, these genes corresponding to the polynucleotide sequences of SEQ ID NOs: 1-3351. Of particular importance is a selected set of genes, including genes that are differentially expressed in the disease for which the test sample is being screened.

As used herein with respect to differential gene expression analysis and diagnosis / prognosis, a “reference sequence” or “reference polynucleotide” refers to a selected set of polynucleotides, which has been selected. The set comprises at least one or more of the differentially expressed polynucleotides described herein. Multiple reference sequences may be included as reference sequences, preferably including positive and negative control sequences. A more suitable reference sequence is GenB
found in ank, Unigene, and other nucleotide sequence databases (including, for example, expressed sequence tags (ESTs), partial and full length sequences)
.

“Reference array” means an array having a reference sequence for use in hybridization with a sample, wherein the reference sequence is the differentially expressed polynucleotide described herein. Of all, at least one of them, or any subset of them. Generally, such arrays contain at least three different reference sequences and may contain any one or all of the differentially expressed sequences provided. The array of interest further includes other sequences of interest, particularly for screening for other gene sequences, such as diseases or disorders (eg, cancer, dysplasia, or other related or unrelated diseases, disorders or conditions). ) Sequences (including polymorphisms). The oligonucleotide sequences in this array are typically at least about 12 nt in length, and can be approximately the length of the sequence provided, or can be extended into flanking regions to produce fragments 100 nt to 200 nt or longer. The reference array may be made according to any suitable method known in the art. For example, a method of making large arrays of oligonucleotides is described in US Pat.
, 134,854 and US Pat. No. 5,445,934. Using a computer control system, a heterogeneous array of monomers is converted into a heterogeneous array of polymers via simultaneous coupling at many reaction sites. Alternatively, microarrays are made by deposition of pre-synthesized oligonucleotides on a solid substrate, as described, for example, in PCT published application WO 95/35505.

As used herein, a “reference expression pattern” or “REP” refers to a gene (especially a selected cell type (eg, normal cells, cancer cells, cells exposed to environmental stimuli, etc.)). Relative expression level of a selected set of differentially expressed genes, which correlate with. A "test expression pattern" or "TEP" is a gene (especially a differentially expressed gene) in a test sample (eg, a cell in an unknown disease state or a suspected disease state in which mRNA is isolated). ) Refers to the relative expression level of the selected set.

REPs can be made in various ways, according to methods well known in the art. For example, REP hybridizes a control sample to an array having a selected set of polynucleotides, particularly a selected set of differentially expressed polynucleotides, and acquires hybridization data from the array. , And can be made by storing the data in a format that allows easy comparison of REP and TEP. Alternatively, all expressed sequences in a control sample can be isolated and sequenced, for example, by isolating mRNA from the control sample, converting the mRNA to cDNA, and sequencing the cDNA. The sequence information obtained approximately or accurately reflects the identity and relative number of expressed sequences in the sample. The sequence information can then be stored in a format that allows easy comparison of REP and TEP, such as a computer readable format. REPs can be normalized before or after data storage and / or can be treated to selectively remove sequences of expressed genes that may be less important or complicate analysis (eg, housekeeping). Some or all of the sequences associated with the gene can be excluded from the REP data).

TEP, for example, hybridizes a test sample to an array having a selected set of polynucleotides, particularly a selected set of differentially expressed polynucleotides, and obtains hybridization data from the array. Then
It can then be made in a manner similar to RFP by storing the data in a format that allows easy comparison of TEP and REP. The REP and TEP used in the comparison can be made at the same time, or the TEP can be compared to a previously made and stored REP.

In one embodiment of the invention, comparing TEP and REP comprises hybridizing a test sample with a reference array, where the reference array is for use in hybridization with the sample. With one or more reference sequences. A reference sequence comprises all of the differentially expressed polynucleotides described herein, at least one of them, or any subset thereof. Hybridization data for the test samples were acquired, the data were normalized, and the TEPs generated were generated using an array with the same or similar selected set of differentially expressed polynucleotides. It is compared to the REP made. Probes corresponding to sequences that are differentially expressed between the two samples show reduced or increased hybridization efficiency for one of the samples compared to the other.

Methods for collecting data from hybridization of a sample with a reference array are well known in the art. For example, the reference sample and the test sample polynucleotides can be generated using a detectable fluorescent label, and hybridization of the polynucleotides in the sample can be performed using, for example, a microscope or a light source to illuminate a substrate. And can be detected by scanning the microarray for the presence of detectable label. The photon counter detects the fluorescence from the substrate, while the xy translation.
The stage changes the position of the substrate. Confocal detection devices that can be used in the method of the present invention are described in US Pat. No. 5,631,734. Scanning laser microscopes are described by Shalon et al., Genome Res. (1996) 6
: 639. A scan using the appropriate excitation line is performed for each fluorophore used. Then the digital image created from the scan
Combined for subsequent analysis. For any particular array element,
The ratio of the fluorescent signal from one sample (eg the test sample) is compared to the fluorescent signal from another sample (eg the reference sample) and the relative signal intensities are determined.

Methods for analyzing the data collected from hybridization to arrays are well known in the art. For example, if detection of hybridization involves a fluorescent label, data analysis may include determining fluorescence intensity as a function of substrate position from the collected data, outliers (ie, from a predetermined statistical distribution). Deviating data) and calculating the relative binding affinity of the target from the remaining data. The data obtained can be presented as an image, with intensities in each region varying according to the binding affinity between target and probe.

Generally, a test sample is a TEP made from a test sample that is derived from a reference sample (eg, cancer, a particular stage of cancer, a sample associated with dysplasia, a sample affected by a disease other than cancer, a normal sample). Are classified as having a gene expression profile that corresponds to a gene expression profile associated with a diseased or non-diseased condition by comparison with one or more REPs that are produced. TEP and RE
The criteria for a match, or a substantial match, with P are expression of the same or substantially the same set of reference genes, and substantially the same level (eg, with the reference sequence selected after normalization of the sample). Expression of these reference genes is not significantly different between samples for relevant signals, or a difference in signal intensity for a given reference sequence of at least about 25% to not more than about 40%.
In general, a pattern match between TEP and REP is a match in expression of at least one, all of them, or any subset thereof of the differentially expressed genes of the invention ( Preferably at a qualitative or quantitative expression level).

Pattern matching can be done manually or using computer programs. A method for the preparation of a substrate matrix (eg an array),
Methods for designing oligonucleotides for use with such matrices, methods for labeling probes, methods for hybridization conditions,
Methods for scanning hybridized matrices, and for analysis of the patterns produced (including comparative analysis) are described, for example, in US Pat.
00,992.

Cancer Diagnosis, Prognosis, and Management Polynucleotides and their gene products of the present invention include genetic or biochemical markers (eg, blood or tissue) that detect the earliest changes along the carcinogenic pathway. In) and / or to monitor the efficacy of various therapeutic and prophylactic interventions. For example, the level of expression of a given polynucleotide may be indicative of a poorer prognosis, and thus warrants more aggressive chemotherapy or radiation therapy to the patient and vice versa. Correlation of response and outcome to treatment in patients with novel surrogate tumor-specific characteristics may define prognostic indicators that allow for the design of a therapy tailored based on the tumor's molecular profile. These therapies include antibody targeting and gene therapy.
Determining the expression of a polynucleotide, and comparing the patient's profile to known expression in normal tissue and disease variants, both in terms of treatment specificity and in terms of patient's pain-free level. , Enables the determination of the best possible treatment for a patient. Surrogate tumor markers such as polynucleotide expression can also be used for better classification, and thus for diagnosing and treating different forms of cancer and disease states. Two widely used classes in oncology that may result from the identification of expression levels of the polynucleotides of the invention are staging of cancer disorders and grading of the nature of cancerous tissue.

The polynucleotides of the present invention allow patients with or susceptible to cancer to detect potentially malignant events at the molecular level before they are detectable at the gross morphological level. Can be useful for monitoring. In addition, the polynucleotides of the present invention identified as being important for one type of cancer may also have relevance for the development or risk of developing another type of cancer, eg, where the polynucleotide Is differentially expressed across various cancer types. Thus, for example, expression of a polynucleotide of clinical relevance for metastatic colon cancer may also be
It may have clinical relevance for gastric cancer or endometrial cancer.

Staging. Staging is a process used by physicians to describe how advanced a cancerous condition is in a patient. Generally, if the cancer does not spread to any lymph nodes and can be detected only in the area of the primary lesion, it is referred to as stage I. If the cancer has spread only to the closest lymph nodes, it is called stage II. In stage III, the cancer has generally spread to lymph nodes proximal to the site of the primary lesion. Cancer that has spread to distant parts of the body (eg, liver, bones, brain, or other sites) is stage IV and is the most advanced stage.

The polynucleotides of the invention may facilitate fine tuning of the staging process by identifying markers for cancer aggressiveness (eg, metastatic potential) and presence in different areas of the body. Thus, stage II cancers with polynucleotides exhibiting high metastatic potential cancers can be used to convert borderline stage II tumors into stage III tumors, justifying more aggressive treatments. Conversely, the presence of polynucleotides with a lower metastatic potential allows for a more conservative tumor staging.

Cancer grading. Grade is a term used to describe how closely a tumor resembles the same type of normal tissue. The appearance of the tumor under a microscope is
It is used to identify tumor grade based on parameters such as cell morphology, cell composition, and other markers of differentiation. As a general rule, a tumor's grade corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors being more aggressive than well-differentiated or low-grade tumors. Is. The following guidelines are generally used to grade tumors: 1) GX grade cannot be assessed; 2) G1 well differentiated; G2 moderately well differentiated 3) G3 poorly differentiated; 4) G4 undifferentiated. Not only can the polynucleotides of the present invention help this to determine the state of differentiation of tumor cells, but they are also valuable in determining tumor aggression (eg, metastatic potential). It may be of particular value in determining the grade of a tumor, since factors other than can be identified.

Detection of lung cancer. The polynucleotides of the present invention can be used to detect lung cancer in a subject. Although there are more than a dozen different types of lung cancer, the two major types of lung cancer are small cell carcinoma and non-small cell carcinoma, which cover approximately 90% of all lung cancer cases. . Small cell carcinoma (also called oat cell carcinoma) usually begins in one of the larger bronchi, grows very quickly, and tends to grow by the time of diagnosis. Non-small cell lung cancer (NSCLC) is composed of three common subtypes of lung cancer. Epidermoid carcinoma (also called squamous cell carcinoma) usually starts in one of the larger bronchi and grows relatively slowly. The size of these tumors can range from very small to very large. Adenocarcinoma begins to grow near the outer surface of the lung and can vary in both size and growth rate. Some slowly growing adenocarcinomas are described as alveolar cell carcinomas. Large cell carcinoma begins near the surface of the lung, grows rapidly, and when diagnosed, the growth is usually very large. Other less common forms of lung cancer are carcinoids, cylinders, mucoepidermoid carcinomas, and malignant mesothelioma.

Polynucleotides of the invention (eg, normal cells versus polynucleotides that are differentially expressed in cancerous lung cells (eg, high or low metastatic tumor cells), or cancerous. Polynucleotides that are differentially expressed between lung cell types (eg, highly metastatic vs. low metastatic), to distinguish lung cancer types,
And can be used to identify cancer-specific characteristics of a patient and to select an appropriate treatment. For example, if a patient's biopsy expresses a polynucleotide associated with low metastatic potential, this may justify leaving a relatively large portion of the patient's lungs in surgery to remove the lesion. Alternatively, relatively small lesions with expression of polynucleotides associated with high metastatic potential are present in lung tissue and / or surrounding lymph nodes even if metastases cannot be identified through pathological examination. May justify a more radical removal of.

Detection of breast cancer. The majority of breast cancers are subtypes of adenocarcinoma, which can be summarized as follows: 1) ductal carcinoma in situ (DCIS) (including comedone cancer); 2) invasive (or invasive). Ductal carcinoma (IDC); 3) Lobular carcinoma in situ (LCIS); 4) Lobular (or invasive) lobular carcinoma (ILC); 5) Inflammatory breast cancer; 6) Medullary carcinoma; 7) Mucinous carcinoma; 8) Paget's disease of the nipple; 9) lobular tumor, and 10) tubular adenocarcinoma.

Expression of the polynucleotides of the invention can be used in the diagnosis and management of breast cancer and to distinguish between breast cancer types. Breast cancer detection can be determined using expression levels of any suitable polynucleotide of the invention, either alone or in combination. Determining the aggressive nature and / or metastatic potential of breast cancer also compares the levels of one or more polynucleotides of the invention and the levels of other sequences known to be altered in cancerous tissues (eg, ER expression) can be determined by comparison. In addition, the development of breast cancer is differentially expressed by the ratio of expression of differentially expressed polynucleotides to the level of steroid hormones (eg, testosterone or estrogen), or differentially to other hormones (eg, growth hormone, insulin). It can be detected by examining the rate of expression of the expressed polynucleotide. Therefore, expression of a particular marker polynucleotide distinguishes between normal and cancerous breast tissue,
It can be used to distinguish between breast cancers with cells of different origin, such as between breast cancers with different potential metastatic rates.

Detection of colon cancer. Polynucleotides of the invention that exhibit the appropriate expression pattern can be used to detect colon cancer in a subject. Colorectal cancer is one of the most common neoplasms in humans, and is probably the most frequent form of hereditary neoplasm. Prevention and early detection are important factors in controlling and curing colorectal cancer. Colorectal cancer begins as polyps, which are small, benign outgrowths of cells that form in the lining of the colon. Over a period of several years, some of these polyps accumulate additional mutations and become cancerous. Multiple familial colorectal cancer disorders have been identified and are summarized as follows: 1) familial adenomatous polyposis (FAP); 2) Gardner's syndrome; 3) hereditary non-polyposis colon cancer (HNPCC). And 4) Familial colorectal cancer in Ashkenazi Jews. Expression of the appropriate polynucleotides of the invention can be used in the diagnosis, prognosis, and management of colorectal cancer. Detection of colon cancer can be determined using the expression level of any of these sequences, either alone or in combination with the level of expression. Determining the aggressive nature and / or metastatic potential of colon cancer involves comparing the levels of one or more polynucleotides of the invention and the total level of another sequence known to be altered in cancer tissue (eg, p53, DCC ras, lo
r FAP expression) can be determined by comparison (eg, Fear).
on ER et al., Cell (1990) 61 (5): 759; Hamilton.
SR et al., Cancer (1993) 72: 957; Bodmer W et al., Nat.
Genet. (1994) 4 (3): 217; Fearon ER, Ann.
NY Acad Sci. (1995) 768: 101). For example, the development of colon cancer can be detected by testing the ratio of either the polynucleotide of the invention to the level of an oncogene (eg, ras) or tumor suppressor gene (eg, FAP or p53). Therefore, the expression of a particular marker polynucleotide distinguishes between normal and cancerous colon tissue,
It can be used to distinguish between colon cancers with different origins of cells, such as between colon cancers with different potential metastatic rates.

Use of Polynucleotides to Screen for Peptide Analogs and Antagonists Polypeptides encoded by the polynucleotides of the present invention and the corresponding full-length genes may be labeled as receptors among the encoded polypeptides. It can be used to screen peptide libraries to identify binding partners. Peptide libraries can be synthesized according to methods known in the art (eg, US Pat. No. 5,010,175 and WO 91/178).
23). Agonists or antagonists of the polypeptides of the invention can be screened using any available method known in the art such as signal transduction, antibody binding, receptor binding, mitogen assay, chemotaxis assay and the like. obtain. Assay conditions should ideally be similar to those in which native activity is shown in vivo, ie under physiological pH, temperature, and ionic strength. Suitable agonists or antagonists exhibit potent inhibition or enhancement of 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 may require concentrations equal to or greater than the native concentration, while inhibitors capable of irreversibly binding the polypeptide are nearly native. It can be added at a concentration as high as about 100%.

Such screens and experiments can be performed with novel polypeptide binding partners, such as receptors, encoded by a gene or cDNA corresponding to a polynucleotide of the invention, and at least one peptide of the novel binding partner. It may lead to the identification of agonists or antagonists. Such agonists and antagonists can be used to modulate, enhance, or inhibit receptor function in cells where the receptor is native or in cells which retain the receptor as a result of genetic engineering. Furthermore, where the novel receptor shares biologically significant characteristics with known receptors, information on agonist / antagonist binding can be obtained by improving the agonist / antagonist properties of known receptors.
It may facilitate the development of antagonists.

Pharmaceutical Compositions and Therapeutic Uses Pharmaceutical compositions of the invention include a polypeptide, antibody, or polynucleotide of the invention (antisense nucleotides and ribozymes in a therapeutically effective amount. ) May be included. As used herein, the term "therapeutically effective amount"
Refers to the amount of therapeutic agent to treat, reduce, or prevent the desired disease or condition, or to exhibit a detectable therapeutic or prophylactic effect. Effects can be detected by, for example, chemical markers or antigen levels. The therapeutic effect is also
Includes reductions in physical symptoms such as decreased body temperature. The precise effective amount for a subject will depend on the subject's size and health, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. Therefore, it is not useful to pre-determine the exact effective amount. However, the effective dose for a given situation is
It will be determined by routine experimentation and is within the judgment of the clinician. For purposes of this invention, an effective dose will generally be about 0.0 in the individual to whom it is administered.
1 mg / kg to 50 mg / kg, or 0.05 mg / kg to about 10 mg / kg
DNA construct of.

The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to therapeutic agents such as antibodies or polypeptides, genes,
And other carriers for the administration of 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 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 carriers in therapeutic compositions may include liquids such as water, saline, glycerol and ethanol. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like, may also be present in such vehicles. Typically, the therapeutic composition is
Injectables, either prepared as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of pharmaceutically acceptable carriers. Pharmaceutically acceptable salts may also be present in pharmaceutical compositions (eg mineral salts such as hydrogen chloride, hydrobromide, phosphate, sulphate, etc .; and acetate, propione). Acid salt,
Salts of organic acids such as malonates, benzoates, etc.). A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceuticals.
Sciences (Mack Pub. Co., New Joursey 1
991).

Methods of Delivery Once formulated, the compositions of the invention can (1) be administered directly to the subject (eg, as a polynucleotide or polypeptide); or (2) the subject. It can be delivered ex vivo to cells from the body (eg, as in ex vivo gene therapy). Direct delivery of the compositions is generally accomplished by parenteral injection (eg, subcutaneous, intraperitoneal, intravenous or intramuscular, intratumoral, or intrathecal tissue injection). Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hypospray.
Osspray). Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and are described, for example, in International Publication No. WO93 / 1477.
No. 8 is described. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoietic stem cells, lymphoid cells, macrophages, dendritic cells, or tumor cells. In general, delivery of nucleic acids for ex vivo and in vitro applications includes, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion,
Electrophoresis, encapsulation of polynucleotides in liposomes, and DNA into the nucleus
Can be achieved by direct microinjection, all well known in the art.

Once the gene corresponding to a polynucleotide of the invention is found to correlate with proliferative disorders such as neoplasia, dysplasia, and hyperplasia, the disorder is provided by the provided polynucleotide, The corresponding polypeptide, or other corresponding molecule (
For example, it is more responsive to treatment by administration of therapeutic agents based on antisense, ribozymes, etc.).

The dosage and means of administration of the pharmaceutical compositions of the present invention are based on the specific quality of the therapeutic composition, the condition, age and weight of the patient, disease progression, and other relevant factors. It is determined. For example, administration of the polynucleotide therapeutic composition agents of the present invention includes local or systemic administration, including injection, oral administration, particle gun (par).
single gun), or administration by catheterization, and local administration. Preferably, the therapeutic polynucleotide composition contains a promoter operably linked to at least 12, 22, 25, 30, or 35 contiguous nt polynucleotides of the polynucleotides disclosed herein. Expression constructs. Various methods can be 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 is injected several times at several different locations within the body of the tumor. Alternatively, the artery supplying the tumor is identified and the therapeutic composition is injected into such artery to deliver the composition directly to the tumor. Tumors with necrotic centers are aspirated, and the composition is injected directly into the now empty center of the tumor. The antisense composition is administered directly to the surface of the tumor, for example by topical application of the composition. X-ray imaging is used to aid in some of the above delivery methods.

Receptor-mediated targeted delivery of therapeutic compositions containing antisense polynucleotides, subgenomic polynucleotides, or antibodies to specific tissues can also be used. Receptor-mediated DNA delivery techniques are described, for example, in Finde.
is et al., Trends Biotechnol. (1993) 11: 202; C
hiou et al., Gene Therapeutics: Methods Ands.
Applications Of Direct Gene Transfer
(JA Wolff Ed.) (1994); Wu et al. Biol. Chem
． (1988) 263: 621; Wu et al., J. Am. Biol. Chem. (19
94) 269: 542; Zenke et al., Proc. Natl. Acad.
Sci. (USA) (1990) 87: 3655; Wu et al. Biol.
Chem. (1991) 266: 338. Therapeutic compositions containing a polynucleotide are provided for topical administration in gene therapy protocols,
Doses range from about 100 ng to about 200 mg DNA. DNA concentration ranges of about 500 ng to about 50 mg, about 1 mg to about 2 mg, about 5 mg to about 500 g, and about 20 mg to about 100 mg can also be used during gene therapy protocols. Factors such as methods of action (eg, to enhance or inhibit the level of the encoded gene product), transformation and expression potency are necessary for the ultimate potency of the antisense subgenomic polynucleotide. It is a matter of concern that affects the doses taken. If better expression is desired over a larger area of tissue, a higher amount of antisense subgenomic polynucleotide, or the same amount re-administered in a continuous protocol of administration, or, for example, at different tumor sites Multiple doses to adjacent or near tissue parts may be required to achieve positive therapeutic results. In all cases, routine experimentation in clinical trials will determine the specific range for optimal therapeutic effect. Suitable uses, doses and administrations for polynucleotide-related genes encoding polypeptides or proteins having anti-inflammatory activity are described in US Pat. No. 5,654.
, 173.

The therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (generally Jolly, Cancer Gene Thera.
py (1994) 1:51; Kimura, Human Gene Thera.
py (1994) 5: 845; Connelly, Human Gene Th.
erase (1995) 1: 815; and Kaplitt, Nature G.
Enetics (1994) 6: 148). 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.

Viral-based vectors for delivery of desired polynucleotides and expression in desired cells are well known in the art. Exemplary virus-based vehicles include recombinant retroviruses (eg, WO90 / 07936;
WO94 / 03622; WO93 / 25698; WO93 / 25234; US Patent No. 5,219,740; WO93 / 11230; WO93 / 10218; US Patent No. 4,777,127; British Patent No. 2,200,651; EP 0
345 242; and WO 91/02805), alphavirus-based vectors (eg, Sindbis virus vector, Semliki Forest virus (see
ATCC VR-67; ATCC VR-1247), Ross River virus (A
TCC VR-373; ATCC VR-1246), and Venezuelan Equine Encephalitis Virus (ATCC VR-923; ATCC VR-1250; ATCC).
VR 1249; ATCC VR-532), and adeno-associated virus (AA).
V) vectors (eg WO94 / 12649, WO93 / 03769; WO9
3/19191; WO94 / 28938; WO95 / 11984, and WO9.
5/00655), but is not limited thereto. Cur
iel. Hum. Gene Ther. (1992) 3: 147, administration of DNA linked to a killed adenovirus can also be used.

Non-viral delivery vehicles and methods can also be used. This method includes, but is not limited to, polycationic condensed DNA (eg Cu, linked only to killed adenovirus or unlinked).
riel, Hum. Gene Ther. (1992) 3: 147); ligand-ligated DNA (eg, Wu, J. Biol. Chem. 264: 1).
6985 (1989)); eukaryotic delivery vehicle cells (eg, US Pat. No. 5,814,482; WO 95/07994; WO 96/170).
72; WO 95/30763; and WO 97/42338).
, And nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA transfer methods are described in WO 90/11092 and US Pat. No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in US Pat. No. 5,422,120; WO 95/13796; W.
O 94/23697; WO 91/14445; and European patent 052496.
8 are described. A further approach is described in Philip, Mol. Cell
Biol. 14: 2411 (1994), and Woffendin, Pro.
c. Natl. Acad. Sci. (1994) 91: 1581.

Additional non-viral delivery suitable for use includes Woffendin et al., P.
roc. Natl. Acad. Sci. USA 91 (24): 11581 (1
Mechanical delivery systems, such as the approach described in 994). In addition, the coding sequences and products of such expression can be delivered through the deposition of photopolymerized hydrogel materials, or the use of ionizing radiation (US Pat. No. 5,206,152).
Issue and WO 92/11033). Other conventional methods for gene delivery that may be used for delivery of coding sequences include, for example, the hand-hold gene transfer particle gun.
Legun) (see, eg, US Pat. No. 5,149,655); use of ionizing radiation to activate transferred genes (eg, US Pat. No. 5,206,152 and WO 92/11033).

The invention will now be illustrated with reference to the following examples, which describe particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as limiting the invention in any way.

Example 1 Example 1 Source of Novel Polynucleotide Biological Material Expressed by Biological Material and Overview of This Polynucleotide Cell line and human normal and tumor tissues Was used to construct a cDNA library from mRNA isolated from cells and tissues. Most sequences were approximately 275-300 nucleotides in length. This cell line is Km12L
4-A cell line, highly metastatic colon cancer cell line (Morika, WAK, et al., Ca
ncer Research (1988) 48: 6863). KM
The 12L4-A cell line is delivered from the KM12C cell line. KM12C cell line (
Lack of metastasis (low metastasis) was established in cultures from Dukes' stage B2 surgical preparations (Morikawa et al. Cancer Res. (1988) 4
8: 6863). KML4-A is a highly metastatic substrain derived from KM12C (Ye.
atman et al., Nucl. Acids. Res. (1995) 23: 4007;
Bao-Ling et al., Proc. Annu. Meet. Am. Assoc. Ca
ncer. Res. (1995) 21: 3269). KM12C and KM12
C-derived cell lines (eg, KM12L4, KM12L4-A, etc.) are well recognized in the art as model cell lines for the study of colon cancer (eg, Morikawa et al., Supra; Radinsky et al., Clin. Cancel
r Res. (1995) 1:19; Yeatman et al. (1995) supra; Ye.
atman et al., Clin. Exp. Metastasis (1996) 14: 2.
46). These and other cell lines and tissues are listed in Table 6.

The sequence of the isolated polynucleotide is first masked using the XBLAST masking program to eliminate low complexity sequences (Claverie “Eff.
active Large-Scale Sequence Similari
ty Searches ": Computer Methods for Ma
cromolecular Sequence Analysis, Dooli
Title edited by Meth. Enzymol. 266: 212-227 Acade
mic Press, NY, NY (1996); especially Claverie, “Au
tomato DNA Sequencing and Analysis
Techniques "Adams et al., Ed., Chap. 36, p. 267 Aca
demic Press, San Diego, 1994 and Claveri
e et al., Comput. Chem. (1993) 17: 191). In general, masks, due to their low complexity, have multiple “hi” s based on related small target sequence exclusions, similarity to repeat regions common to multiple sequences (eg, Alu repeats).
It does not affect the final search results, except for the removal of "t". The sequences remaining after masking are then labeled with BLASTN vs. Used for Genbank search. Sequences that showed greater than 70% duplication, 99% identity, and p-value less than 1 × 10 ⁻⁴⁰ were discarded in this search. The sequences from this search also matched the global parameters, but were discarded if the sequences were from ribosomes or vectors.

The sequences obtained by the above search were classified into three groups (shown below, 1, 2 and 3), and BLASTX vs. Searched by NRP (non-redundant protein) database search: (1) unknown (not applicable by Genbank search), (2) weak similarity (identity greater than 45% and p-value 1 × 1).
0 less than ^-5 is), and (3) high similarity (greater overlap 60%, greater than 80% identity, and 1 × 10 p value of less than ^-5). Over 70% overlap, 99
Sequences with greater than% identity and p-value less than 1 × 10 ⁻⁴⁰ were discarded.

The remaining sequences were classified as unknown (not applicable), weak similarity and high similarity (parameters above). Two searches were performed on these sequences. at first,
BLAST vs. An EST database search was performed and sequences with greater than 99% overlap, greater than 99% similarity, and p-value less than 1 × 10 ⁻⁴⁰ were discarded. P less than 1 × 10 ⁻⁶⁵ when compared to database sequences of human origin
Sequences with values were also removed. Second, BLASTN vs. Patent G
Implementing the eneSeq database and greater than 99% identity, 1x1
Sequences with p-values less than ^0-40 and greater than 99% duplication were discarded.

The remaining sequences were screened using other rules and redundancy in the dataset. A sequence having a p-value less than 1 × 10 ^{-1 11} in relation to database sequences of human origin, in particular eliminated. Finally, 3351 sequences listed in the attached sequence listing were obtained. Each identified polynucleotide represents a sequence from at least some mRNA transcripts. The polynucleotides determined to be new have been assigned the SEQ ID NOs.

The new polynucleotides have been assigned SEQ ID NOS: 1-3351. The first 1847 DNA sequences corresponding to the new polynucleotides are shown in the sequence listing in Table 1. DNAs corresponding to the novel polynucleotides of SEQ ID NOs: 1848-3351
The sequences are shown in the sequence listing in Table 2. The DNA sequences in Table 2 (numbered SEQ ID NOS: 1-1504) correspond to SEQ ID NOS: 1848-3351 in the Sequence Listing. For example,
Table 2 SEQ ID NO: 2 is SEQ ID NO: 1848, Table 2 SEQ ID NO: 2 is SEQ ID NO: 18
49, and so on. Each DNA sequence in Table 4 is 184 based on its sequence number in the sequence listing.
7 Uniquely identified by a small number. Tables 1 and 2 show: 1) the SEQ ID NO assigned to each sequence, or the corresponding number for use herein; 2) the sequence used as the internal identifier of this sequence. Name; 3) the name assigned to the clone (from which the sequence was isolated); and 4) the number of clusters (to which the sequence was assigned) (cluster ID; if cluster ID is 0, this sequence is Not assigned to any cluster).

Since the provided polynucleotides represent partial mRNA transcripts, two or more polynucleotides of the invention may represent the same mRNA transcript and different regions of the same gene. Thus, if two or more SEQ ID NOs are identified as belonging to the same clone, either sequence can be used to obtain the full length mRNA or gene.

Example 2: Results of a public database search to identify the function of the gene product SEQ ID NOS: 1-3351 were used in all three reading frames to determine the best alignment of the individual sequences. translated. These amino acid and nucleotide sequences are commonly referred to as query sequences, which are arranged against the individual sequences. Queries and individual sequences can be found at http: // www. ncbi. nlm.
nih. Aligned using the BLAST program available over the World Wide Web at gov / BLAST /. Again, this sequence was masked against the various regions that interfered with the search for repetitive or polyA sequences using the XBLAST program for masking low complexity described in Example 1 above.

Tables 3 and 4 (inserted before the claims) show the alignment results. Table 3 shows
Includes alignment information for SEQ ID NOs: 1-1847, and Table 4 lists SEQ ID NOs: 1848-3.
351 alignment information. The DNA sequences in Table 4 (numbered SEQ ID NOS: 1-1504) correspond to SEQ ID NOS: 1848-3351. Each DNA sequence in Table 4 is uniquely identified with a number 1847 less than its SEQ ID NO. Tables 3 and 4 show each sequence by its SEQ ID NO or the corresponding number, the closest and explanation from the Genbank and Non-Redundant Protein searches, and the p-value search results.

The optimal alignment for the protein or DNA sequences for each of SEQ ID NOs: 1-1847 is included in Table 3, and the optimal alignment for each of SEQ ID NOs: 1848-3351 is included in Table 4. The activity of the polypeptides encoded by SEQ ID NOs: 1-3351 is the same as or similar to the most closely related ones reported in Table 3 or 4. The closest relative's accession number has been reported and provides a reference to the activity exhibited by this closest relative. The search programs and databases used for this alignment also show the calculation of p-values.

The full-length sequences or fragments of the closest polynucleotide sequences can be used as probes and primers to identify and isolate full-length sequences of SEQ ID NOs: 1-3351. The closest one may indicate the tissue or cell type to be used to construct the library for the full length sequence of SEQ ID NOs: 1-3351.

Example 3 Protein Family Members The sequences (SEQ ID NOs: 1-3351) were used to perform profile searches as described herein above. Some of the polynucleotides of the invention have the characteristics of polypeptides belonging to known protein families (thus indicating novel members of these protein families) and / or contain known functional domains. , Was found to encode a polypeptide (Table 5).
. "Start" and "Stop" in Table 3 indicate the position within the individual sequences that align with the query sequence having the indicated SEQ ID NO. Orientation refers to the orientation of the query sequence with respect to the individual sequences, where positive direction (positive) indicates that the alignment is in the same direction as the sequences provided in the sequence listing (left to right), and reverse direction. (Inverse) indicates that the alignment has a sequence complementary to the sequence provided in the sequence listing.

Some polynucleotides showed multiple profile hits. This is because, for example, a particular sequence contains overlapping profile regions and / or this sequence contains two different functional domains. These profile hits are described in more detail below.

(Ank Repeat (ANK)) SEQ ID NOs: 187, 1268, 1804, 181
9, 1830, 1839, 2652, 3015 and 3267 represent polynucleotides encoding an Ank repeat containing protein. The ankyrin motif is a 33 amino acid sequence named after the protein ankyrin, which has a 24 tandem 33 amino acid motif. Ank repeats were originally identified in the cell cycle regulatory protein cdc10 (Breden et al., Nature.
e (1987) 329: 651). Examples of proteins containing ankyrin repeats include ankyrin, myotropin, I-κB protein, cell cycle protein cdc10, Notch receptor (Matsuno et al., Developmen.
t (1997) 124 (21): 4265); major histocompatibility complex class I.
II region G9a (or BAT8) (Biochem J. 290: 811-).
818, 1993), FABP, GABP, 53BP2, Lin12, glp-.
1, SW14, and SW16. The function of ankyrin repeats is compatible with its role in protein interactions (Bork, Proteins (19
93) 17 (4): 363; Lambert and Bennet, Eur. J.
Biochem. (1993) 211: 1; Kerr et al., Current Op.
． Cell Biol. (1992) 4: 496; Bennet et al., J. Am. Bio
l. Chem. (1980) 255: 6424).

(ATPases Associated with Various Cellular Activities (ATPases)) SEQ ID NO: 431
, 639, 2135, 2684, 2859, 3197, and 3266 correspond to sequences encoding a novel member of the "ATPase associated with diverse cellular activities" (AAA) protein fanmilly. This AAA protein family is composed of multiple ATPases that share a conserved region of approximately 220 amino acids containing the ATP binding site (Froehlich et al., J Cel.
l Biol. (1991) 114: 443; Erdmann et al., Cell (1
991) 64: 499; Peters et al., EMBO J .; (1990) 9:17
57; Kunau et al., Biochimie (1993) 75: 209-224;
Confalonieri et al., BioEssays (1995) 17: 639;
http: // yeamob. pci. chemie. uni-tuebing
en. de / AAA / Description. html). Proteins belonging to either of these families contain one or two AAA domains. In general, the AAA domain in these proteins acts as an ATP-dependent protein clamp (Confalonieri et al. (1995) B.
ioEssays 17: 639). In addition to the ATP-binding "A" and "B" motifs located in the N-terminal half of this domain, a highly conserved region is located in the central part of the domain, which is important for the development of signature patterns. Was used. This consensus pattern is [LIVMT] -x- [L
IVMT] [LIVMF] -x [GATMC]-[ST]-[NS] -x (
4)-[LIVM] -D-x-A- [LIFA] -x-R.

(Bromodomain) SEQ ID NO: 1814 represents a polynucleotide encoding a polypeptide having a bromodomain region (Ha
ynes 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. Bromodomains are thought to be involved in protein-protein interactions and may be important for the assembly or activity of multicomponent complexes associated with transcriptional activity. The consensus pattern over the main 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) -Yx (12,13)-[LIVM] -x (
2) -N- [SACF] -x (2)-[FY].

(Basic region and leucine zipper transcription factor (BZIP)) SEQ ID NO: 410
, 552, 768, 822, 836, 1288, 1365, 1454, 1540
, 1549, 1556, 1557, 1563, 1622, 1630, 1704,
1808, 2363, 2424, 3147, 3152, 3158 and 3208
Represents a polynucleotide encoding a basic region and a novel member of the family of leucine zipper transcription factors. Eukaryotic DNA binding transcription factor bZIP
Super Family (Hurst, Protein Prof. (1995) 2
: 105; and Ellenberger, Curr. Opin. Struct
． Biol. (1994) 4:12) contain proteins containing basic region-mediated sequence-specific DNA binding followed by leucine zippers required for dimerization. The consensus pattern for this protein family is [KR] -x (1,
3)-[RKSAQ] -N-x (2)-[SAQ] (2) -x- [RKTAEN
Q] -x-R-x- [RK].

(EFhand) SEQ ID NOs: 820, 1755 and 3285 correspond to polynucleotides encoding novel proteins in the family of EFhand proteins. Many calcium-binding proteins belong to the same evolutionary family and share one 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 12 residue loop flanked by 12 residue α-helical domains. In the EF hand loop, calcium-iodine is a pentagonal bipyramidal.
It is coordinated in a three-dimensional configuration. The six residues involved in binding are at positions 1, 3, 5, 7,
9 and 12; these residues are designated by X, Y, Z, -Y, -X and -Z. Invariant Glu or Asp at position 12 is Ca (bidentate ligand)
Provide two oxygens to coordinate with. Consensus pattern is complete EF
Hand loop, and the first residue that follows the loop and is always hydrophobic: D-
x- [DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-
{GP}-[LIVMC]-[DENQSTAGC] -x (2)-[DE]-[
LIVMFYW].

(Ets Domain (Ets Nterm)) SEQ ID NO: 1811 is ET
1 shows a polynucleotide encoding a polynucleotide having N-terminal homology in the S domain. This family of proteins contains the conserved domain "ETS domain" involved in DNA binding. This domain appears to recognize a purine-rich sequence; it is approximately 85-90 amino acids in length and is rich in aromatic and positively charged residues (Wasylyk et al., Eur).
． J. Biochem. (1993) 211: 718). This ets gene family encodes a novel class of DNA binding proteins, each of which binds a specific DNA sequence and interacts specifically with sequences containing the common core trinucleotide sequence GGA. including. In addition to the ets domain,
The native ets protein contains other sequences that can regulate the biological specificity of the protein. Ets genes and proteins are involved in various essential biological processes including cell proliferation, differentiation and development, and three members are involved in tumor processes.

(G-protein α subunit (G-α)) SEQ ID NO: 1846 represents a polynucleotide encoding a novel polypeptide of the G-protein α subunit family. Guanine nucleotide-binding proteins (G-proteins) are membrane-associated proteins that couple extracellularly activated integral membrane receptors to intracellular effectors (eg, ion channels and enzymes that alter the concentration of second messenger molecules). It is a family of proteins. G-proteins are three subunits (α, β,
And γ). The α subunit binds GTP and exhibits GTPase activity. The G-protein α subunit is 350-400 amino acids in length and has a molecular weight in the range 40-45 kDa. Seventeen different types of α subunits have been identified in mammals, and based on both sequence similarity and function, there are four major groups: α-s, α-q, α-i, and α. -12 (Simon et al., Science (1993) 252: 8.
02). They are often N-terminally acylated, usually by myristic and / or palmitoylate, and these fatty acid modifications are important for membrane association and high affinity interactions with other proteins. Can be.

(Helicase with Conserved C-Terminal Domain (Helicase C)) SEQ ID NO: 14
96, 2826 and 2871 represent polynucleotides encoding novel members of the DEAD / H helicase family. Many eukaryotic and prokaryotic proteins have been characterized on the basis of their structural similarities (Schmi
d S. R et al., Mol. Microbiol. (1992) 6: 283; Li
nder P et al., Nature (1989) 337: 121; Wassarma.
n D. A, et al., Nature (1991) 349: 463). All are involved in ATP-dependent nucleic acid unwinding. All DEAD box family members of the above proteins share a number of conserved sequence motifs, some of which are specific to the DEAD family and others of which are AT
Shared by P-binding proteins or by proteins belonging to the helicase "superfamily" (Hodgman TC, Nature (
1988) 333: 22 and Nature (1988) 333: 578 (Er.
rata). One of these motifs is the "DEAD Box".
, Which represents a special version of the B motif of the ATP-binding protein. Some other proteins that belong to a subfamily with His instead of the second Asp are therefore "DE-AH-box" proteins (Wassa).
rman D.L. A et al., Nature (1991) 349: 463; Harosh.
I et al., Nucleic Acids Res. (1991) 19: 6331;
Koonin E. V. et al. Gen. Virol. (1992) 73: 9.
89. The following characteristic patterns are used to identify members for both subfamilies: 1) [LIVMF] (2) -DEAD- [R
KEN] -x- [LIVMFYGSTN]; and 2) [GSAH] -x- [L
IVMF] (3) -DE- [ALIV] -H- [NECR].

(Homeobox Domain (Homeobox)) SEQ ID NOS: 1676, 1820
And 1821 represent polynucleotides encoding proteins with homeobox domains. This homeobox is a protein domain of 60 amino acids (Gehring In: Guidebook to the H
oemobox Genes, DOUBLE D edit, pp. 1-10, Oxford
rd University Press, Oxford, (1994); Bu.
erglin In: Guidebook to the Homebox
Genes, pages 25-72, Oxford University Press
, Oxford, (1994); Gehring, Trends Bioche.
m. Sci. (1992) 17: 277-280; Gehring et al., Ann.
u. Rev. Genet. (1986) 20: 147-173; Schofie.
ld, Trends Neurosci. (1987) 10: 3-6), first identified in many Drosophila homeotic and segmentation proteins. It is very well preserved in many other animals, including vertebrates. This domain binds DNA with a helix-turn-helix type structure. Some proteins, including homeobox domains,
Play an important role in development. Most of these proteins are sequence-specific D
It is an NA-binding transcription factor. This homeobox domain is also very similar to the region of yeast mating type proteins. These act as master switches in yeast differentiation by controlling gene expression in a cell-type specific manner,
It is a sequence-specific DNA binding protein.

A schematic representation of this homeobox domain is shown below. Helix-Turn-
The helix region is indicated by the symbols "H" (helix), and "t" (turn).

[0183]

[Chemical 1] This pattern detects homeobox sequence 24 residues long and spans positions 34-57 of the homeobox domain. The consensus pattern is as follows: [LIVMFYG]-[ASLVR] -x (2)-[LIVMSTACN].
] -X- [LIVM] -x (4)-[LIV]-[RKNQESTAY]-[
LIVFSTNKH] -W- [FYVC] -x- [NDQTAH] -x (5)-
[RKNAIMW].

(MAP Kinase Kinase (mkk)) SEQ ID NOs: 29, 31, 196, 317
5, 3190 and 3281 represent members of the novel MAP kinase kinase family. MAP kinase (MAPK) is involved in signal transduction and is important in controlling cell cycle and cell proliferation. MAP kinase kinase (
MAPKK) is a bispecific protein kinase that phosphorylates and activates MAP kinase. MAPKK homologs have been found in yeast, invertebrates, amphibians, and mammals. Furthermore, the MAPKK / MAPK phosphorylation switch constitutes a basic module that is activated in different pathways in yeast and in vertebrates. MAPKKs are essential transducers that signals must pass through before they reach the nucleus. For an overview, see, for example, Biologique Biol. Cell (1993) 79
: 193-207; Nishida et al., Trends Biochem Sci.
(1993) 18: 128-31; Ruderman Curr Opin C.
Lell Biol (1993) 5: 207-13: Dhanasekaran et al., Oncogene (1998) 17: 1447-55; Kiefer et al., Bi.
Ochem Soc Trans (1997) 25: 491-8; and Hil.
l. See Cell Signal (1996) 8: 533-44.

(Protein Kinase (Protkinase)) SEQ ID NOS: 1157, 147
8, 1496, 2286, 2969 and 3190 represent polynucleotides encoding protein kinases. Protein kinases catalyze the phosphorylation of proteins by various pathways and are involved in cancer. Eukaryotic protein kinases (Hanks SK, et al., FASEB J. (1995) 9: 576; Hunt.
er T, Meth. Enzymol. (1991) 200: 3: Hanks.
S. K et al., Meth. Enzymol. (1991) 200: 38; Hanks.
, S. K, Curr. Opin. Struct. Biol. (1991
) 1: 369; Hanks S .; K et al., Science (1988) 241: 4.
2) are enzymes belonging to a very broad family of proteins that share a conserved catalytic core common to both serine / threonine and tyrosine protein kinases. There are many conserved regions in the catalytic domain of protein kinases. The first region, located at the N-terminal end of the catalytic domain, is a glycine-rich stretch of residues near the lysine residue, which is involved in ATP binding. The second region is located in the central part of the catalytic domain and contains a conserved aspartic acid residue that is important for the catalytic activity of the enzyme (Knightton DR et al., Sci.
ence (1991) 253: 407). The protein kinase profile is
It contains two characteristic patterns for this second region: one specific for serine / threonine kinases and the other specific for tyrosine kinases. The third profile is aligned (Hanks SK et al., FASEB J. (1
995) 9: 576) and covers the entire catalytic domain.

The consensus pattern is as follows: 1) [LIV] -G- {P} -G
-{P}-[FYWMGSTNH]-[SGA]-{PW}-[LIVCAT]
-{PD} -x- [GSTACLIVMFY] -x (5,18)-[LIVMF
YWCSTAR]-[AIVP]-[LIVMFAGCKR] -K, where K binds ATP; 2) [LIVMFYC] -x- [HY] -x-D-
[LIVMFY] -Kx (2) -N- [LIVMFYCT] (3), where D
Is an active site residue; and 3) [LIVMFYC] -x- [HY] -x.
-D- [LIVMFY]-[RSTAC] -x (2) -N- [LIVMFYC]
, Where D is an active site residue.

If the protein analyzed contains two 0 of the above protein kinase features:
The probability that it is a protein kinase is close to 100%.

(Ras Family Protein (ras)) SEQ ID NOs: 1688 and 3258
Shows a polynucleotide encoding a novel member of the ras family, a small GTP / GDP binding protein (Valencia et al., 1991, Bi.
Chemistry 30: 4637-4648). Ras family members are generally specific guanine nucleotide exchange factor (GEF) and specific GTPase activating protein (GA) as stimulators of overall GTPase activity.
P) is required. The highest degree of sequence conservation of ras-related proteins is found in the four regions directly involved in guanine nucleotide binding. First two
One constitutes the majority of the phosphate and Mg2 + binding sites (PM sites) and is located in the first half of the G-domain. The other two regions are involved in guanosine binding,
It is located in the latter half of the C-terminal side of this molecule. The motifs and conserved structural features of ras-related proteins are reviewed in Valencia et al., 1991, Bioc.
Chemistry 30: 4637-4648. The major consensus patterns of ras proteins are: D-T-A-G-G-Q-E-K- [LF].
-G-G-LR- [DE] -G-Y-Y.

(Thioredoxin Family Active Site (Thioredox)) SEQ ID NO: 16
77 indicates a polynucleotide encoding a protein having a thioredoxin family active site. Thioredoxin is a small protein of about 100 amino acid residues that participates in various redox reactions through the reversible oxidation of active center disulfide bonds (Holmgren A. Annu. Rev. Biochem.
(1985) 54: 237; Gleason F .; K. Et al., FEMS Micr
obiol. Rev. (1988) 54: 271; Holmgren A .; J
． Biol. Chem. (1989) 264: 13963; Eklund H et al., Proteins (1991) 11:13). Thioredoxin exists in either a reduced or oxidized form in which two cysteine residues are linked in an intramolecular disulfide bond. Thioredoxin is present in prokaryotes and eukaryotes, and the sequences surrounding redox-active disulfide bonds are well conserved. All PDIs are 2 or 3 of the thioredoxin domain (ERp72)
Including copy. The consensus pattern is: [LIVMF]-[LIVMSTA]
-X- [LIVMFYC]-[FYWSTHE] -x (2)-[FYWGTN]
-C- [GATPLVE]-[PHYWSTA] -C-x (6)-[LIVMF
YWT] (where the two Cs form a redox active bond).

(Trypsin) SEQ ID NO: 1410 corresponds to a novel serine protease of the trypsin family. The catalytic activity of serine proteases from the trypsin family is provided by a charge relay system involving an aspartic acid residue that hydrogen bonds to histidine, which itself hydrogen bonds to serine. The sequences near the active site serine and histidine residues are well conserved in this family of proteases (Brenner S, Nature (
1988) 334: 528). The consensus pattern for the trypsin protein family is: 1) [LIVM]-[ST] -A- [STAG] -HC
, Where H is an active site residue; and 2) [DNSTAGC]-[GSTA
PIMVQH] -x (2) -G- [DE] -SG- [GS]-[SAPHV]
-[LIVMFYWH]-[LIVMFYSTANQH], where S is an active site residue. All sequences known to belong to this family are detected by the consensus sequence above, except for the 18 different proteases that the first conserved glycine has lost. If the protein contains both serine and histidine active site features, the probability that it is a trypsin family serine protease is 100%.

(WD domain, G-β repeat (WD domain)) SEQ ID NOs: 1336, 1380
, 1711, 1762, 1909, 2218, 3047, 3108, and 32.
92 represents a novel member of the WD domain / G-β repeat family. β-transducin (G-β) is a guanine nucleotide binding protein (G-G) that acts as an intermediate in the transduction of signals generated by transmembrane receptors.
Is one of the three subunits (α, β, and γ) of (protein) (
Gilman, Annu. Rev. Biochem. (1987) 56: 615.
). The α subunit binds and hydrolyzes GTP; the functions of the β and γ subunits are less well defined, but are required for GDP displacement by GTP and for membrane anchoring and receptor recognition. It seems In higher eukaryotes, G-β exists as a small multigene family of highly conserved proteins of approximately 340 amino acid residues. Structurally, G-β consists of eight tandem repeats of approximately 40 residues, each containing a central Trp-Asp motif (this type of repeat is sometimes referred to as the WD-40 repeat). WD domain / G-β
The consensus pattern for the repeat family is: [LIVMSTAC]-[LIVM
FYWSTAGC]-[LIMSTAG]-[LIVMSTAGC] -x (2)
-[DN] -x (2)-[LIVMWSTAC] -x- [LIVMFSTAG]
-W- [DEN]-[LIVMFSTAGCN].

(Wnt family of developmental signaling proteins (Wnt dev sig
n)) SEQ ID NO: 1538 corresponds to a novel member of the wnt family of developmental signaling proteins. Wnt-1 (formerly known as int-1) is a developmental member of this family (Nusse R, Trends
Genet (1988) 4: 291), thought to play a role in intracellular communication, and appear to be an important signaling molecule in the development of the central nervous system (CNS). All wnt family proteins share the following features characteristic of secreted proteins: a signal peptide, several potential N-glycosylation sites, and 22 conserved cysteines likely involved in disulfide bonds. The wnt protein appears to adhere to the plasma membrane of secretory cells and therefore signal over only a small cell diameter. Consensus pattern (3
Based on a highly conserved region containing one cysteine) is as follows:
C-K-C-H-G- [LIVMT] -S-G-x-C.

(Protein Tyrosine Phosphatase (Y Phosphatase)) SEQ ID NO: 141
7 represents a polynucleotide encoding a protein tyrosine kinase. Tyrosine-specific protein phosphatase (EC 3.1.348) (PTPas
e) (Fischer et al., Science (1991) 253: 401; Cha.
rbonneau et al., Annu. Rev. Cell Biol. (1992) 8
: 463; Troubridge, J .; Biol. Chem. (1991) 26
6: 23517; Tonks et al., Trends Biochem. Sci. (1
989) 14: 497; and Hunter, Cell (1989) 58:10.
13) catalyzes the removal of phosphate groups attached to tyrosine residues. These enzymes are very important in controlling cell growth, proliferation, differentiation and transformation. Multiple forms of PTPase have been characterized and can be divided into two categories: soluble PT.
A transmembrane receptor protein containing Pase and PTPase domains. Structurally, all known receptors PTPase consist of an extracellular domain of variable length, followed by a transmembrane region and a C-terminal catalytic cytoplasmic domain. The PTPase domain consists of about 300 amino acids. A search for two conserved cysteines showed that they are absolutely necessary for activity. Moreover, many conserved residues in their immediate vicinity were also shown to be important. The consensus pattern for PTPase is [LIVMF] -HC-x (2) -G-x (3)-.
[STC]-[STAGP] -x- [LIVMFY]; C is the active site residue.

(Zinc finger, C2H2 type (Zincfing C2H2)) SEQ ID NOs: 308, 807, 1324, 1503, 1527, 3081, 3193 and 3
306 corresponds to a polynucleotide encoding a novel member of the C2H2-type zinc finger protein family. Zinc finger domain (Kl
ug et al., Trends Biochem. Sci. (1987) 12: 464;
Evans et al., Cell (1988) 52: 1; Payre et al., FEBS Le.
tt. (1988) 234: 245; Miller et al., EMBO J. et al. (198
5) 4: 1609; and Berg, Proc. Natl. Acad. Sci.
USA (1988) 85:99) is a nucleic acid binding protein structure. In addition to the conserved zinc ligand residue, many other positions have also been shown to be important for the structural integrity of the C2H2 zinc finger (Rosenfeld et al., J. Bi.
omol. Struct. Dyn. (1993) 11: 557). The best conserved position is found in the four residues after the second cysteine: this position is generally an aromatic or aliphatic residue. The consensus pattern for C2H2 zinc fingers is: Cx (2,4) -Cx (3)-[LIVMF
YWC] -x (8) -H-x (3,5) -H. Two Cs and two Hs are zinc ligands.

(Src Homology 2) SEQ ID NOs: 186, 2591, 3307, and 3339 represent polynucleotides encoding novel members of the family of Src Homology 2 (SH2) proteins. The Src homology 2 (SH2) domain is a protein domain of approximately 100 amino acid residues that was first identified as a conserved sequence region between the oncoproteins Src and Fps (Sadowski I., et al.
Mol. Cell. Biol. 6: 4396-4408 (1986)). Similar sequences are found in many other intracellular signaling proteins (Russel
R. B. Et al., FEBS Lett. 304: 15-20 (1992)). SH
The 2 domain functions as a preparative module of the intracellular signaling cascade by interacting with high affinity with phosphotyrosine-containing target peptides in a sequence-specific and phosphorylation-dependent manner (Marangere LEM.
, Pawson T .; J. Cell Sci. Suppl. 18: 97-10
4 (1994); Pawson T .; , Schlessinger J .; , Cu
rr. Biol. 3: 434-442 (1993); Mayer B .; J. , B
Altimore D. , Trends Cell. Biol. 3: 8-13 (
1993); Pawson T .; , Nature 373: 573-580 (1
995)).

The SH2 domain has a conserved 3D structure consisting of two α-helices and 6-7 β-strands. The core of this domain is formed by a contiguous β-curve consisting of two linked β-sheets (Kuriyan J., Cowburn.
D. Curr. Opin. Struct. Biol. 3: 828-837 (
1993)). The total profile for detecting SH2 domains is 9
Based on a structural alignment consisting of 8 gapless blocks and 7 linker regions, 2 match positions.

(Src Homology 3) SEQ ID NOs: 234, 1832 and 1835 represent polynucleotides encoding novel members of the family of Src homology 3 (SH3) proteins. The Src homology 3 (SH3) domain is a conserved sequence in the noncatalytic portion of some cytoplasmic protein tyrosine kinases (eg, Sr
c, Abl, Lck) is a small protein domain first identified as approximately 60 amino acid residues (Mayer BJ, et al., Nature 332: 27).
2-275 (1988)). Since then, a wide variety of intracellular or membrane-bound proteins have been found (Musaccio A. et al., FEBS Let).
t. 307: 55-61 (1992); Pawson T. et al. , Schlessi
nger J. Curr. Biol. 3: 434-442 (1993); Ma.
yer B. J. , Baltimore D .; , Trends Cell Bi
ol. 3: 8-13 (1993); Pawson T. et al. , Nature 373
: 573-580 (1995)).

The SH3 domain is arranged as two closely packed anti-parallel β-sheets 5
Or it has a characteristic fold consisting of 6 β-strands. The linker region may include short helices (Kuriyan J., Cowburn D., Curr.
Opin. Struct. Biol. 3: 828-837 (1993)).

The function of the SH3 domain may be to mediate the assembly of specific protein complexes via binding to proline-rich peptides (Morton C
． J. , Campbell I .; D. Curr. Biol. 4: 615-61
7 (1994)).

Generally, the SH3 domain is found as the signal copy in a given protein, although a significant number of proteins with two SH3 domains and 3
Or there are some with 4 copies.

Type III fibronectin. SEQ ID NOs: 746 and 1192 represent polynucleotides encoding novel members of the family of type III fibronectin proteins. Multiple receptors for lymphokines, hematopoietic growth factors, and growth hormone-related molecules have been found to share a common binding domain (Bazan JF, Biochem. Biophys. Res. Co.
mmun. 164: 788-795 (1989); Bazan J. et al. F. , Pr
oc. Natl. Acad. Sci. U. S. A. 87: 6934-6938 (
1990); Cosman D .; Et al., Trends Biochem. Sci.
15: 265-270 (1990); d'Andrea A. et al. D. , Fasma
n G. D. Lodish H. et al. F. , Cell 58: 1023-102.
4 (1989); d'Andrea A. et al. D. , Fasman G .; D. , Lo
dish H. F. Curr. Opin. Cell Biol. 2: 648-
651 (1990)).

The conserved region constitutes all or part of the extracellular ligand binding region and is approximately 200 amino acid residues in length. In the growth hormone receptor, there are two known pairs of cysteines involved in disulfide bonds at the N-terminus of this domain.

[0203]

[Chemical 2] Two patterns detect this receptor family. The first pattern is
The second pattern is from the first N-terminal disulfide loop and the second pattern is a tryptophan-rich pattern located at the C-terminus at the end of the extracellular region.

A consensus for this protein family is: C- [LVFYR]-
x (7,8)-[STIVDN] -Cxw (where the two Cs are linked by a disulfide bond). The second consensus for this protein family is: [STGL] -x-W- [SG] -x-WS.

LIM domain containing proteins. SEQ ID NOs: 1269, 1309, 1360, and 1386 represent polynucleotides encoding novel members of the family of LIM domain-containing proteins. Many proteins contain a conserved cysteine-rich domain of about 60 amino acid residues (Freyd G. et al., Nature.
e 344: 876-879 (1990); Baltz R. et al. Et al, Plant
Cell 4: 1465-1466 (1992); Sanchez-Garci.
a I. , Rabbitts T .; H. , Trends Genet. 10: 3
15-320 (1994)).

There are seven conserved cysteine residues and one histidine in the LIM domain. The sequence followed by these conserved residues is Cx (2) -Cx (16
, 23) H-x (2)-[CH] -x (2) -Cx (2) -Cx (16,2)
1) -C-x (2,3)-[CHD]. The LIM domain binds two zinc ions (Michelsen JW, et al., Proc. Natl. Ac.
ad Sci. U. S. A. 90: 4404-4408 (1993)). LIM
Does not bind DNA, but rather acts as an interface for protein-protein interactions. The consensus for this protein family is:
C-x (2) -C-x (15,21)-[FYWH] -H-x (2)-[CH]
It is -x (2) -Cx (2) -Cx (3)-[LIVMF]. Five Cs and one H binds zinc.

The C2 domain (protein kinase C-like. SEQ ID NOs: 1325 and 2282 represent polynucleotides that encode novel members of the family of C2 domain-containing proteins. Some isozymes of protein kinase C (PKC) have about: It contains a domain known as C2 of 116 amino acid residues, which is located between two copies of the C1 domain, which binds phorbol ester and diacylglycerol, and the protein kinase catalytic domain (Azzi A. et al. Et al., Eur. J. Biochem. 208: 547-557 (
1992); Stabel S .; , Semin. Cancer Biol. 5:
277-284 (1994)).

The C2 domain is involved in calcium-dependent phospholipid binding (Davleto).
v B. A. , Suedhof T .; C. J. Biol. Chem. 268:
26386-26390 (1993)). Other putative functions for the C2 domain include binding to inositol-1,3,5-tetraphosphate, as domains related to the C2 domain have also been found in proteins that do not bind calcium. (Fukuda M., et al., J. Biol. Chem. 26.
9: 29206-29211 (1994)).

The consensus pattern for the C2 domain is located in the conserved part of this domain and the connecting loop is located between β-strands 2 and 3. The profile for the C2 domain covers the entire domain. The consensus for this protein family is: [ACG] -x (2) -Lx (2,3) -Dx (
1, 2)-[NGSTLIF]-[GTMR] -x- [STAP] -D- [PA
]-[FY].

Serine proteases, trypsin family, active site. SEQ ID NO: 1410 represents a polynucleotide that encodes a novel member of the family of serine proteases (trypsin proteins). The catalytic activity of serine proteases from the trypsin family is provided by a charge relay system containing an aspartic acid residue hydrogen bonded to histidine, which is itself hydrogen bonded to serine. The sequences near the active site serine and histidine residues are well conserved in this family of proteases (Brenner S., Nature 334: 528-530 (1988)).

A consensus for this protein family is: [LIVM]-[ST
] -A- [STAG] -HC [H is the residue of the active site]. The second consensus for this protein family is: [DNSTAGC]-[G
STAPIMVQH] -x (2) -G- [DE] -SG- [GS]-[SAP
HV]-[LIVMFYWH]-[LIVMFYSTANQH] [S is the active site residue].

RNA recognition motif domain (RRM, RBD, or RNP). Sequence number 1
464 and 1514 represent polynucleotides encoding novel members of the family of RNA recognition motif domain proteins (Bandziulis JJ et al., Genes Dev. 3: 431-437 (1989); Dre.
yfuss G. Et al., Trends Biochem. Sci. 13: 86-9
1 (1988)).

Within the putative RNA-binding domain, there are two regions that are highly conserved. The first region is a 6-residue hydrophobic segment (termed the RNP-2 motif); the second region is the octapeptide motif (RNP-1 or RNP).
NP-CS). The positions of both motifs in this domain are shown in the schematic diagram below.

[0214]

[Chemical 3] The RNP-1 motif was used as a consensus pattern for this type of domain. The consensus for this protein family is: [RK]
-G- {EDRKHPCG}-[AGSCI]-[FY]-[LIVA] -x-
[FYLM].

Phosphatidylinositol-specific phospholipase C, Y domain. SEQ ID NO: 1707 represents a polynucleotide encoding a novel member of a phosphatidylinositol-specific phospholipase C, Y domain family of proteins. Phosphatidylinositol-specific phospholipase C (EC 3.1.4.1)
1) is a eukaryotic intracellular enzyme that plays an important role in the signal transduction process (Meldrum E. et al., Biochim. Biophys.
Acta 1092: 49-71 (1991)). This enzyme catalyzes the hydrolysis of 1-phosphatidyl-D-myo-inositol-3,4,5-triphosphate to the second messenger molecules diacylglycerol and inositol-1,4,5-triphosphate. To do. This catalytic process is tightly regulated by reversible phosphorylation and binding of regulatory proteins (Rhee SG, Cho.
i K. D. , Adv. Second Messenger Phosphop
rotation Res. 26: 35-61 (1992); Rhee S .; G. ，
Choi K. D. , J Biol. Chem. 267: 12393-1239
6 (1992); Sternweis P .; C. , Smrcka A .; V. , T
rends Biochem. Sci. 17: 502-506 (1992)).

All eukaryotic PI-PLCs contain two regions of homology, these are "X-
Called "box" and "Y-box". The order of these two regions is the same (NH2-XY-COOH), but their intervals are different. In most isoforms, the distance between these two regions is only 50-100 residues, whereas in the gamma isoform, one PH domain, two SH2 domains, and one SH3 domain are two. It is inserted between two PLC-specific domains. These two conserved regions have been shown to be important for catalytic activity. At the C-terminus of the Y-box, there is a C2 domain probably involved in Ca-dependent membrane binding.

Serine carboxypeptidase. SEQ ID NO: 1744 represents a polynucleotide encoding a novel member of the serine carboxypeptidase family of proteins. Carboxypeptidases are metallocarboxypeptidases or serine carboxypeptidases (EC3.4.16.5 and EC3.4.16.
It can be any of 6). The catalytic activity of serine carboxypeptidases is provided by a charge relay system containing an aspartic acid residue hydrogen-bonded to histidine, which itself is hydrogen-bonded to serine, similar to the catalytic activity of serine proteases of the trypsin family. (Liao DI, Remington S.
． J. J. Biol. Chem. 265: 6528-6531 (1990)).
.

The sequences surrounding the active site serine and histidine residues are highly conserved in all these serine carboxypeptidases. The consensus for this protein family is: [LIVM] -x- [GTA] -ES
-Y- [AG]-[GS] [S is the residue of the active site]. The second consensus for this protein family is: [LIVF] -x (2)-[L
IVSTA] -x- [IVPST] -x- [GSDNQL] [SAGV]-[S
G] -Hx- [IVAQ] -Px (3)-[PSA], where H is the active site residue.

Dsrm double-stranded RNA binding motif. SEQ ID NO: 1818 is a dsrm double-stranded R
1 depicts a polynucleotide encoding a novel member of NA binding motif protein. In eukaryotic cells, numerous RNA binding proteins play key roles in the post-transcriptional regulation of gene expression. Characterization of these proteins has led to the identification of several RNA binding motifs. Several human and other vertebrate genetic disorders are caused by aberrant expression of RNA binding proteins (CG Burd and G. Dreyfuss, Science 265:
615-621 (1994)).

Proteins containing the double-stranded RNA binding motif bind to specific RNA targets. The double-stranded RNA binding motif is illustrated in humans by a protein kinase induced by interferon, which is part of the cellular response to dsRNA.

SEQ ID NOs: 2577, 3183, and 3195 encode members of the four-transmembrane integral membrane protein family. This family consists of type III proteins, which are integral membrane proteins that contain an N-terminal membrane anchor domain that is not cleaved during biosynthesis, and function as translocation signals and membrane anchors. These proteins also have three additional transmembrane regions. The consensus pattern is G-x (3)-[LIVMF] -x (2)-[GSA.
]-[LIVMF] (2) -GC-x- [GA]-[STA] -x (20- [
eG] -x (20- [CwN]-[LIVM] (2).

SEQ ID NO: 2944 encodes a polypeptide having calpain large subunit, domain III. Calpain plays various biological roles,
It is a family of intracellular proteases. Calpain 3 (also known as p94) is predominantly expressed in skeletal muscle and plays a role in limb girdle muscular dystrophy type 2A (Sorimachi, H. et al., Biochem. J. et al.
328: 721-732, 1997).

SEQ ID NOs: 1911 and 1980 encode polypeptides having a C3HC4 type zinc finger domain (RING finger). This domain is
It is a 40-60 residue cysteine-rich domain that binds diatomic zinc and is believed to be involved in mediating protein-protein interactions. Mammalian proteins of this family include: V (D) J recombination activating protein, which activates rearrangement of immunoglobulin and T cell receptor genes; breast cancer type 1 susceptibility protein ( BRCA1); bmi-1
Protooncogene; cbl protooncogene; and mel-18 protein, which is a transcriptional repressor that is expressed in various tumor cells and recognizes and binds specific DNA sequences. This consensus pattern is Cx-H-x- [LIV
MFY] -Cx (2) -C- [LIVMYA].

SEQ ID NO: 3274 encodes a eukaryotic transcription factor with a forkhead domain of about 100 amino acid residues. Proteins in this group are transcription factors that include: mammalian transcription factors HNF-3-α, -β, and -γ; interleukin enhancer binding factor; and HTLF, which is a human T-cell leukemia virus. Binds to regions of long terminal repeats). This consensus pattern is [KR] -P- [PTQ]-[FYLVQH] -S- [FY] x (2)-[L
IVM] -X (3,4)-[AC]-[LIM].

SEQ ID NO: 3345 encodes a polypeptide having a PDZ domain. Several dozen signaling proteins are known as PDZ domains, 80-1
Belonging to this group of proteins, which have 00 residue repeats. Some of these proteins interact with the C-terminal tetrapeptide motif X-Ser / Thr / X-Val-COO- of ion channels and / or receptors (
Ponting, C.I. P. , Protein Sci. 6; 464-468, 1
997).

SEQ ID NO: 3351 encodes a polypeptide of the family of phorbol ester / glycerol binding proteins. Phorbol ester (PE) is an analog of diacylglycerol (DAG) and is a strong tumor promoter. DAG activates a family of serine-threonine protein kinases known as protein kinase C. The N-terminal region of protein kinase C binds PE and DAG and contains one or two copies of a cysteine-rich domain of approximately 50 amino acid residues. Other proteins with this domain include diacylglycerol kinase; the vav oncogene; and N-chimerin, a brain-specific protein. DAG /
The PE binding domain binds two zinc ions via the 6 cysteines and 2 histidines that are conserved in this domain. This consensus pattern is H-x- [LIVMFYW] -x (8,1l) -Cx (2) -C-.
x- (3)-[LIVMFC] -x (5,10) -Cx (2) -Cx (4)
-[HD] -x (2) -C-x (5,9) -C.

SEQ ID NO: 2216 encodes a polypeptide having a WW / rsp5 / WWP domain. The protein is named for the presence of a conserved aromatic position (generally tryptophan) and a conserved proline. Proteins having this domain include dystrophin, vertebrate YAP protein, and IQGAP (human GTPase activating protein acting on rat). This consensus pattern is Wx (9,11)-[VFY]-[FY
W] -x (6,7)-[GSTNE]-[GSTQCR]-[FYW] -x (2
) -P.

SEQ ID NO: 2428 encodes a member of the bispecific phosphatase family with a catalytic domain, and SEQ ID NOS: 2281 and 2310 encode members of the protein tyrosine phosphatase family. These families are related and classified as tyrosine-specific protein phosphatases. These enzymes catalyze the removal of phosphate groups from tyrosine residues,
And it is important in controlling cell growth, proliferation, differentiation, and conversion. This consensus pattern is [LIVMF] -H-C-x (2) -G-x- (3).
-[STC]-[STAGP] -x- [LIVMFY].

[0229]

[Table 1]

[0230]

[Table 2]

[0231]

[Table 3]

[0232]

[Table 4]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/19 C12N 1/21 4H045 1/21 C12Q 1/68 A 5/10 G01N 33/53 D C12Q 1 / 68 MG01N 33/53 33/566 33/574 Z 33/566 A61K 31/7088 33/574 C12N 15/00 ZNAA // A61K 31/7088 5/00 A 38/00 A61K 37/02 (81) designation Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW) ), E (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH , CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU , SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Williams, Lewis T. United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Escobed, James United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Innis, Michael A .. United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Garcia, Pablo Dominguez United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Klinger, Julie United States California 94662-8097, Emily Bill, P. Oh. Box 8097, Chiron Corporation (72) Inventor Kassam, Altough United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Rainhard, Christoph United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Randazo, Filippo United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Kennedy, Guilliacy. United States California 94662-8097, Emilyville, P. Oh. Box 8097, Chiron Corporation (72) Inventor Pot, David United States California 94662-8097, Emily Bill, P. Oh. Box 8097, Chiron Corporation (72) Inventor Lanson, George United States California 94662-8097, Emily Bill, P. Oh. Box 8097, Chiron Corporation (72) Inventor Darmanac, Ladge United States California 94086, Sunnyvale, Armano Avenue 675 (72) Inventor Kirken Yakov, Radmer United States California 94086, Sunnyvale, Armano Avenue 675 (72) Inventor Darmanac, Snezana United States California 94086, Sunnyvale, Almanau Avenue 675 (72) Inventor Dixon, Mark United States California 94086, Sunnyvale, Armano Avenue 675 (72) Inventor Labat, Ivan United States California 94086, Sunnyvale, Armano Avenue 675 (72) Invention Reshko Withits, Dina United States California 94086, Sunnyvale, Armano Avenue 675 (72) Inventor Kita, David United States California 94086, Sunnyvale, Armano Avenue 675 (72) Inventor Garcia, Veronica United States California 94086, Sunnyvale, Almaneau Avenue 675 (72) Inventor Jones, Lee William USA California 94086, Sunnyvale, Armano Avenue 675 (72) Inventor Strache Crane, Bargit United States California 94086, Sunnyvale, Armano Avenue 675 F Term (reference) 4B024 AA12 BA80 CA04 CA09 CA20 DA01 DA02 DA05 DA11 DA12 GA11 HA11 HA13 HA14 4B063 QA QA19 QQ53 QR08 QR32 QR35 QR40 QR56 QR62 QS16 QS25 QS34 QS36 QX02 4B065 AA01X AA58X AA72X AA87X AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4C084 AA01 AA13 CA27 CA53 ZB26 4C086 AA15 AA02 A16 MAA02 A16 AA02 A16 MA14 0 CA40 DA75 EA20 EA50 FA71 FA74

Claims (15)

[Claims] 1. A library of polynucleotides, wherein the library comprises sequence information for at least one of SEQ ID NOs: 1-3351. 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 according to claim 1, wherein the library comprises a polynucleotide corresponding to a gene differentially expressed in a cancer cell having a high metastatic potential as compared with a control cell. , The control cells are normal cells or cells with low metastatic potential, the expression is greater in metastatic tissue and the sequences are SEQ ID NOs: 14, 137, 151, 152, 171, 200, 254, 2
62, 271, 348, 412, 472, 507, 520, 530, 588, 6
23, 637, 660, 678, 680, 700, 714, 774, 812, 8
34, 901, 937, 976, 1168, 1333, 1352, 1520, 1
524, 1546, 1550, 1574, 1580, 1590, 1599, 16
07, 1622, 1706, 1752, 1768, 1769, 1780, 178
1, 1799, 1803, 1811, 1851, 1856, 1867, 1872
, 1875, 1884, 1919, 1923, 1939, 1975, 2024,
2045, 2060, 2071, 2118, 2119, 2128, 2135, 2
177, 2181, 2184, 2185, 2190, 2193, 2232, 22
39, 2283, 2311, 2314, 2338, 2378, 2393, 239.
4, 2395, 2398, 2460, 2490, 2505, 2514, 2540
, 2542, 2597, 2607, 2640, 2657, 2669, 2670,
2674, 2679, 2684, 2707, 2724, 2757, 2776, 2
804, 2818, 2906, 2959, 2964, 2968, 2976, 29
80, 2987, 3010, 3043, 3047, 3050, 3071, 307
2, 3092, 3095, 3097, 3140, 3157, 3173, 3187
3203, 3210, 3212, 3220, 3236, 3249, 3264,
A library selected from the group consisting of 3284, 3288, 3305, 3309, 3318, 3330, 3331, and 3335. 5. The library of claim 1, wherein the library comprises a polynucleotide corresponding to a gene that is differentially expressed in normal colon tissue as compared to colon cancer tissue, said expression. Is larger in cancerous tissue and the sequence is SEQ ID NO: 7, 164, 734, 836, 928, 965, 987, 102.
6, 1044, 1119, 1226, 1227, 1251, 1316, 1429
, 1442, 1540, 1553, 1560, 1577, 1588, 1610,
A library selected from the group consisting of 1620, 1626, 1673, 2416, 2749, 2976, 3129 and 3132. 6. The library of claim 1, wherein the library comprises a polynucleotide corresponding to a gene that is differentially expressed in normal colon tissue compared to colon cancer tissue, the expression being Is larger in normal tissue than in cancer tissue and the sequence is SEQ ID NO: 105, 198, 465, 489, 745, 859.
, 976, 1011, 1045, 1138, 1226, 1251, 1253, 1
392, 1474, 1559, 1571, 1589, 1591, 1607, 16
08, 1643, 1753, 1764, 1766, 1782, 1811, 274
9, 2784, 2790, 2805, 2976, 3128, 3129, 3146
A library selected from the group consisting of: 3150, and 3151. 7. The library of claim 1, wherein the library comprises a polynucleotide corresponding to a gene that is differentially expressed in normal human prostate cells as compared to human prostate cancer cells, The expression is greater in normal cells than in cancer cells and the sequences are SEQ ID NOs: 53, 446, 1410, 1754,
1801, 1845, 2060, 2143, 2632, 2899, and 333
A library selected from the group consisting of 8. 8. The library of claim 1, wherein the library comprises polynucleotides corresponding to genes that are differentially expressed in normal human prostate cells as compared to human prostate cancer cells, The expression is greater in cancer cells than in normal cells and the sequences are SEQ ID NOs: 86, 93, 687, 1269, 15
81, 1647, 1649, 1710, 1717, 1772, 1960, 298.
A library selected from the group consisting of 7, 3128, 3132, 3150, 3222, and 3268. 9. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to the identified sequences of SEQ ID NOs: 1-3351, or degenerate variants or fragments thereof. 10. A recombinant host cell comprising the polynucleotide of claim 9. 11. A polynucleotide encoded by the polynucleotide of claim 9,
Isolated polypeptide. 12. An antibody that specifically binds the polypeptide of claim 11. 13. A vector comprising the polynucleotide of claim 9. 14. A method for detecting a differentially expressed gene that correlates with the cancerous state of a mammalian cell, the method comprising: in a test sample derived from a cell suspected of having cancer, Detecting at least one differentially expressed gene product, said gene product comprising SEQ ID NOs: 14, 137, 151, 152, 171, 200, 254, 262, 271,
348, 412, 472, 507, 520, 530, 588, 623, 637,
660, 678, 680, 700, 714, 774, 812, 834, 901,
937, 976, 1168, 1333, 1352, 1520, 1524, 154
6, 1550, 1574, 1580, 1590, 1599, 1607, 1622
, 1706, 1752, 1768, 1769, 1780, 1781, 1799,
1803, 1811, 1851, 1856, 1867, 1872, 1875, 1
884, 1919, 1923, 1939, 1975, 2024, 2045, 20
60, 2071, 2118, 2119, 2128, 2135, 2177, 218
1, 2184, 2185, 2190, 2193, 2232, 2239, 2283
, 2311, 2314, 2338, 2378, 2393, 2394, 2395,
2398, 2460, 2490, 2505, 2514, 2540, 2542, 2
597, 2607, 2640, 2657, 2669, 2670, 2674, 26
79, 2684, 2707, 2724, 2757, 2776, 2804, 281
8, 2906, 2959, 2964, 2968, 2976, 2980, 2987
, 3010, 3043, 3047, 3050, 3071, 3072, 3092,
3095, 3097, 3140, 3157, 3173, 3187, 3203, 3
210, 3212, 3220, 3236, 3249, 3264, 3284, 32
88, 3305, 3309, 3318, 3330, 3331, and 3335, wherein the detection of the differentially expressed gene product is encoded by a gene corresponding to the sequence of at least one of the test samples. A method of correlating with a cancerous state of a cell from which the. 15. A method for detecting a differentially expressed gene that correlates with the cancerous state of a mammalian cell, the method comprising: in a test sample derived from a cell suspected of having cancer, Detecting at least one differentially expressed gene product, wherein the gene product is SEQ ID NO: 7, 164, 734, 836, 928, 965, 987, 1026, 1044.
, 1119, 1226, 1227, 1251, 1316, 1429, 1442,
1540, 1553, 1560, 1577, 1588, 1610, 1620, 1
626, 1673, 1960, 2416, 2749, 2976, 2987, 31
28, 3129, 3132, 3150, 3222, and 3268, wherein the detection of the differentially expressed gene product is encoded by a gene corresponding to the sequence of at least one of the cells from which the test sample was derived. Methods that correlate with the cancer status of.