JP2003530816A - Human genes and gene expression products - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel human polynucleotides and variants thereof, polypeptides encoding them and variants thereof, the genes corresponding to these polynucleotides, and the proteins expressed by the genes. The invention also relates to diagnostic and therapeutic agents using such novel human polynucleotides, their corresponding genes or gene products (eg, these genes and proteins), including probes, antisense constructs, And antibodies.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to polynucleotides of human origin and 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 was isolated, etc. Are key to identifying the genetic factors responsible for the phenotype that correlate 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, their encoded polypeptides and their variants, to the genes corresponding to these polynucleotides, and by their genes. Regarding expressed proteins. 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 corresponds to a polynucleotide containing sequence information of at least one of SEQ ID NOs: 1 to 1079.

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 relates to polynucleotides containing the disclosed nucleotide sequences,
It relates to full-length cDNA, mRNA genomic sequences, and genes corresponding to these sequences and degenerate variants thereof, and to polypeptides and polypeptide variants encoded by the polynucleotides of the invention. The following detailed description includes 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, polynucleotide and gene structures. Identification of motifs,
Identification of the function of the gene product encoded by the gene corresponding to the polynucleotide of the invention, as a probe as well as mapping and tissue profiling (
use of the provided polynucleotides during profiling, use of the corresponding polypeptides and other gene products for raising antibodies, and of polynucleotides and their encoded gene products for therapeutic and diagnostic purposes. Describe the use.

(Polynucleotide Composition) The scope of the present invention relating to a polynucleotide composition includes SEQ ID NOs: 1 to 107.
A polynucleotide having the sequence set forth in any one of 9; other biological materials described herein or otherwise hybridized under stringent conditions, particularly conditions of high stringency. Polynucleotides obtained from biological sources (especially human origin); Genes corresponding to the provided polynucleotides; Provided polynucleotides and variants of their corresponding genes, especially the biology 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) However, it is not necessarily limited to these. Other nucleic acid compositions contemplated within the scope of the present invention will be readily apparent to those of 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, particularly human colon, breast, and / or lung tissue. A novel nucleic acid composition of the invention for a particular purpose comprises the sequences set forth in any one of SEQ ID NOs: 1-1079 and their identified sequences. "Identifying sequence (identifying sequence)
) "Is at least about 10 nt long and about 2 to uniquely identify the polynucleotide sequence.
A contiguous sequence of residues 0 nt long, usually at least about 50 nt to about 100 nt long, eg 90% for any contiguous nucleotide sequence greater than about 20 nt.
Less than about 80% and usually less than about 85% sequence identity. Therefore, the novel nucleic acid composition of the present invention comprises a full-length cDNA or mRNA containing an identifying sequence of contiguous nucleotides of any one of SEQ ID NOs: 1-1079.

Polynucleotides of the invention also include polynucleotides that have sequence similarity and sequence identity. Nucleic acids with sequence similarity are detected by hybridization under low stringency conditions, eg, 50 ° C. and 10 × SSC (0.9M saline / 0.09M sodium citrate), and 1 × S.
Retains binding when subjected to washing at 55 ° C in SC. Sequence identity can be determined by stringent conditions, eg, hybridization at 50 ° C. or above and 0.1 × SSC (9 mM saline / 0.9 mM sodium citrate). Hybridization methods and conditions are well known in the art, see, eg, USPN 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 1-1079)
Bind to. By using probes, in particular labeled probes of DNA sequences, homologous or related genes can be isolated. Sources of homologous genes are
It can be any species, eg primate species, especially humans: rodents (eg rat and mouse); dogs, cats, cows, sheep, horses, yeasts, nematodes, etc.

Preferably, hybridization is performed using at least 15 contiguous nucleotides (nt) of at least one of SEQ ID NOs: 1-1079. That is, when at least 15 consecutive nts of one of the disclosed SEQ ID NOs are used as probes, the probe will preferentially hybridize with a nucleic acid containing a complementary sequence and the selected probe It allows the identification and recovery of nucleic acids that hybridize uniquely to. Probes from more than one SEQ ID NO: may hybridize to the same nucleic acid if the cDNA from which they correspond corresponds to one mRNA. Probes greater than 15 nt, eg, about 18 nt to about 1
A 00 nt probe can be used, but 15 nt represents sufficient sequence for unique identification.

Polynucleotides of the invention also include naturally occurring variants of the nucleotide sequence (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 they show a non-match. Generally, allelic variants are 15-
Including 25% bp non-conformance, and 5-15%, or 2-5%, or 1-2%
It can include as little as bp non-matching, as well as single bp non-matching.

The present invention also includes homologs corresponding to the polynucleotides of SEQ ID NOs: 1-1079, wherein the source of homologous genes is any mammalian species, eg, primate species, especially humans; It can be a tooth (eg rat); dog, cat, cow, sheep, horse, yeast, nematode, etc. Among mammalian species, such as human and mouse, homologs generally have substantial sequence similarity, eg, at least 75%, usually at least 90%, more usually at least 95% sequence identity between nucleotide sequences. Sequence similarity is calculated based on the reference sequence, which may be a subset of longer sequences, such as conserved motifs, coding regions, flanking regions, etc. A reference sequence is usually at least about 18 contiguous nts in length, more usually at least about 30 nts in length, and can be extended to the complete sequence being compared. Algorithms for sequence analysis are known in the art (eg gapped BLAST (Altschul et al., Nucleic Acids.
Res. (1997) 25: 3389-3402)).

Generally, variants of the invention will be at least about 65%, preferably at least about 7%.
Have a sequence identity of greater than 5%, more preferably at least about 85%, and M
It can be at least about 90% or more as determined by the Smith-Waterman homology search algorithm as performed in the PSRCH program (Oxford Molecular). For purposes of the present invention, the preferred method of calculating percent identity is the Smith-Wate using:
The rman algorithm. The overall DNA sequence identity was determined by the following search parameters: gap open penalty.
, 12; and gap extension penalties.
nity, using an affine gap search with 1, must not exceed 65% as determined by the Smith-Waterman homology search algorithm as performed in the MPSRCH program (Oxford Molecular). I won't.

The nucleic acids of the invention encode cDNA or genomic DNA, and fragments thereof, particularly biologically active gene products, and / or are useful in the methods disclosed herein (eg, in 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, as well as 3'non-coding region and 5'non-coding region. Usually mRNA species have contiguous exons, with intron intervention, and, if present, removed by nuclear RNA splicing, producing a contiguous open reading frame encoding the polypeptide of the invention.

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

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 chemically synthesizing oligonucleotides according to conventional methods, restriction enzyme digestion, PCR amplification and the like. The isolated polynucleotides and nucleotide fragments of the invention are at least about 10, about 15, about 20, about 35, about 50, selected from the polynucleotide sequences as set forth in SEQ ID NOS: 1-1079.
It comprises about 100, about 150 to about 200, about 250 to about 300, or about 350 consecutive nts. For the most part, fragments are at least about 15 nt, usually at least 18 nt or 25 nt, and up to at least about 50 consecutive n.
It is a fragment of t length or longer. 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: 1-1079.

Probes specific for the polynucleotides of the present invention can be made using the polynucleotide sequences disclosed in SEQ ID NOs: 1-1079. The probe is preferably at least about 12 of the corresponding contiguous sequences of SEQ ID NOS: 1-1079.
, 15, 16, 18, 20, 22, 24, or 25 nt fragments and can be less than 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 is, for example, a radioactive tag, a biotinylated tag,
Or it can be labeled with a fluorescent tag. Preferably, the probe is SEQ ID NO: 1-107.
Designed based on the identified sequence of one of the nine polynucleotides. More preferably, the probe is designed based on a continuous 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, the unmasked regions are selected, as indicated by the polynucleotide outside the poly-n stretch of masked sequence generated by the masking program).

The polynucleotides of the present invention are generally isolated and obtained with substantial purity, except for intact chromosomes. Usually, the polynucleotide is DNA or R
Obtained as being substantially free of other naturally-occurring nucleic acid sequences, as any of the NAs, and generally being at least about 50%, usually at least about 90% pure,
And is typically "recombinant," for example flanked by one or more nucleotides that are not normally associated in the naturally occurring chromosome.

The polynucleotide of the present invention may be provided as a linear molecule or in a circular molecule, and may be provided in an autonomously replicating molecule (vector) or in a molecule without a replication sequence. . Expression of polynucleotides is by themselves
Alternatively, it may be regulated by other regulatory sequences known in the art. Polynucleotides of the invention can be prepared by a variety of techniques available in the art (eg, transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acid, liposome-mediated DNA transfer, DNA coating). Trafficking of modified latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate mediated transfection, etc.) and can be introduced 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 1079 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: 1-1079, or at least 12, 1
Polynucleotides having those moieties containing 5, 18, or 20 nt may be probed as described in USPN 5,654,173,
Used as a hybridization probe to detect hybridizing members of a cDNA library using cloning methods and clone selection techniques. A library of cDNA is made from selected tissues, such as normal or tumor tissues, or from mammalian tissues, eg, treated with a pharmaceutical agent. Preferably, the tissue is the same as the 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 c
DNA 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 identification 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. When the provided polynucleotides are isolated from a cDNA library, the library is a human colon cell, more preferably a human colon cancer cell m.
RNA, even more preferably from highly metastatic colon cells Km 12L4-A.

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: 1-1079. In one embodiment, the 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 larger than the polynucleotide provided and which preferably contain the complete coding sequence of the native message. cDNA
To confirm that all were obtained, RNA protection experiments are performed as follows. Hybridization of full-length cDNA to mRNA protects RNA from RNase degradation. If the cDNA is not full length, the portion of the mRNA that does not hybridize is subject 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. S
ambrook et al., Molecular Cloning: A Laborat
ory Manual, 2nd edition, (1989) Cold Spring Har
bor Press, Cold Spring Harbor, NY. Partial c
To obtain additional sequences 5'to the ends of the DNA, 5'RACE (PCR Protocols: A Guide to Methods and Ap) was obtained.
applications, (1990) Academic Press, Inc.
) Can be done.

Genomic DNA is isolated using a polynucleotide provided in a manner similar to the isolation of full length cDNA. Briefly, the provided polynucleotide, or a portion thereof, is used as a probe for a library of genomic DNA. Preferably, this library is obtained from the cell types used to produce the polynucleotides of the invention. However, this is not mandatory. Most preferably, this genomic DNA is obtained from the biological material described in the examples herein. Such libraries may be present in vectors suitable for carrying large segments of the genome (eg P1 or YAC as described in detail in Sambrook et al., 9.4-9.30). In addition, genomic sequences can be isolated from human BAC libraries. This is, for example, Research Genetic
s, Inc. , Huntville, Alabama, USA. To obtain additional 5'or 3'sequences, chromosomal walking is performed as described in Sambrook et al., Resulting in the isolation of flanking and overlapping fragments of genomic DNA. These are mapped and stitched together using restriction digests and DNA ligases as are 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. Performed on many cell types to determine which cell line expresses the gene of interest at the highest level using either method, preferably Northern blot. 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 viral or expression vectors. Typically, a library of mRNAs containing poly (A) tails can be generated using poly (T) primers. Similarly, a cDNA library 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 sequence from the full length cDNA corresponding to the polynucleotide of the invention. Such PCR methods include gene trapping and RACE methods. Gene trapping involves inserting a member of a cDNA library into a vector. The vector is then denatured to produce a single chain molecule. Next, a substrate-bound probe (eg,
Biotinylated oligos) are used to trap the cDNA insert of interest. The biotinylated probe can be linked to an avidin-conjugated solid substrate. The PCR method is
It can be used to amplify this trapped cDNA. Labeled probe sequences are based on the polynucleotide sequences of the invention to trap sequences corresponding to the full-length gene. Random primers specific for the library vector can be used to amplify this trapped cDNA. Such gene trapping techniques are described in Gruber et al., WO 95/04745 and Gru.
Ber et al., US Pat. No. 5,500,356. Kits for performing gene trapping experiments are commercially available (eg, Life Tech).
Nologies, Gaithersburg, Maryland, USA).

“Rapid amplification of cDNA ends,” or RACE, is a PCR method that amplifies cDNA from a number of different RNAs. The cDNA is attached to an oligonucleotide linker and amplified by PCR with two primers. 1
One primer is based on a sequence derived from the polynucleotide of the invention, which is desired for the full length sequence. And the second primer contains a sequence that hybridizes with an oligonucleotide linker for amplifying the cDNA. A description of this method is given in WO 97/19110. In a preferred embodiment of RACE,
Common primers are designed to anneal with any adapter sequence that is ligated to the ends of the cDNA (Apte and Siebert, Biotechni.
ques (1993) 15: 890-893; Edwards et al., Nuc. Ac
ids Res. (1991): 5227-5232). Single gene-specific R
When the ACE primer pairs with the generic primer, preferential amplification of the sequence occurs between the single gene-specific primer and the generic primer. Commercially available cDNA pools modified for use in RACE are available.

Another PCR-based method does not require specific information of the cDNA sequence and produces a full-length cDNA library with immobilized ends. This method uses a lock-docking primer (I-VI) (one primer, polyTV (I-II).
I) anchors over the poly A tail of the eukaryotic mRNA resulting in first-strand synthesis, and the second primer, polyGH (IV-VI), is added by terminal deoxynucleotidyl transferase (TdT). Immobilized on a poly C tail) (see, for example, WO 96/40998).

The promoter region of the gene is generally located 5'to the start site for RNA polymerase II. Hundreds of promoter regions contain a "TATA box" (eg, sequences such as TATTA or TATAA), which are susceptible to mutations. The promoter region can be obtained by performing 5'RACE using primers derived from the coding region of the gene. 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 has been obtained, the DN encoding the variant
A 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 of optional changes in amino acids to achieve changes in protein structure and / or function.

As an alternative method for 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. Thus, the present invention corresponds to at least 15 contiguous nucleotides of 15 nucleotide length (one of SEQ ID NOs: 1-1079) suitable for one or more biological manipulations, including replication and expression of nucleic acid molecules. ) To the maximum length. The present invention comprises (a) a nucleic acid having the size of the complete gene and comprising at least one of SEQ ID NOs: 1-1079; (b) also comprising at least one additional gene, allowing expression of the fusion protein. A nucleic acid of (a) operably linked for: (c) an expression vector comprising (a) or (b); (d)
A plasmid containing (a) or (b); and (e) a recombinant virus particle containing (a) or (b), but not limited thereto. Once provided with the polynucleotides disclosed herein, the construction and preparation of (a)-(e) is well known to those of skill in the art.

Nucleic acid sequences comprising at least 15 contiguous nucleotides of at least one of SEQ ID NOS: 1-1079, preferably nucleic acid sequences comprising the entire sequence of at least one of SEQ ID NOS: 1-1079 are not limited thereto. Not A, T, G
, And / or C (with respect to DNA) and A, U, G, and / or C
It can be any sequence (for RNA) or their modified bases, including inosine and pseudouridine. The choice of sequence will be based on the desired function and may be dictated by the desired coding region, desired intron-like region, and desired regulatory region. When the entire sequence of any one of SEQ ID NOs: 1-1079 is present within a nucleic acid, the resulting nucleic acid is referred to herein as a polynucleotide comprising the sequence of any one of SEQ ID NOs: 1-1079.

(Expression of Polypeptide Encoded by Full-length cDNA or Full-length Gene) A provided polynucleotide (for example, a polynucleotide having one sequence of SEQ ID NOs: 1 to 1079), a corresponding cDNA, or a full-length gene is , Used to express partial or complete gene products. Sequence number 1-1079
A polynucleotide construct having the sequence of may also be synthetically produced. Alternatively, single-step assembly of genes and entire plasmids from multiple members of oligodeoxyribonucleotides is described, for example, in Stemmer et al., Gene (Am.
sterdam) (1995) 164 (1): 49-53.
In this method, assembly PCR (synthesis of long DNA sequences from multiple members of an oligodeoxyribonucleotide (oligo)) is described. This method
DNA shuffling (Stemmer, Nature (1994) 370: 3
89-391) and does not rely on DNA ligase, but instead to construct increasingly longer DNA fragments during the assembly process.
Rely on A polymerase.

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 United States Dept. of HHS, Nat
ional Institute of Health (NIH) Guidel
Purified under current rules as described in the ins 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 them 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 metastasis 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 the preparation of vectors containing the desired sequence are well known in the art.

The polynucleotides set forth in SEQ ID NOS: 1-1079 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 nascent-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 of the above host cells, or other suitable host cells or organisms, are used to replicate and / or express the polynucleotides or nucleic acids of the invention, the resulting replicated Nucleic acids, RNA, expressed proteins or polypeptides are within the scope of the invention as a product of the host cell or organism. 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 Screened Against Publicly Available Databases Provided polynucleotides, cDNAs, or translations of the nucleotide sequence of the complete gene are individually labeled. Can be aligned with known sequences of. The similarity to individual sequences can be used to determine the activity of the polypeptide encoded by the polynucleotide of the invention. Also, sequences exhibiting similarities 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 flanking polynucleotide sequences can be used as probes and primers to identify and isolate full-length sequences corresponding to the provided polynucleotides. The closest neighbors may indicate a tissue or cell type that can be used to construct a library for the full length sequence corresponding to the provided polynucleotide.

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 three frames may be sufficient (with a few specific exceptions as described in the Examples). But there is). These amino acid sequences are commonly referred to as "query sequences," which are aligned with the individual sequences. 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. The databases are Genbank, EMBL, and DNA Database.
of Japan (DDBJ).

The query sequence and individual sequences can be aligned using the methods and computer programs described above, and BLAST 2.0 (http: //www.n.
cbi. nlm. nih. Gov / BLAST / available on the World Wide Web). Altschul et al., Nucleic Acids Res. (
1997) 25: 3389-3402. Another alignment algorithm is Fasta (Genetics Computing Group (GCG
) Available in packages, Madison, Wisconsin, USA, Ox
ford Molecular Group, Inc. Is a wholly owned subsidiary). Other techniques for alignment are described in Doolittle, supra. Preferably, alignment programs that allow gaps in the sequences are utilized to align the sequences. Smith-Waterman is one type of algorithm that allows for gaps in sequence alignments. Meth. Mol. Biol. (1
997) 70: 173-187. Also, the GAP program using the Needleman and Wunsch alignment methods can be utilized to align sequences. An alternative search strategy uses MPSRCH software.
This is done on a MASPAR computer. MPSRCH is used by Smith-Waterm to score sequences on a massively parallel computer.
Use the an algorithm. This approach improves the ability to identify sequences that are distantly associated 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. All sequences published at the filing date of this application, either by DNA sequence databases or protein sequence databases, including patent databases (eg, GeneSeq), are incorporated herein by reference. Sequences deposited in these databases at the filing date of the present application, but which have not been made public since the filing date of the present application, are also incorporated herein by reference.

The results of the alignment of individual and query sequences can be divided into three categories: high similarity, weak similarity, and no similarity. Individual alignment results, ranging from high similarity to weak similarity, provide the basis for determining polypeptide activity and / or structure. Parameters for categorizing individual results include: percentage of aligned region length where the strongest alignment is found, percent sequence identity, and p-value. The percentage of alignment region length is found in the region of the strongest alignment (eg, the contiguous region of an individual sequence that contains the largest number of residues that are identical to the residues in the corresponding region of the aligned query sequence). It is calculated by counting the number of residues in each individual sequence issued. This number is divided by the total residue length of the query sequence to calculate the percentage. For example, a query sequence of 20 amino acid residues may be
Can align with amino acid regions. The individual sequences are amino acid residue 5 of the query sequence,
9-15 and 17-19. Thus, the region of strongest alignment is the 11 amino acid stretch, which is the region extending to residues 9-19. The percentage of alignment region length is as follows: 11 (length of region of strongest alignment) 2
Divided by 0 (query array length), or 55%.

Percent sequence identity counts the number of amino acid matches between the query and individual sequences, and the total number of matches is the number of individual sequence residues found in the region of the strongest alignment. Calculated by dividing. Therefore, the percent identity in the above example is 10 matches divided by 11 amino acids, or about 90.9%.
Is.

The p-value is the probability that the alignment happened by chance. For simple alignment, the p-value is Kar
Lin et al., Proc. Natl. Acad. Sci. (1990) 87: 226.
4 and Karlin et al., Proc. Natl. Acad. Sci. (1993
) 90. The p-value for multiple alignments using the same query sequence is
Altschul et al., Nat. Genet. (1994) 6: 119 and can be calculated using the heuristic approach. Alignment programs such as the BLAST program can calculate p-values (see also Altschul et al., Nucleic Acids Res. (1997) 25: 3389-3402).

Another factor to consider in determining identity or similarity is localization of similarity or identity. A strong local alignment may indicate similarity, even if the alignment length is short. Sequence identity distributed over the length of the query sequence may also indicate similarity between the query and profile sequences. The boundaries of the regions where the sequences align are described in Doolittle, supra; BLAST 2.0 (eg, Altschul et al., Nucleic Aci.
ds Res. (1997) 25: 3389-3402) or according to the FAST program; or by determining the region of highest sequence identity.

High Similarities In alignment results that are generally 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%; much more typically at least about 60% of the total residue length of the query sequence. Generally, the percent of length of the alignment region can be as high as about 62%; more usually as high as about 64%; much more usually as high as about 66%. Further, for high similarity, regions of alignment will typically have at least about 75% sequence identity; more typically, at least about 78%; and even more typically, at least about 80% sequence. Shows identity.
Usually, percent sequence identity is as high as about 82%; more usually about 84%.
Much higher; much more usually can be as high as about 86%.

The p-value is used in conjunction with these methods. If high similarity is found, p
If the value is about 10 ^-2 or less; more usually about 10 ^-3 or less; much more usually about 10 ^-4 or less, the query sequence has a high degree of similarity to the profile sequence. Then be considered. More typically, the p-value will not exceed about 10 ^-5 ; more typically it will be about 10 ^-10 or less; much more typically, for the query sequences to be considered highly similar. Is about 10 ⁻¹⁵ or less.

Weak Similarities In general, when the alignment result is considered to be a weak similarity, there is no minimum percentage of the length of the alignment region or the minimum length of the alignment. A more sufficient indication of weak sequence similarity is that the region of alignment is typically at least about 15 amino acid residues in length; more typically at least about 20; even more typically; at least about 25 amino acids in length. To be considered in case. Generally, the length of the alignment regions will be about 30 amino acid residues; more usually about 40; even more usually about 60 amino acid residues. Moreover, for weak similarity, regions of the alignment typically exhibit at least about 35% sequence identity; more typically at least about 40%; even more typically; at least about 45% sequence identity. Show. Generally, percent sequence identity can be as high as about 50%; more usually as high as about 55%; even more usually as high as about 60%.

If low similarity is found, the query sequence is usually less than or equal to about 10 ⁻³ if the p-value is typically less than or equal to about 10 ⁻² ; If equal; even more usually; less than or equal to about 10 ^−4, it is considered to have weak sequence similarity with the profile sequence. More typically, for query sequences that are considered to be weakly similar, the p-value does not exceed about 10 ⁻⁵ : more usually; does not exceed about 10 ⁻¹⁰ or is equal to this; Even more usually; not more than or equal to about 10 ^-15 .

Similarity (Determined by Sequence Identity Alone) Sequence identity alone can be used to determine the similarity of query sequences to individual sequences and can indicate the activity of that sequence. Such an alignment preferably allows 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%; and even more typically,
At least about 25%; and even more typically at least about 50%, is associated with the profile sequence. As a measure of similarity, sequence identity alone is most useful 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. More typically, similarity is based on sequence identity alone when the query sequence is preferably 100 residues long; more preferably 120 residues long; even more preferably 150 amino acid residues long. It can be concluded.

(Alignment with Profile Sequences and Multiple Aligned Sequences) Provided polynucleotide translations can be aligned with amino acid profiles that define either a protein family or a common motif. Also, translations of the provided polynucleotides can be aligned to multiple sequence alignments (MSA) containing the polypeptide sequences of protein family or motif members. The 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, sequences that show identity and similarity to chemokine profiles or MSA may exhibit chemokine activity.

Profiles can be manually designed by (1) creating MSA, which is an alignment of amino acid sequences of members belonging to the family, and (2) constructing a statistical representation of the alignment. Such methods are described, for example, in Birney et al., Nucl.
． Acid Res. (1996) 24 (14): 2730-2739. MSAs for several protein families and motifs are publicly available. For example, http: // genome. wastel. edu
/ Pfam / contains MSAs of 547 different families and motifs.
These MSAs are also described by Sonnhammer et al., Proteins (1997).
) 28: 405-420. Other sources on the World Wide Web include http: // www. embl-heidelberg.
de / argos / ali / ali. html; or ALI @ EMBL-HEIDELBERG. A message may be sent to the DE. A brief description of these MSAs can be found in Pascarella et al., Prot. En
g. (1996) 9 (3): 249-251. Techniques for constructing profiles from MSA are described by Sonnhammer et al., Supra; Bir.
ney et al., supra: and "Computer Methods for Polymer Sequence Analysis," Meth.
od in Enzymology (1996) 266, Doolittle,
Academic Press, Inc. San Diego, California
nia, USA.

The similarity between the query sequence and the protein family or motif is (a
) By comparing the query sequence against the profile, and / or (
b) Can be determined by aligning the query sequence with family or motif members. Typically, a program like Searchwise
Used to compare query sequences against a statistical representation of multiple alignments, also known as profiles (see Birney 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 the query sequence with members of a family or motif also known as MSA. Sequence alignments can be created using any of a variety of software tools. Examples include PileUP, which creates multiple sequence alignments and is described by Feng et al. Mol. Evol. (
1987) 25: 351. Another way GAP is Needl
eman et al. Mol. Biol. (1970) 48: 443. GAP is best suited for the overall alignment of sequences. Be of the third method
stFit is described in Smith et al., Adv. Appl. Math. (1981) 2:
It works by inserting gaps to maximize the number of matches using the 482 local homology algorithm. Generally, the following factors are used to determine whether there is a similarity between the query sequence and the profile or MSA: (1) the number of conserved residues found in the query sequence, ( 2) Percentage of conserved residues found in the query sequence, (3) number of frameshifts, and (4) spacing between conserved residues.

If some alignment programs that both translate and align the sequences translate the nucleotide sequences to produce the best alignment, any number of frameshifts can be made. The fewer frameshifts required to make an alignment, the stronger the similarity or identity between the query sequence and the profile or MSA. For example, the weak similarity that results from no frameshifting may be a better indication of the activity or structure of the query sequence than the strong similarity that results from two frameshifting. 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 are found in the alignment of the query sequence with the profile or MSA.

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

Typically, the residue of a polypeptide is an amino acid or single amino acid class that is at least about 40% of all class members; more typically at least about 50%; Is conserved if found at a particular location in at least about 60% of the members. Usually, residues are at least about 7 members of a class or single amino acid family or motif member.
0%; more usually at least about 80%; even more usually at least about 90%;
Even more usually, it is conserved if found in at least about 95%.

Residues are considered to be conserved if three unrelated amino acids; more usually two unrelated amino acids are found at particular positions in some or all of the members. These residues are unrelated amino acids at least about 40% of members of all classes; more typically at least about 50%;
Even more typically, it is conserved if found at a particular location in at least about 60% of the members. Usually, the residue is at least about 70% of the members of the family or motif in the class or single amino acid; more usually at least about 80%; even more usually at least about 90%; even more usually at least Conserved if found in about 95%.

The query sequence is at least about 25% of the conserved residues of the profile or MSA; more usually at least about 30%; even more usually; at least 40.
If included, the query sequence has similarity 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%. Sequence is profile sequence or MS
It has a stronger similarity to A.

(Identification of Secretory Polypeptide and Membrane-bound Polypeptide) Both the secretory polypeptide and the membrane-bound polypeptide of the present invention are particularly important. For example, secretory polypeptide levels 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 eliciting an immune response. Such antigens include all or part of the extracellular region of the membrane-bound polypeptide. Since both secretory and membrane-bound polypeptides contain fragments of contiguous hydrophobic amino acids,
Algorithms that predict hydrophobicity can be used to identify such polypeptides.

Signal sequences are usually encoded by both secretory and membrane-bound polypeptides in order to direct the polypeptide to the surface of cells. Signal sequences usually include stretches of hydrophobic residues. Such signal sequences can be folded into a helical structure. Membrane-bound polypeptides typically include at least one transmembrane region that carries a stretch of hydrophobic amino acids that can cross the membrane. Some transmembrane regions also show helical structures. Hydrophobic fragments within the polypeptide can be identified by using computer algorithms. Examples of such algorithms include Hopp and Woods, Proc. Natl
． Acad. Sci. USA (1981) 8: 3824-3828; Kyte and Doolittle, J. et al. Mol. Biol. (1982) 157: 105.
-132; and RAOAR algorithm, Degli Esposti et al., E.
ur. J. Biochem. (1990) 190: 207-219.

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

Identification of Function of Full-Length Gene Expression Product Ribozymes, antisense constructs, and dominant negative mutants were used to determine the function of expression products of genes corresponding to the polynucleotides provided herein. Can be used. These methods and compositions are particularly useful when the novel polynucleotides provided do not show significant and substantial homology to sequences encoding genes of known function. Antisense molecules and ribozymes can be constructed from synthetic polynucleotides. Typically, the phosphoramidite method of oligonucleotide synthesis is used. Beaucage et al., Tet. Lett
． (1981) 22: 1859 and U.S. Pat. No. 4,668,777. Automated equipment for synthesis is available to synthesize oligonucleotides using this chemical. An example of such a device is the Applie
d Biosystems a division of Perkin El
mer Corp. , Foster City, California, USA
Biosearch 8600.392 and 394; and Per.
cceptive Biosystems, Framingham, Massac
Husetts, Expedite by USA. Synthetic RNA, phosphate analog oligonucleotides, and chemically derivatized oligonucleotides can also be produced and covalently attached to other molecules. RNA
Oligonucleotides can be synthesized using, for example, RNA phosphoramidites. This method is described in Applied Biosystems, 392 and 39.
It can be performed in an automated synthesizer such as a Type 4, Foster City, California USA.

Phosphorothioate oligonucleotides can also be synthesized for antisense construction. Sulfuration reagents (eg, tetraethylthiuram in acetonitrile (
thiraum) disulfide (TETD) can be used to convert internucleotide cyanoethyl phosphites to phosphorothioate triesters within 15 minutes at room temperature. TETD replaces the indole reagent, but the other reagents used in standard phosphoramidite chemistry remain the same. Such a synthesis method is described, for example, in Applied Biosystems, Model 39.
2 and Model 394 can be used to automate.

Oligonucleotides of up to 200 nucleotides, more typically 100 nucleotides, more typically 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, Sa
See mbrook et al., supra. Trans-cleaving catalytic RNA (ribozyme)
Is an RNA molecule that possesses endonuclease 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. Cleavage events destabilize mRNA and prevent protein expression. Importantly, ribozymes can be used to inhibit the expression of genes of unknown function for the purpose of determining the unknown function in in vitro or in vivo situations by detecting phenotypic effects. One commonly used ribozyme motif is the hammerhead, for which the substrate sequence requirements are minimal. The design of hammerhead ribozymes, as well as therapeutic uses for ribozymes, is described by Usman et al., Current.
Opin. Struct. Biol. (1996) 6: 527. Methods for the production of ribozymes (including heparin-structured ribozyme fragments), methods of increasing ribozyme specificity, etc. are known in the art.

The hybridizing region of ribozyme includes Horn and Urdea, Nucle.
ic Acids Res. (1989) 17: 6959, or prepared as a branched structure. The basic structure of ribozymes can also be chemically altered in methods well known to those skilled in the art, and chemically synthesized ribozymes can be administered as synthetic oligonucleotide derivatives modified by monomeric units. In the therapeutic setting, liposome-mediated delivery of ribozymes is described by Birikh et al., Eur. J. Biochem (
1997) 245: 1 to improve cell uptake.

Antisense nucleic acids are designed to specifically bind RNA, and RNA-D
Resulting in the formation of NA or RNA-RNA hybrids, DNA replication, reverse transcription,
Alternatively, the translation of messenger RNA is stopped. Antisense polynucleotides based on the selected polynucleotide sequence may interfere with expression of the corresponding gene. Antisense polynucleotides are typically made intracellularly by expression from an antisense construct containing the antisense strand as the transcribed strand. Antisense polynucleotides based on the disclosed polynucleotides bind and / or prevent the translation of mRNA containing sequences complementary to the antisense polynucleotide. The expression products of control cells and cells treated with the antisense construct are compared to detect the protein product of the gene corresponding to the polynucleotide on which the antisense construct is based. The protein is isolated and identified using conventional biochemical methods.

Given the broad background literature and clinical experience in antisense therapy, one of skill in the art can use selected polynucleotides of the present 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", it is desirable to test the polynucleotide as an antisense compound in the corresponding cancer cells.

As an alternative method for identifying the function of the genes corresponding to the polynucleotides disclosed herein, dominant negative mutations can be readily made for the corresponding proteins that are active as homomultimers. . The mutant polypeptide interacts with the wild-type polypeptide (made from the other allele) and forms a non-functional multimer. Thus, the mutation is in the substrate binding domain, catalytic domain, or cellular localization domain. Preferably, the mutant polypeptide may be overproduced. A point mutation having such an effect is produced. Moreover, the function of different peptides of varying lengths to the ends of the protein can give rise to dominant negative mutants. Common strategies are available to generate dominant negative mutants (eg Her.
Skowitz, Nature (1987) 329: 219). Such techniques can be used to create loss-of-function mutations that 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 SEQ ID NOs: 1 to 1079 or a variant thereof.

In general, as used herein, the term "polypeptide" is encoded by the full-length polypeptide encoded by the indicated polynucleotide, the gene indicated by the indicated polynucleotide. Refers to both polypeptides, and parts or fragments thereof. "Polypeptide" also includes variants of naturally occurring proteins, where such variants are homologous or substantially similar to naturally occurring proteins and are naturally occurring. It 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 is at least about 80%, usually at least about 90% with the differentially expressed polypeptide of the invention as measured by BLAST 2.0 using the parameters described above. , And more usually, sequences having at least about 98% sequence identity. Variant polypeptides are naturally glycosylated or are not naturally glycosylated (ie, the polypeptides have a glycosylation pattern that differs from that found in the corresponding naturally occurring protein). ).

The present invention also includes homologues (or fragments thereof) of the disclosed polypeptides, wherein the homologues are isolated from other species (ie, other animal or plant species), Such homologues are usually of mammalian species (eg, mouse,
Rodents such as rats: livestock (eg horses, cows, dogs, cats); and humans. By "homolog" is meant a polymorph having at least about 35%, usually at least about 40%, and more usually at least about 60% amino acid sequence identity to a particular specifically expressed protein as identified above. Peptide is meant where sequence identity is BLA using the parameters described above.
Determined using the ST 2.0 algorithm.

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, the protein of the invention is present in a composition enriched for that protein as compared to a control. As such, a purified polypeptide is provided, wherein purified means that the protein is present in a composition that is substantially free of differentially expressed polypeptide, where "substantially" By "absent" is meant that less than 90%, usually less than 60%, and more usually less than 50% of the composition 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 includes 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. The substitution or deletion of the above cysteine residues may be a substitution for minimizing misfolding. Conservative amino acid substitutions are those that preserve the overall charge, hydrophobicity / hydrophilicity, and / or steric bulk of the amino acid being replaced. Variants retain the enhanced biological activity of specific regions of the protein (eg, functional domains and / or regions associated with the consensus sequence if the polypeptide is a member of the protein family). Or can be designed to have. The choice of amino acid changes for the generation of variants is based on the accessibility of the amino acids (internal vs. external) (eg Go et al. Int. J. Peptide Prote.
in Res. (1980) 15: 211), the thermal stability of variant polypeptides (eg, Querol et al., Prot. Eng. (1996) 9: 2).
65), the desired glycosylation site (eg Olsen and Th).
omsen, J .; Gen. Microbiol. (1991) 137: 579), the desired disulfide bridge (eg, Clarke et al., Bioch.
emissry (1993) 32: 4322; and Waarchuchuk et al., P.
protein Eng. (1994) 7: 1379), the desired metal binding site (eg, Toma et al., Biochemistry (1991) 30:
97, and Haezerbrook et al., Protein Eng. (199
3) 6: 643), as well as desired substitutions within the proline loop (eg Mas
ul et al., Appl. Env. Microbiol. (1994) 60: 3579.
) Can be based on. Cysteine-depleted muteins are described in US Pat. No. 4,959,3
It may be produced as described in No. 14.

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 more than about 1000 amino acids in length. No, here the fragment has a stretch of amino acids which is identical to the polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 1-1079 or homologues 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 In general, a library of polynucleotides is a collection of sequence information, which information is in biochemical form (eg, as a collection of polynucleotide molecules), or electronically. Provided in any form (eg, as a collection of polypeptide nucleotide sequences stored in a computer-readable form, as 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. Generally, the disease marker is all cells affected by the 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). Is a representation of the gene product present in. 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, it is either over-expressed or repressed in breast ductal cells affected by cancer.

The nucleotide sequence information for the library may be embodied in any suitable form, such as electrical or biochemical form. For example, a library of sequence information embodied in electrical form is, 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 and non-malignant cancer cells (
Or normal cells) and / or vi) dysplastic cells compared to normal cells are differentially expressed (eg, overexpressed or repressed).
Contains available computer data files (or collections of nucleic acid molecules in biochemical form) containing representative nucleotide sequences of genes. Other combinations and comparisons of cells affected by various diseases or stages of disease are
It will be readily apparent to one skilled in the art. The 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 the entire genes in the library or fragments thereof, as described in more detail below. Can respond.

Polynucleotide libraries of the invention generally include sequence information for multiple polynucleotide sequences, wherein at least one polynucleotide has any of the sequences SEQ ID NOs: 1-1079. By plural is meant at least 2, usually at least 3, and can include all of SEQ ID NOS: 1-1079. The length and number of polynucleotides in a library will vary with the nature of the library, eg, whether the library is an oligonucleotide array, a cDNA array, a computer database of sequence information, etc.

When 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 NO: 1
The nucleic acid sequence of any of the 1079 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,
RAM and ROM); and hybrids of these categories (eg,
Magnetic / optical storage media), but is 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 the product 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 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, Syb
gapped BLAST in the asase system (Altschul et al., Nucl
eic Acids Res. (1997) 25: 3389-3402) and BLAZE (Brutlag et al., Comp. Chem. (1993) 17:20.
3) The search algorithm uses open reading frames (O
Can be used to identify ORFs within the genome that contain homology to (RF).

As used herein, "computer-based system" refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware of the computer-based system of the present invention comprises 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 a 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 fragments or regions of the genome that match a particular target sequence or target motif. A variety of known algorithms
Publicly known and commercially available (eg MacPattern (E
MBL), BLASTN, and BLASTX (NCBI)). A "target sequence" can be any polynucleotide, or amino acid sequence of 6 or more contiguous nucleotides, or 2 or more amino acids, preferably about 10-100 amino acids, or about 30-300 nucleotides. A variety of comparison means can be used to achieve comparison of sequence information from a sample using data storage means (eg, analyzing target sequences, target motifs, or relative expression levels). One of skill in the art will readily appreciate that any one of the publicly available homology search programs can be used as a search tool for the computer-based systems of the present invention to achieve target sequence and motif comparisons. Can be recognized. Computer programs for analyzing expression levels in samples and controls also
Are known in the art.

“Target structural motif” or “target motif” refers to any rationally selected sequence or combination of sequences, where a sequence is a tertiary form formed upon folding of the target motif. Selection is based on original conformation or on the consensus sequence of regulatory or active sites. A wide variety of target motifs are known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, heparin structures, promoter sequences, and other expression elements such as binding sites for transcription factors.

A variety of structural formats for input and output means can be used to input and output information in the computer-based system of the present invention.
One format for the output means assesses 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: 1-1.
A biochemical library of 079 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: 1-1079 is represented in an array. The array has at least two different nucleic acid targets on one of the surfaces of the substrate,
By article of manufacture having at least one substrate is meant here the number of different nucleic acids can be quite high, typically at least 10 nucleotides, usually at least 20 nucleotides and often at least 25 nucleotides.
A wide 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, analog libraries of polypeptides are also provided, wherein the polypeptides of the library are at least a portion of the polypeptides encoded by SEQ ID NOs: 1-1079. Represents

Utility Uses of Polynucleotide Probes in Mapping and Tissue Profiling Polynucleotide probes generally comprise at least 12 contiguous nucleotides of a polynucleotide as shown in the Sequence Listing, for a variety of purposes ( For example, chromosomal mapping of polynucleotides 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 have at least 5, 10, or 20-fold higher detection signaling than background hybridization provided with other unrelated sequences. Should be provided.

Detection of Expression Level 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 by hybridizing to a particular size mRNA species. The amount of hybridization is quantified, for example, to detect 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 initiate the reaction. This primer is 3'to or within the polynucleotide of the sequence listing.
And the 5'sequence. 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 is also Sambr
Look et al., “Molecular Cloning: A Laboratory”.
Manual "(New York, Cold Spring Harbor)
It can be detected by traditional blotting techniques (eg, Southern blot, Northern blot, etc.) described in Laboratory, 1989) (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 invention can be used to identify the chromosome where the corresponding gene resides. Such a mapping may be useful in identifying the function of a polynucleotide-related gene by virtue of its proximity to other genes of known function. Function can also be assigned to the polynucleotide-related gene 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 facilitates comparative genomic hybridization to allow global genomic evaluation of changes in relative copy number DNA. (For example, Vald
es et al., Methods in Molecular Biology (199).
7) 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 Genetics, (
1995) 33: 63-99; Walter et al., Nature Genetic.
s (1994) 7:22; Walter and Goodfellow, Tren.
See ds in Genetics (1992) 9: 352. Panel on Radiative Hybrid Mapping, Research Genetics
, Inc. , Huntsville, Alabama, USA. A database of markers using various panels is available at http: // F / sh
gc-www. stanford. edu; and http: // www-ge
nome. wi. mit. edu / cgi-bin / contig / rhmap
per. Pl is available via the internet. 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 can be found at http: // www. sph. umich. edu / g
It is available via the internet at group / statgen / software. 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 Profiling The expression of specific mRNA corresponding to a provided polynucleotide can be altered in different cell types and can 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, a branched DNA probe assay, or a blotting technique that utilizes nucleic acid probes that are substantially identical or complementary to the polynucleotides listed in the Sequence Listing is used to determine the presence of the corresponding cDNA or mRNA. Or the absence may be determined.

Tissue typing is used to identify the developing organ or tissue source of metastatic lesions by identifying the expression of specific markers of that organ or tissue. When the polynucleotide is expressed only in a specific tissue type,
And when a metastatic lesion was found to express the polynucleotide, a developmental source of the lesion was identified. Expression of a particular polynucleotide can be assayed by detection of either the corresponding mRNA or protein product. As will be readily apparent to any forensic practitioner, the sequences disclosed herein are useful in distinguishing human from non-human tissue. In particular, these sequences
For example, it is useful for distinguishing human tissue from avian, reptile, and amphibian tissues.

(Use of Polymorphism) The polynucleotide of the present invention can be used for forensic analysis, genetic analysis,
It can be used in mapping and diagnostic applications, where the corresponding region of the gene is a polymorphism 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, It is not limited to these.

Antibody Production The expression products of the polynucleotides of the invention and the corresponding mRNAs, cDNAs, or complete genes have been prepared and used for experimental, diagnostic, and therapeutic purposes. Can be used to trigger For polynucleotides to which the corresponding gene has not been assigned, this provides an additional method for identifying the corresponding gene. The polynucleotide or related cDNA is expressed and antibodies are prepared as described above. These antibodies are specific for an 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. Can be obtained or bound to it.

Methods for the production of antibodies that specifically bind a selected antigen are well known in the art. An immunogen for raising antibodies is prepared by mixing the polypeptide encoded by the polynucleotide of the present invention with an adjuvant.
And / or can be prepared by making a fusion protein with a larger immunogenic protein. Polypeptides can also be covalently linked to other larger immunogenic proteins such as keyhole limpet hemocyanin. Immunogens are typically intradermal, subcutaneous, or intramuscular to produce antibodies,
It is administered to laboratory animals such as rabbits, sheep, and mice. Monoclonal antibodies can be made by isolating splenocytes and fusing myeloma cells to form hybridomas. Alternatively, the selected polynucleotide is directly administered and expressed in vivo, for example, by intramuscular injection. The expressed protein produces a variety of protein-specific immune responses, including the production of antibodies, which is comparable to administration of the protein.

Preparation of polyclonal and monoclonal antibodies specific for the polypeptides encoded by the selected polynucleotides is made using standard methods known in the art. The antibody specifically binds to an epitope present on the polypeptide encoded by the polynucleotide disclosed in the Sequence Listing. Typically, at least 6, 8, 10, or 12 contiguous 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 human polypeptides encoded by provided polypeptides, when used in Western blots or other immunochemical assays, are more likely than detection signals provided with other proteins. It should provide at least 5, 10, or 20 times higher detection signal. Preferably, antibodies specific for the polypeptides of the invention do not bind detectable levels to other proteins in immunochemical assays and are capable of immunoprecipitating certain polypeptides from solution.

The present invention also contemplates naturally occurring antibodies specific for the polypeptides of the present invention. For example, serum antibodies to polypeptides of the invention in the human population can be obtained by methods well known in the art, for example by 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.

Polynucleotides or Arrays for Diagnostic Polynucleotide arrays provide a high throughput technique by which a large number of polynucleotide sequences in a sample can be assayed. This technique can be used as a diagnostic and as a tool for testing differential expression, eg, to determine the function of the encoded protein. Arrays can be made by spotting polynucleotide probes on a substrate (eg, glass, nitrocellulose, etc.) in a two-dimensional matrix or array with the probes attached. The probe can be bound 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 the probe. The double-stranded polynucleotide comprises a labeled sample polynucleotide bound to a probe polynucleotide and can be detected once the unbound portion of the sample has been washed away.
Techniques for constructing arrays, and methods of using these arrays are described in EP7.
99,897; WO97 / 29212; WO97 / 27317; EP785,2.
80; WO97 / 02357: US Patent No. 5,593,839; US Patent No. 5
, 578,832; EP 728,520; U.S. Pat. No. 5,599,695;
EP 721 016; US Pat. No. 5,556,752; WO95 / 22058.
And US Pat. No. 5,631,734. 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 differential expression of polynucleotides between test cells and control cells (eg, cancer cells and normal cells). For example, high expression of a particular message in cancer cells may not be observed in the corresponding normal cells, indicating a cancer-specific gene product. Exemplary uses of arrays are further described, for example, in Papalarado et al., Sem. Ra
direction Oncol. (1998) 8: 217; and Ramsay.
Nature Biotechnol. (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 for identifying abnormal or diseased tissues 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 compared in molecular weight, amino acid sequence or nucleotide sequence, or relative amount is a gene in human tissue suspected of being affected, Alternatively, it indicates a change in a gene that regulates the gene. Examples of the detection of differential expression and 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 the expression level of mRNA or protein corresponding to a polynucleotide of the invention in fetal tissue to the relevant level in normal fetal tissue. Fetal tissues used for this purpose include, but are not limited to, amniotic fluid, 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 or size of the same product of a fetal polynucleotide-related gene or mRNA, or changes in the relative amounts of molecular weight, amino acid sequence, or fetal protein, can be due to germline in the fetal polynucleotide-related gene. , Which indicates a genetic predisposition to the disease. In general, the diagnostic, prognostic, and other methods of the invention based on differential expression are
In a test sample obtained from a patient suspected of having a disease (eg, breast cancer, lung cancer, colon cancer, and / or metastatic form thereof) or susceptible to the disease,
Detection of levels or amounts of gene products, particularly differentially expressed gene products, and their levels found in normal cells (eg, cells substantially free of cancer) and / or other control cells. Comparison with the level (
For example, to distinguish cancer cells from cells suffering from dysplasia). In addition, the severity of the disease is determined by the level of differentially expressed gene product detected in the sample and the level of differential gene product correlated with varying degrees of severity of the disease. Can be evaluated by comparing with. 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, an open reading frame encoding a gene product (eg, a polypeptide), and / or introns of such genes, and flanking those involved in the regulation of expression. 5'and 3 '
It is intended to include polynucleotides that may include non-coding nucleotide sequences (up to about 20 kb beyond the coding region, but probably more 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 2
Differences in expression levels that correlate with a decrease in expression levels of 5%, usually at least about 50% to 70%, and more usually at least about 90% or more, result in a differentially expressed gene of interest (ie, compared to a control sample). Genes that are under-expressed or down-regulated in test samples). Furthermore, at least about 25%, usually at least about 5%, as compared to the control sample.
A difference in expression levels that correlates with an increase in expression of 0% to 75%, more usually at least about 90%, can be at least about 1 and 1/2 fold, usually at least about 2 fold to about 10 fold, And, there can be an increase of about 100-fold to about 1000-fold, which is an indicator of the gene of interest that is differentially expressed (ie, overexpressed or upregulated gene).

“Differentially expressed polynucleotide” as used herein means a nucleic acid molecule (RNA or DNA) that comprises a sequence that represents a differentially expressed gene,
For example, the differentially expressed polynucleotide uniquely identifies the differentially expressed gene such that detection of the differentially expressed polynucleotide in the sample is correlated with the presence of the differentially expressed gene in the sample. It includes a sequence (eg, an open reading frame encoding a gene product). "Differentially expressed polynucleotide" also includes fragments of the disclosed polynucleotides (eg, fragments that retain biological activity), and homologous, substantially similar, or substantially homologous to the disclosed polynucleotides. Are meant to include nucleic acids identical to (eg, having about 90% sequence identity).

As used herein, “diagnosis” generally refers to a determination of a patient's susceptibility to a disease or disorder, a determination as to whether a subject is currently suffering from the disease or disorder, And determining prognosis of a subject suffering from a disease or disorder (eg, identifying 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, in situ carcinoma (eg, in situ ductal carcinoma), estrogen receptor (ER) -positive breast cancer, ER-negative breast cancer, or other forms and / or stages of breast cancer), lung cancer (small cell). Carcinoma, non-small cell carcinoma, mesothelioma, and other forms and / or stages of lung cancer) and colon cancer (eg, adenomatous polyps, colorectal carcinoma, and other forms and / or stages of colon cancer) Including a diagnosis of the subject in the context.

“Sample” or “biochemical sample” as used throughout this specification
Is generally from a sample of biological fluid or tissue, in particular tissue, in particular diseases for which diagnostic applications are designed (eg, ductal adenocarcinoma).
It is meant to refer to a sample or the like obtained from a cell type that correlates with oma)). “Sample” is also meant to include derivatives and fractions (eg, cell lysates) of such sample. If the sample is solid tissue, cells of the tissue can be dissociated or tissue sections can be analyzed.

The methods of the invention useful in diagnosis or prognosis typically involve the selection of selected differentially expressed gene products in a sample of interest to determine any relative differences in gene product expression. Including a comparison of the amount with that of the control, where the difference can be measured qualitatively and / or quantitatively. Quantitation can be accomplished, for example, by comparing the level of expression product detected in the sample to the amount of product present in the standard curve. Comparisons; by using techniques such as densitometry with or without the aid of computer processing;
A representative library of cDNA clones of mRNA isolated from test samples was prepared, the clones in the library were sequenced to determine the number of cDNA clones corresponding to the same gene product, and in control samples. By analyzing the number of clones corresponding to the same gene product, relative to the number of clones of the same gene product; or using the array, the relative level of hybridization to a selected sequence or set of sequences Can be done visually and by comparing the hybridization pattern to the control hybridization pattern. 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 WO 97/2731).
7).

In general, the diagnostic assays of the present invention involve the detection of the gene product (eg, mRNA, or polypeptide) of polynucleotide sequences corresponding to the sequences of SEQ ID NOs: 1-1079. The patient from whom the sample is obtained may be clearly healthy, may be predisposed to a disease, as determined by, for example, family history or exposure to certain environmental factors, or an alteration in the gene product of the invention. It can already be identified as having a condition that correlates with the expressed expression.

The diagnostic is at least 1 with the sequences set forth in SEQ ID NOS: 1-1079.
Of the gene product encoded by one, preferably at least two or more, at least three or more, or at least four or more polynucleotides, and can be determined based on the expression level of the detected gene product, and SEQ ID NO: 1 Detecting the expression of genes corresponding to all of the -1079 and / or additional sequences that may act as additional diagnostic markers and / or reference sequences. When the diagnostic method is designed to detect the presence of or the susceptibility of a patient to cancer, the assay preferably comprises the gene product encoded by the gene corresponding to the polynucleotide differentially expressed in the cancer. Includes detection. 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 the various detectable levels can be used in connection with various embodiments of the diagnostic methods of the present invention. Suitable selectable labels include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Texas red, phycoerythrin, allophycocyanin, 6-carboxyfluoroscein (6-FA.
M) 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxyfluorocein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2 '.
, 4 ', 7', 4,7-hexachlorofluorocein (HEX), 5-carboxyfluorocein (5-FAM), or N, N, N ', N',-tetramethyl-6-carboxyrhodamine (TAMRA) )), Radioactive labels (eg, ³² P, ³⁵ S, ³ H, etc.) 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 buffers or labeled components 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 Diagnosis In one embodiment, test samples are assayed for the level of differentially expressed polypeptide. Diagnosis can be accomplished using any of numerous methods for measuring the absence or presence, or altered amount, of differentially expressed polypeptides in a test sample. For example, detection can utilize staining of cells or histological sections with labeled antibodies and can be 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 time 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 in conjunction 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 a variety of 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 can be used (eg, ELISA, Western blot, immunoprecipitation, radioimmunoassay, etc.).

(Detection of mRNA) The diagnostic method of the present invention may also, or alternatively, mRNA encoded by a gene corresponding to the differentially expressed polynucleotide of the present invention.
Detection of. Any suitable qualitative or quantitative method known in the art for detecting a particular mRNA can be used. mRNA is, for example,
Reverse transcriptase P by in situ hybridization in tissue sections
It can be detected by CR 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. M in the sample
RNA expression levels can also be determined by making a library of expressed sequence tags (ESTs) from a sample, where the EST library is representative of the sequences present in the sample (Adams et al. (1991). Science 2
52: 1651). An enumeration of the relative representations of ESTs in the library can be used to approximate the relative representations of gene transcripts in the starting sample. Then
The results of the EST analysis of the test sample are compared with the EST analysis of the reference sample,
The relative expression level of the selected polynucleotide, particularly the polynucleotide corresponding to one or more of the differentially expressed genes described herein, can be determined.
Alternatively, the gene expression in the test sample is determined by serial analysis of gene expression (SAGE).
Methodology (eg, Velculescu et al., Science (1995) 270.
: 484) or a differential display (DD) methodology (see, eg, 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 filtration, 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 a Single Gene in Diagnostic Applications The diagnostic methods of the invention can focus on the expression of a single differentially expressed gene. For example, diagnostic methods can include detecting differentially expressed genes or polymorphisms of such genes associated with disease (eg, polymorphisms in the coding or regulatory regions). Disease-related polymorphisms may include gene deletions or truncations, mutations that alter expression levels and / or affect the activity of the encoded protein, and the like.

Many methods are available for analyzing nucleic acids for the presence of specific 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 quantity for analysis, and a detectable label is added to the amplification reaction ( For example, 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 the sequence of choice, eg, the wild-type sequence. Hybridization with a polymorphic sequence or variant sequence can also be used to determine its presence in a sample (eg, by Southern blot, dot blot, etc.). High polymorphic or variant sequences and control sequences for arrays of oligonucleotide probes immobilized on a solid support, as described in US Pat. No. 5,445,934 or in WO95 / 35505. The hybridization pattern is also
It can be used as a means to identify polymorphic or variant sequences that correlate with disease.
Single-strand conformation polymorphism in gel matrix
informational polymorphism (SSCP) analysis,
Denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis are used to detect conformational changes created by alteration of DNA sequences as alterations in electrophoretic mobility. Alternatively, if the polymorphism creates or destroys a recognition site for a restriction endonuclease, the sample is digested with endonuclease and the product size is fractionated to determine whether the fragment was digested. It Fractionation is performed by gel or capillary electrophoresis, especially acrylamide gels or agarose gels.

Screening for mutations in a gene may be based on the functional or antigenic properties of the protein. Protein truncation 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 have led 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 methods of the present invention include detecting expression of a selected set of genes in a test sample to generate a test expression pattern (TEP). The TEP is compared to a reference expression pattern (REP), which is generated by detection of 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: 1-1079. Of particular interest is a selected set of genes, including genes that are differentially expressed in the disease for which the test sample is screened.

As used herein in differential gene expression analysis and diagnostic / prognostic context, a “reference sequence” or “reference polynucleotide” refers to and is a selected set of polynucleotides. The set comprises at least one or more of the differentially expressed polynucleotides described herein. Multiple reference sequences, preferably including positive and negative control sequences, can be included as reference sequences. A further suitable reference sequence is GenBa
Found in nk, Unigene, and other nucleotide sequence databases (including, for example, expressed sequence tags (ESTs), partial and full length sequences).

By “reference array” is meant an array having a reference sequence for use in hybridization with a sample, wherein the reference sequence is all of the differentially expressed polynucleotides described herein. , At least one of them,
Or include any subset thereof. Usually 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 comprises other gene sequences, particularly other sequences of interest for screening for a disease or disorder (eg, cancer, dysplasia, or other related or unrelated disease, disorder, or condition). Sequences (including polymorphisms) can be included. The oligonucleotide sequences in the array are usually at least 12 nucleotides in length, and can be near the length of the sequences provided, or can be extended into flanking regions to create fragments 100 nucleotides to 200 nucleotides or more in length. The reference array can be generated according to any suitable method known in the art. For example, methods of producing large arrays of oligonucleotides are described in US Pat. No. 5,134,854 and US Pat. No. 5,445,934 using light directed synthesis techniques. A computer controlled system is used to convert a heterogeneous array of monomers 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, for example, as described in PCT Published Application No. WO 95/35505.

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

REPs can be made in a variety of 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, acquires hybridization data from the array, and It can be created 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 obtained by, for example, isolating mRNA from the control sample, converting the mRNA to cDNA, and sequencing the cDNA.
It can be isolated and sequenced. The resulting sequence information 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 processed to selectively remove sequences of expressed genes that are of little interest and can complicate analysis (eg, housekeeping). Some or all of the sequences associated with the gene are REP
Can be excluded from the 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, It can then be made 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 simultaneously 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, wherein the reference array comprises
It has one or more reference sequences for use in hybridization with a sample. 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 R generated using an array with the same or similar selected set of differentially expressed polynucleotides.
Compared to EP. Probes corresponding to sequences that are differentially expressed between the two samples show reduced or increased hybridization potency for one of the samples compared to the other.

Methods for recovery of data from hybridization of a sample with a reference array are well known in the art. For example, the polynucleotides of the reference and test samples can be made using a detectable fluorescent label, and hybridization of the polynucleotides in the sample can be performed using, for example, a microscope and a light source to illuminate the 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 step 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. The digital images produced from the scan are then combined for subsequent analysis. One sample (for any particular array element
For example, the ratio of the fluorescence signal from a test sample is
The fluorescent signal from the reference sample) is compared and the relative signal intensity is determined.

Methods for analyzing the data collected from hybridization to arrays are well known in the art. For example, if the 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, predetermined statistical distributions). Data) that deviates from the above), and calculating the relative binding affinity of the target from the remaining data. The data obtained can be displayed as an image, with the intensity in each region varying according to the binding affinity between the target and the probe.

Generally, a test sample is a TEP made from a test sample that is derived from a reference sample (eg, cancer, a particular condition of cancer, a sample that correlates 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 that correlates with a diseased or non-diseased state 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 sequences, and substantially the same level (eg, with the reference sequence selected after normalization of the sample). Expression of these reference genes with no significant difference between samples for correlated signals, or a difference in signal intensity for a given reference sequence of not less than about 25% to about 40%). Generally, 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 Includes adaptation in qualitative or quantitative expression levels.

Pattern matching can be done manually or using computer programs. Preparation of substrate matrices (eg, arrays), design of oligonucleotides for use with such matrices, probe labeling, hybridization conditions, scanning of hybridized matrices, and analysis of the patterns produced (comparative analysis Methods for including) are described, for example, in US Pat. No. 5,800,992.

Diagnosis, Prognosis, and Management of Cancer The polynucleotides and their gene products of the present invention are genetic or biochemical markers (eg, in blood or tissue) that detect the earliest changes along the carcinogenic pathway. Of) 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, thus warranting more aggressive chemotherapy or radiation therapy to the patient, and vice versa. Correlation with novel surrogate tumor-specific characteristics in relation to treatment response and outcome in patients may define prognostic indicators that allow the design of treatments made based on the molecular profile of the tumor. These therapies include antibody targeting and gene therapy. Determining the expression of a polynucleotide, and comparing a patient's profile to known expression in normal tissue and disease variants, both with respect to treatment specificity and with regard to the patient's pain-free level. , Allows the decision of the best possible treatment for the 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 classes of widely used in oncology that may result from the identification of expression levels of the polynucleotides of the invention are:
It is a staging of cancer disorders and a grading of the nature of the cancerous tissue.

The polynucleotides of the invention have or are susceptible to cancer in order to detect potentially malignant events at the molecular level before they are detectable at the terrible morphological level. It can be useful for monitoring patients. In addition, the polynucleotides of the 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, a polynucleotide herein. Is differentially expressed across various cancer types. Thus, for example, expression of a polynucleotide having clinical relevance for metastatic colon cancer may also 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. Staging aids physicians in determining prognosis, in planning treatment, and in assessing the results of such treatment. Staging systems vary with cancer type, but generally include the following "TNM" systems: tumor type is indicated by T; whether the cancer has metastasized to lymph nodes is N , And whether the cancer has spread to more distant parts of the body is indicated by M. Generally, if cancer is detectable only in the area of the primary lesion without spreading to any lymph nodes, it is referred to as stage I. If the tumor has spread only to the closest lymph nodes, it is called stage II. In stage III, the cancer has generally spread to lymph nodes almost 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 regions of the body. Thus, stage II cancers with polynucleotides that represent highly metastatic cancers can be used to transform borderline stage II tumors into stage III tumors, justifying a more aggressive treatment. . Conversely, the presence of polynucleotides that represent less metastatic cancers allows more conservative staging of tumors.

Cancer Grading Grade is a term used to describe how closely a tumor resembles the same type of normal tissue. The microscopic appearance of tumors is used to identify tumor grade based on parameters such as cell morphology, cell organization, 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 there. 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. The polynucleotides of the present invention not only help this in determining the status of differentiation of tumor cells, but they are also valuable in determining tumor aggressiveness (eg, metastatic potential), other than differentiation. Can be of particular value in determining the grade of a tumor as it can identify

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 cells and non-small cells, which comprises approximately 90% of all lung cancer cases. Small cell carcinoma (also called oat cell carcinoma) usually starts 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 sizes. Adenocarcinomas begin 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 carcinoma. Large cell carcinomas start near the surface of the lung, grow rapidly, and when diagnosed, the growth is usually very large. Other less common forms of lung cancer are carcinoids, cylinders, mucoepidermoid mesothelioma and malignant mesothelioma.

Polynucleotides of the invention, eg, in normal cells versus cancerous lung cells (eg, high or low metastatic potential tumor cells) or between cancerous lung cell types (eg, high metastatic versus low). (Metastatic), differentially expressed polynucleotides are used to distinguish types of lung cancer, as well as to identify cancer-specific features in a patient, and to select the appropriate treatment. , Can be used. For example, if a patient's biopsy expresses a polynucleotide associated with low metastatic potential, this may justify leaving a larger portion of the patient's lungs in surgery to remove the lesion. Alternatively, smaller lesions with the expression of polynucleotides associated with high metastatic potential may be 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.

Breast Cancer Detection The majority of breast cancers are subtypes of adenocarcinomas and 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 (LCI)
S); 4) invasive (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 carcinoma.

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 the 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 tissue (eg, , ER expression). In addition, the development of breast cancer depends on the steroid hormone (
For example, it can be detected by testing the ratio of the expression of the differentially expressed polynucleotide to the level of testosterone or estrogen) or to other hormones (eg growth hormone, insulin). Thus, expression of the specific marker polynucleotide differentiates between normal and cancerous breast tissue, and thus between breast cancers with cells of different origin, resulting in different potential metastasis rates. It can be used to distinguish between associated breast cancers, and so on.

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 small polyps, which are benign growths 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 diseases 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 James. 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 alone or in combination with the level of expression. Determining the aggressive nature and / or metastatic potential of colon cancer is the comparison of the levels of one or more polynucleotides of the invention, and another sequence known to be altered in cancer tissue (eg p53, Expression of DCC ras and lor FAP)
Can be determined by comparing the total levels of (eg, Fearon ER
Et al., Cell (1990) 61 (5): 759; Hamilton SR et al., C.
ancer (1993) 72: 957; Bodmer W et al., Nat Gene.
t. (1994) 4 (3): 217; Fearon ER, Ann NY Ac.
ad Sci. (1995) 768: 101). For example, colon cancer development can be detected by testing the ratio of any polynucleotide of the invention to the level of an oncogene (eg, ras) or tumor suppressor gene (eg, FAP or p53). Thus, the expression of the specific marker polynucleotides differentiates between normal and cancerous colon tissue, and thus between colon cancers with cells of different origin, different potential metastasis rates. It can be used, for example, to distinguish between colon cancers with.

Detection of Prostate Cancer Polynucleotides displaying their appropriate differential expression patterns and their corresponding genes and gene products can be used to detect prostate cancer in a subject. Over 95% of primary prostate cancers are adenocarcinomas. Signs and symptoms include: frequent urination (especially at night), inability to urinate, difficulty in initiating or controlling urination, weak or interrupted micturition flow, and lower back, hip or upper thigh. Frequent pain or stiffness in.

Many signs and symptoms of prostate cancer can be caused by a variety of other non-cancerous conditions. For example, a common cause of one of many of these signs and symptoms is a benign prostatic hypertrophy, a condition called BPH. In BPH, the prostate may become larger and block flow or urine, or interfere with sexual function. The methods and compositions of the present invention can be used to distinguish between prostate cancer and such non-cancerous conditions. The methods of the invention are combined with conventional methods of diagnosis, eg, digital rectal exam and / or detection of levels of prostate specific antigen (PSA), a substance produced and secreted by the prostate. Can be used.

Use of Polynucleotides to Screen for Peptide Analogs and Antagonists Polypeptides encoded by the polynucleotides of the invention and the corresponding full-length genes are 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 are screened using any available method known in the art such as signal transduction, antibody binding, receptor binding, mitogenic assays, chemotaxis assays 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. An agonist or antagonist that competes for binding to a native polypeptide may require a concentration equal to or greater than the native concentration, while an inhibitor capable of irreversibly binding to a polypeptide is Can be added at a concentration similar to.

Such screens and experiments are performed to detect 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 agonist of the novel binding partner. Alternatively, it may lead to the identification of 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. Moreover, where the novel receptor shares biologically important characteristics with known receptors, information about agonist / antagonist binding may facilitate the development of improved agonist / antagonists of known receptors.

Pharmaceutical Compositions and Therapeutic Uses The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a polypeptide, antibody, or of the claimed invention. Polynucleotides may be included, including antisense nucleotides and ribozymes. The term “therapeutically effective amount” as used herein treats, alleviates a desired disease or condition,
Or, refers to the amount of a therapeutic agent to prevent, or to exhibit a detectable therapeutic or prophylactic effect. Effects can be detected by, for example, chemical markers or antigen levels. The therapeutic effect also includes a decrease 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 amount for a given condition is determined by routine experimentation,
And within the judgment of the clinician. For purposes of this invention, an effective dose is generally about 0.01 mg / kg to 50 mg / kg in the individual to whom it is administered,
Or 0.05 mg / kg to about 10 mg / kg DNA construct.

The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of therapeutic agents such as antibodies or polypeptides, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and 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 agents and the like, may also be present in such vehicles. Typically, therapeutic compositions are prepared as either injectable, liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection. Forms 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, inorganic salts such as hydrogen chloride, hydrobromide, phosphates, sulphates and the like; and acetates, propiones. Salts of organic acids such as acid salts, malonates, benzoates, etc.). A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharma.
available in Scientific Sciences (Mack Pub. Co., NJ 1991).

Methods of Delivery Once formulated, the compositions of the invention can be (1) directly administered to a subject (eg, as a polynucleotide or polypeptide); or (2) a subject. It can be delivered ex vivo to cells from the body (eg, as in ex vivo gene therapy). Direct delivery of the composition is generally by parenteral injection (
For example, subcutaneously, intraperitoneally, intravenously or intramuscularly, intratumorally, or into the interstitial space of tissue). Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hypospray.
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, eg, WO 93/147.
No. 78. Examples of cells useful in ex vivo applications include:
Examples include stem cells, especially 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 nuclear. DN to
A can be achieved by direct microinjection of A, all well known in the art.

Once the gene corresponding to the polynucleotide of the invention is found to correlate with proliferative disorders, such as neoplasia, dysplasia, and hyperplasia, the disorder is the provided polynucleotide, corresponding Are susceptible to treatment by administration of a therapeutic agent based on a polypeptide or other corresponding molecule (eg, antisense, ribozyme, etc.).

The dose and means of administration of the pharmaceutical compositions of the present invention will be based on the specific quality of the therapeutic composition, the condition, age and weight of the patient, disease progression, and other relevant factors. Will be decided. For example, administration of the polynucleotide therapeutic composition agents of the invention includes local or systemic administration, including injection, oral administration, particle gun, or catheterization, and topical administration. Preferably,
The therapeutic polynucleotide composition comprises an expression containing a promoter operably linked to at least 12, 22, 25, 30, or 35 contiguous nt polynucleotides of the polynucleotides disclosed herein. Includes 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
It is injected several times at several different locations within the body of the tumor. Alternatively, the tumor-acting artery is identified and the therapeutic composition is injected into such artery to deliver the composition directly to the tumor. Tumors with necrotic cancer are aspirated,
The composition is then 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 certain 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 Find.
eis et al., Trends Biotechnol. (1993) 11: 202;
Chiou et al., Gene Therapeutics: Methods And
Applications Of Direct Gene Transfer
r (JA Wolff eds.) (1994); Wu et al. Biol. Che
m. (1988) 263: 621; Wu et al., J. Am. Biol. Chem. (1
994) 269: 542; Zenke et al., Proc. Natl. Acad.
Sci. (USA) (1990) 87: 3655; Wu et al. Biol.
Chem. (1991) 266: 338. Therapeutic compositions containing polynucleotides are administered in the range of about 100 ng to about 200 mg of DNA for topical administration in gene therapy protocols. About 500
Concentration ranges of ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during gene therapy protocols. Factors such as methods of action (eg, for enhancing or inhibiting levels of the encoded gene product), transformation and expression potency are required for maximal potency of the antisense subgenomic polynucleotides. It is a consideration that affects the dosage that is given. 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 Several doses to adjacent or near tissue parts may be required to achieve a positive therapeutic result. In all cases, routine experimentation in clinical trials will determine a particular range for optimal therapeutic effect. Suitable uses, doses, and administrations for polynucleotide-related genes encoding polypeptides or proteins with 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 Ther.
apy (1994) 1:51; Kimura, Human Gene Ther.
apy (1994) 5: 845; Connelly, Human Gene T.
herapy (1995) 1: 815; and Kaplitt, Nature.
Genetics (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;
See U.S. Patent No. 4,777,127; British Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alphavirus-based vectors (e.g., Sindbis virus vector, Semliki Forest). Virus (ATCC VR-67; ATCC VR-1247), Ross River virus (
ATCC VR-373; ATCC VR-1246), and Venezuelan Equine Encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (A).
AV) vector (eg, WO94 / 12649, WO93 / 03769; WO
93/19191; WO94 / 28938; WO95 / 11984, and WO
95/00655), but is not limited thereto. Cu
riel. Hum. Gene Ther. (1992) 3: 147, administration of DNA linked to a killed adenovirus is also
Can be used.

Non-viral delivery vehicles and methods can also be used, polycationic enriched DN, with or without ligation to killed adenovirus alone.
A (eg Curiel, Hum. Gene Ther. (1992) 3:
147); ligand-ligated DNA (eg, Wu, J. Biol.
． Chem. (1989) 264: 16985); eukaryotic cell delivery vehicle cells (eg, US Pat. No. 5,814,482; WO95 / 07).
994; WO96 / 17072; WO95 / 30763; and WO97 / 42
338), as well as, but not limited to, nuclear charge neutralization, or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA introduction methods are described in WO 90/11092 and US Pat. No. 5,580,85.
No. 9 is described. Liposomes that can act as gene delivery vehicles are
U.S. Pat. No. 5,422,120; WO95 / 13796; WO94 / 236
97; WO 91/14445; and EP 0524968. A further approach is described by Philip, Mol. Cell. Biol. (
1994) 14: 2411, and Woffendin, Proc. Natl
． Acad. Sci. (1994) 91: 1581.

Additional non-viral delivery suitable for use is described by Woffendin et al., Pr.
oc. Natl. Acad. Sci. USA (1994) 91 (24)
: 11581, including mechanical delivery systems. In addition, the coding sequences and the products of expression of such sequences can be delivered through the deposition of photopolymerized hydrogel materials or the use of ionizing radiation (eg, US Pat.
206, 152 and WO 92/11033). Other conventional methods for gene delivery that may be used for delivery of coding sequences include, for example:
Hand-held gene transfer particle gun (see, eg, US Pat. No. 5,149,655)
Use of ionizing radiation for activation of transferred genes (see, eg, US Pat. No. 5,206,152 and WO 92/11033).

The invention will now be illustrated by 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.

Examples The following examples are provided primarily for purposes of illustration. It will be apparent to those skilled in the art that the formulations, dosages, methods of administration and other parameters of the present invention can be further modified or substituted in various ways without departing from the spirit and scope of the invention. In contrast, it is easily apparent.

Example 1 Source of Biological Material and Overview of Novel Polynucleotides Expressed by Biological Material A cDNA library was constructed using the human colon cancer cell line Km12L4-A (Morika).
wa et al., Cancer Research (1998) 48: 6863), KM.
12C (Morikawa et al., Cancer Res. (1988) 48:19.
43-1948), or MDA-MB-231 (Brinkley et al., Can.
cer Res. (1980) 40: 3118-3129) and these cells were used to construct a cDNA library from mRNA isolated from the cells. The sequences expressed by these cell lines were isolated and analyzed; most sequences were approximately 275-300 nucleotides in length. K
The M12L4-A cell line is derived from the KM12C cell line. The KM12C cell line
Poor metastasis (low metastasis), established in cultures from Duke stage B2 surgical specimens (Morikawa et al. Cancer Res. (19).
88) 48: 6863). KML4-A is a highly metastatic substrain derived from KM12C (Yatman et al., Nucl. Acids. Res. (19
95) 23: 4007; Bao-Ling et al., Proc. Annu. Meet
． Am. Assoc. Cancer. Res. (1995) 21:32
69). KM12C and KM12C-derived cell lines (eg, KM12L4, K
M12L4-A) is well recognized in the art as a model cell line for the study of colon cancer (eg Morikawa et al., Supra; Radin.
sky et al., Clin. Cancer Res. (1995) 1:19; Yea
tman et al. (1995) supra; Yetman et al., Clin. Exp. Me
astasis (1996) 14: 246). MDA-MB-2
The 31 cell line was originally isolated from pleural effusion (Cailleau, J. Natl.
Cancer Inst. (1974) 53: 661), highly metastatic, and poorly differentiated adenocarcinoma stage (gr) in nude mice consistent with breast cancer.
ade) II is formed.

The sequence of the isolated polynucleotide was sequenced using the XBLAST masking program (
Claverie "Effective Large-Scale Sequence"
nce Similarity Searches "Computer Met
hods for Macromolecular Sequence Ana
Lysis, edited by Doolite, Meth. Enzymol. 266: 2
12-227 Academic Press, NY, NY (1996); in particular, Claverie, "Automated DNA Sequencing.
and Analysis Techniques "Adams et al., Ed., Chapter 36, p. 267 Academic Press, San Diego, 1994,
And Claverie et al., Comput. Chem. (1993) 17: 1
91)) was used to mask first and eliminate low complexity sequences.
In general, masking excludes relatively small sequences of interest due to the low complexity of the sequences, and multiple “hits” based on similarity to repeat regions common to multiple sequences (eg, Alu repeats). It does not affect the final search results, except for the exclusion of "." Masking resulted in the exclusion of 43 sequences. The remaining sequences were then used in a BLASTN vs. GenBank search; sequences showing greater than 70% overlap, 99% identity, and p-value less than 1 × 10 ⁻⁴⁰ were excluded.
The sequences from this search were also excluded if the global parameters were matched, but the sequences were either ribosome-derived or vector-derived.

Sequences obtained from previous searches were grouped into three groups (1, 2, and 3 below) and searched in a BLASTX vs. NRP (non-redundant protein) database search: (1) unknown (No hit in GenBank search), (2)
Weak similarity (greater than 45% identity and p-value less than 1 × 10 ⁻⁵ ), and (
3) High similarity (> 60% duplication,> 80% identity, and p-value <1 x ^10-5 ). Sequences with greater than 70% overlap, greater than 99% identity, and p-value less than 1 × 10 ⁻⁴⁰ were excluded.

The remaining sequences were classified as unknown (no hit), weak similarity, and high similarity (parameters as above). Two searches were performed on these sequences. First, a BLAST vs. EST database search was performed and sequences with> 99% overlap,> 99% similarity, and p-value <1 × 10 ⁻⁴⁰ were excluded. Sequences with p-values less than 1 × 10 ⁻⁶⁵ when compared to database sequences of human origin were also excluded. Second, BLASTN vs. Patent G
done eneSeq database and greater than 99% identity, 1 × 10 ^-40
Sequences with p-values less than, and greater than 99% duplication were excluded.

The remaining sequences were subjected to screening using other rules and redundancy in the dataset. P less than 1 × 10 ⁻¹¹¹ for database sequences of human origin
Sequences with values were specifically excluded. The final results provided 982 sequences, listed as SEQ ID NOS: 1-982 in the attached sequence listing and summarized in Table 1A (inserted before the claims). Each identified polynucleotide represents a sequence from at least a partial mRNA transcript.

Table 1A provides: 1) the SEQ ID NO assigned to each sequence for use herein; 2) the filing date of the US priority patent application in which the sequence was first filed;
3) the agent reference number assigned to the priority application (for internal use); 4) the sequence number assigned to the sequence in the priority application; 5) the sequence name used as the internal identifier of the sequence; and 6) the sequence The name assigned to the isolated clone. Two or more polynucleotides of the invention may represent the same mRNA transcript and different regions of the same gene, as the provided polynucleotides represent partial mRNA transcripts. Thus, if two or more SEQ ID NOs are identified as belonging to the same clone, either sequence can be used to obtain full length mRNA or full length gene.

In order to confirm the sequences of SEQ ID NOs: 1 to 982, an automated device retreat was used.
val) system was used to recover clones from the library and the inserts of the recovered clones were re-sequenced. These "validation (validation
) "Sequences are provided in the sequence listing as SEQ ID NOs: 983-996, and"
A summary of "confirmation" sequences is provided in Table 1B (inserted before the claims). Table 1B provides: 1) the SEQ ID NO assigned to each sequence for use herein; 2) the sample name assigned to the resulting "confirmed"sequence; and 3) the indicated "confirmed". The name of the clone containing the sequence. The "confirmation" sequences can be correlated with the original sequences, which are confirmed by reference to Table 1A. The “confirmation” sequence is often longer than the original polynucleotide sequence and therefore provides additional sequence information. All confirmation sequences can be obtained from either the corresponding clones or the cDNA libraries described herein (eg, using primers designed from the sequences provided in the sequence listing).

Example 2: Results of a public database search to identify the function of the gene product SEQ ID NOs: 1-1079 were translated in all three reading frames and the nucleotide sequence and translated amino acids Sequence the GenBank (
Nucleotide sequences) or Non-Redundant Protein (amino acid sequence) databases were used as query sequences to search for homologous sequences. The query sequence and individual sequences can be found at http: //
ww. ncbi. nlm. nih. world wi at gov / BLAST /
Aligned using the BLAST 2.0 program available via de web (Altschul et al., Nucleic Acids Res. (199
7) 25: 3389-3402). Sequences were masked to varying degrees to prevent searches for repetitive or poly A sequences using the XBLAST program to mask low complexity as described above in Example 1.

Tables 2A and 2B (inserted before the claims) provide a summary of alignments with p-values of 1 × 10 ⁻² or less, and the public databases designated sequences of the invention. Shows substantial homology with the sequences of. Table 2A provides the sequence number of the query sequence, the accession number of the GenBank database entry for the homologous sequence, and the p-value for the alignment. Table 2A shows the sequence number of the query sequence and the homologous sequence Non-
It provides the registration number of the Redundant Protein database registration and the p-value of the alignment. The alignments provided in Tables 2A and 2B are the best available alignments for DNA or amino acid sequences at the time just before the filing of the present application. The activity of the polypeptide encoded by the SEQ ID NOs listed in Tables 2A and 2B is substantially the same as or substantially similar to the activity of the reported closest or closely related sequences. It can be assumed that there is. The closest sequence accession number is reported, providing a publicly available reference to the activity and function exhibited by the closest sequence. Public information regarding the activity and function of each closest sequence is incorporated by reference in this application. Also incorporated by reference is the sequence, as well as the putative activity and actual activity of the closest sequences listed in Table 2 and their correlated sequences, and all publicly available uses for putative and actual functions. This is possible information. Also shown are the search programs and databases used for the alignment, and p-value calculations.

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 the corresponding polynucleotides. The closest sequence may indicate the tissue or cell type to be used to construct the library for the full length sequence of the corresponding polynucleotide.

Example 3 Identification of Contiguous Sequences Comprising a Polynucleotide of the Invention The novel polynucleotides are used to facilitate the identification of sequences contiguous with any of the polynucleotides of SEQ ID NOs: 1-982. (E.g., the polynucleotide provides a sequence that results in a 5'extension of another DNA sequence, resulting in the production of a longer, contiguous sequence composed of the given polynucleotide and other DNA sequences). Publicly available databases to determine and owned (pro
The database was screened. Wiring the contig
It was performed using GCG's Gelmerge application from Sconsin University (default setting).

These parameters were used to generate 83 contigated sequences. These contigated sequences are provided as SEQ ID NOs: 997-1079 (see Table 1C). Table 1C provides the publicly available test human consensus (THC) used with the sequence number of the contig sequence, the name of the sequence used to make the contig, and the sequence of the corresponding sequence name that provides the contig. ) Provide the accession number of the sequence. The contigs can be generated by associating the sequence names in Table 1C with the SEQ ID numbers of the polynucleotides used and referring to Tables 1A and 1B.

Contigated sequences (SEQ ID NOs: 997-1079) represent longer sequences that include another polynucleotide sequence of the invention. The contigated sequences were then translated in all three reading frames and the BLAST program as described above was used to determine the best alignment with the individual sequences. This sequence was masked using the XBLAST program for masking low complexity as described above in Example 1. As described in more detail below, some contiguous sequences have the characteristics of polypeptides belonging to known protein families (thus indicating new members of these protein families).
, And / or a known functional domain (see Example 4 and Table 3 below). Therefore, the present invention provides
A fragment of such a polynucleotide that retains a biological activity associated with the protein family and / or functional domain identified herein,
Fusions, as well as variants are included.

Example 4: Protein Family Members SEQ ID NOS: 1-1079 were used to perform a profile search, as described herein above. Some of the polynucleotides of the invention have the characteristics of polypeptides belonging to known protein families (thus indicating new members of these protein families), and / or
Alternatively, it has been found to encode a polypeptide containing a known functional domain. Table 3 (inserted before the claims) shows the sequence number of the query sequence, a brief description of the profile hits, the position of the query sequence within the individual sequences (designated as "start" and "stop"). , And the orientation of the query sequence for individual sequences (
Direction, "Dir"), where the forward direction (for) indicates that the alignment is in the same direction (left to right) as the sequences provided in the sequence listing, and the reverse direction (rev) is , Shows that the alignment has a sequence complementary to the sequence provided in the sequence listing.

Some polynucleotides showed multiple profile hits, including profile regions with overlapping query sequences, and / or this sequence containing two different functional domains. Each profile hit in Table 3 is described in more detail below. Abbreviations for profiles (provided in brackets) are:
Abbreviations used to identify profiles in the Pfam and Prosite databases. The Pfam database can be any of the following URLS: http: // pfam. wastel. edu / index. ht
ml; http: // www. sanger. ac. uk / Software /
Pfam /; and http: // www. cgr. ki. It can be accessed via se / Pfam /. The Prosite database is http: // www
w. expasy. It can be accessed at ch / prosite /. Public information available in the Pfam and Prosite databases for various profiles includes, but is not limited to, the activity, function, and consensus sequences of various protein families and protein domains, and is incorporated herein by reference. Incorporated.

(14-3-3 Family (1433; Pfam Pfam Registration No. PF
00244)) SEQ ID NO: 1053 corresponds to the sequence encoding the 14-3-3 protein family member. The 14-3-3 protein family was first identified as being highly abundant in mammalian brain tissue and is preferentially located in neurons, a closely related acidic homodimer protein of approximately 30 kD. (Aitken et al., Trends Biochem. Sci. (1
995) 20: 95-97; Morrison Science (1994) 2.
66: 56-57; and Xiao et al., Nature (1995) 376: 18.
8-191). The 14-3-3 protein has multiple biological activities, which include important roles in signaling pathways and the cell cycle. 1
4-3-3 proteins interact with kinases (eg PKC or Raf-1) and may also function as protein kinase-dependent activators of tyrosine hydroxylase and tryptophan hydroxylase. 1
The 4-3-3 protein sequence is very well conserved and contains two highly conserved regions: the first is an 11 residue peptide located in the N-terminal portion; the second is It is a 20 amino acid region located in the C-terminal part. The consensus pattern is as follows: 1) R-NL- [LIV] -S- [V
G]-[GA] -Y- [KN] -N- [IVA]; 2) Y-K- [DE] -S-
T-L-I- [IM] -Q-L- [LF]-[RHC] -DN- [LF] -T
-[LS] -W- [TAN]-[SAD].

(Ank Repeat (ANK: Pham Accession No. PF0023)) SEQ ID NO: 311 shows a polynucleotide 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. The Ank repeat was originally identified as the cell cycle regulatory protein cdc10 (Breeden et al., N.
ature (1987) 329: 651). Examples of proteins containing ankyrin repeats include ankyrin, myotropin, I-κB protein, cell cycle protein cdc10, Notch receptor (Matsun).
O, et al., Development (1997) 124 (21): 4265); G9a (or BAT8) (Bi) in the class III region of the major histocompatibility complex.
ochem 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-protein interactions (B
ork, Proteins (1993) 17 (4): 363; Lambert and Bennet, Eur. J. Biochem. (1993) 211: 1; K
err et al., Current Op. Cell Biol. (1992) 4:49.
6; Bennet et al. Biol. Chem. (1980) 255: 6424.
).

ATPases Associated with Various Cellular Activities (ATPases; PFam Accession No. PF0004) SEQ ID NOs: 1035, 1058 and 1072 encode members of the ATPase (AAA) family that are associated with diverse cellular activities. Corresponds to the array you want. The AAA protein family is composed of multiple ATPases that share a conserved region of approximately 220 amino acids containing the ATP binding site (Fro
ehlich et al. Cell. Biol. (1991) 114: 443; Er.
dmann et al., Cell (1991) 64: 499; Peters et al., EMBO J. et al. (1990) 9: 1757; Kunau et al., Biochimie (199).
3) 75: 209-224; Confalonieri et al., BioEssays.
(1995) 17: 639; http: // yeamob. pci. chemi
e. uni-tuebingen. de / AAA / Description. h
tml). The AAA domain may be present in 1 or 2 copies and acts as an ATP-dependent protein clamp (Confalonieri et al. (1
995) BioEssays 17: 639), and contains a highly conserved region located in the central portion of the domain. The consensus pattern is: [LIVMT] -x- [LIVMT]-[LIVMF] -x- [GATMC].
-[ST]-[NS] -x (4)-[LIVM] -D-x-A- [LIFA]-
x-R.

Basic Region and Leucine Zipper Transcription Factor (BZIP; Pfam Accession No. PF00170) SEQ ID NO: 918 sets forth a polynucleotide encoding a novel member of the base region and leucine zipper transcription factor family. The bZIP superfamily of eukaryotic DNA-binding transcription factors (Hurst, Prote
in Prof. (1995) 2: 105; and Ellenberger et al.,
Curr. Opin. Struct. Biol. (1994) 4:12) contain a basic region that mediates sequence-specific DNA binding, followed by a protein containing a leucine zipper that is required for dimerization. The consensus pattern for this protein family is: [KR] -x (1,3)-[RKSAQ
] -N-x (2)-[SAQ] (2) -x- [RKTAENQ] -x-R-x-
[RK].

(EFhand; Pfam accession number PF00036) SEQ ID NO: 242 corresponds to a polynucleotide encoding a member of the EF-hand protein family, the calcium-binding domain belonging to many evolutionary families. Shared by calcium-binding proteins (Kawasaki et al.,
Protein. Prof. (1995) 2: 305-490). This domain is a 12-residue loop flanked on each side by a 12-residue α-helix domain, where the calcium ion is coordinated into a pentagonal, bipyramidal conformation. The six residues involved in binding are at positions 1, 3, 5, 7, 9 and 12; these residues are represented by X, Y, Z, -Y, -X and -Z. Shown. The invariant Glu or Asp at position 12 is 2 for linking Ca
It provides two oxygens (two ligands). The consensus pattern contains the complete EF hand loop, as well as the first residue following the loop and that seems to be always hydrophobic: Dx- [DNS]-{ILVFYW}-[
DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQ
STAGC] -x (2)-[DE]-[LIVMFYW].

(Ets Domain (Ets Nterm; Pfam Registration No. PF110178
)) SEQ ID NO: 547, the sequence which confirms it, shows a polynucleotide encoding a polypeptide having N-terminal homology in the ETS domain. Proteins in this family are conserved domains (““
ETS domain "). This domain appears to recognize purine-rich sequences; it is approximately 85-90 amino acids long and is rich in aromatic and positively charged residues (Wasylyk et al., Eur. J. . Biochem
． (1993) 211: 718). The 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. Contains the domain. native e in addition to ets domain
The ts protein contains other sequences that may regulate the biological specificity of this protein. The ets genes and proteins are involved in a variety of essential biological processes, including cell proliferation, differentiation and development, and three members are involved in carcinogenic processes.

(FKH; Pfam accession number PF00250) SEQ ID NO: 925 corresponds to the gene encoding the polypeptide containing the forkhead domain. This forkhead domain (also known as the "winged helix") is present in the family of eukaryotic transcription factors, and DNA
It is a conserved domain of about 100 amino acid residues involved in binding (Weige
1 et al., Cell (1990) 63: 455-456; Clark et al., Nature.
e (1993) 364: 412-420). Mammalian genes containing the forkhead domain include genes that encode: 1) transcriptional activators (
For example, HNF-3-α protein, HNF-3-β protein and HNF-
3-γ proteins, which interact with cis-acting regulatory regions of many liver genes; 2) interleukin enhancer binding factor (ILF), which is H
Binds to purine-rich NFAT-like motifs in the IV-1 LTR and interleukin-2 promoter and is involved in both positive and negative regulation of important viral and cellular promoter elements); 3) transcription factor BF
-1 (which plays an important role in establishing subdivisions of regions of brain development and in telencephalon development); 4) human HTLF (which is the human T-cell leukemia virus long terminal repeat (HTLV- I LTR) binds to the purine-rich region); 5) transcription factors FREAC-1 (FKHL5, HFH-8), FREEC-
2 (FKHL6), FREAC-3 (FKHL7, FKH-1), FREAC-
4 (FKHL8), FREAC-5 (FKHL9, FKH-2, HFH-6),
FREAC-6 (FKHL10, HFH-5), FREAC-7 (FKHL11
), FREAC-8 (FKHL12, HFH-7), FKH-3, FKH-4,
FKH-5, HFH-1 and HFH-4); 6) human AFX1 (which is involved in chromosomal translocations that cause acute leukemia); and 7) human FKHR (which is
Involved in chromosomal translocations that give rise to rhabdomyosarcoma). This fork domain is highly conserved and is detected by two consensus patterns: the first corresponds to the N-terminal segment of this domain; the second is the heptapeptide located in the middle segment of this domain. Corresponding to. The consensus pattern is as follows: 1) [KR] -P- [PTQ]-[FYLVQH] -S- [FY] -x (2).
-[LIVM] -x (3,4)-[AC]-[LIM]; and 2) W- [QK
R]-[NS] -S- [LIV] -RH.

Helicase with Conserved C-Terminal Domain (Helicase C; Pfam Accession No. PF00271) SEQ ID NOs: 227 and 1058 show polynucleotides encoding novel members of the DEAD / H helicase family. DEAD
The box family includes many eukaryotic and prokaryotic proteins involved in ATP-dependent unwinding of nucleic acids. All of the DEAD box family members of the above proteins share many conserved sequence motifs, some of which are specific to the DEAD family, while others are due to other ATP binding proteins or to helicase. Shared by proteins belonging to the "superfamily" (Hodgman, N
Nature (1988) 333: 22 and Nature (1988) 333:
578; http: // www. expasy. ch / www / linder /
HELICASES_TEXT. html). One of these motifs is "D
-EAD-box ", which represents a specific version of the B motif of the ATP binding protein. Some other proteins belong to a subfamily that has His in place of the second Asp, and thus "DEA-H-
Referred to as "box" proteins (Wassarman DA, et al. Natu.
re (1991) 349: 463; Harosh I. et al. Nucleic A
cids Res. (1991) 19: 6331; Koonin E .; V. La J
． Gen. Virol. (1992) 73: 989; http: // www. e
xpasy. ch / www / linder / HELICASES_TEXT. h
tml). The following signature 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].

(Kazal Serine Protease Inhibitor Family Signature (Kazal
Pfam accession number PF00050)) SEQ ID NO: 97 corresponds to the polynucleotide of the gene encoding the serine protease inhibitor of the Kazal inhibitor family (Laskowski et al., Annu. Rev. Bioche.
m. (1980) 49: 593-626). The basic structure of Kazal serine protease inhibitors (such as the type of inhibitor) is Pfam accession number P
F00050. Exemplary proteins known to belong to this family include: splenic secretory trypsin inhibitor (PST)
I) (this physiological function is to prevent trypsin-catalyzed premature activation of zymogen in the spleen); mammalian semen acrosin inhibitor bitter;
Dog and cat submandibular double-headed protease inhibitor, which contains two Kazal-type domains, the first is a domain that inhibits trypsin and the second is a domain that inhibits elastase. A mouse prostate secretory glycoprotein induced by androgen (
Shows antitrypsin activity); avian ovomucoid; chicken ovo inhibitor (o
and a hirutrypsin inhibitor Bdelli
n B-3. The consensus pattern is as follows: C-x (7) -C-
x (6) -Yx (3) -Cx (2,3) -C, where the four Cs are involved in the disulfide bond.

(MAP Kinase Kinase (mkk)) SEQ ID NOS: 635 and 992 are MA
1 shows members of the P kinase kinase (mkk) family. MAP kinase (
MAPK) is involved in signal transduction and is important in the control of cell cycle and cell proliferation. MAP kinase kinase (MAPKK) is a bispecific protein kinase that phosphorylates and activates MAP kinase. MA
PKK 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, Biologiq
ue Biol. Cell (1993) 79: 193-207; Nishida.
Et al., Trends Biochem Sci (1993) 18: 128-31;
Ruderman Curr Opin Cell Biol (1993) 5:
207-13: Dhanasekaran et al., Oncogene (1998) 1.
7: 1447-55; Kiefer et al., Biochem Soc Trans (
1997) 25: 491-8; and Hill. Cell Signal (19
96) 8: 533-44.

(Neurotransmitter Gated Ion Channel (neur_chan; Pfam Accession No. PF00065)) SEQ ID NO: 1078 corresponds to a sequence encoding a neurotransmitter gated ion channel. Neurotransmitter-gated ion channels provide the molecular basis for rapid signaling at chemical synapses, and post-synaptic oligomeric transmembranes that transiently form ion channels upon binding of specific neurotransmitter molecules. It is a complex. Five types of neurotransmitter gated receptors are known: 1) nicotinic acetylcholine receptor (AchR); 2).
Glycine receptor; 3) γ-aminobutyric acid (GABA) receptor; 4) serotonin 5HT3 receptor; and 5) glutamate receptor. All known sequences of subunits from neurotransmitter-gated ion channels are structurally related and a large extracellular glycosylated N-terminal ligand binding domain, followed by three hydrophobic transmembrane spanning ion channels. It consists of a region followed by an intracellular region of variable length. The fourth hydrophobic region is found at the C-terminus of the sequence. The consensus pattern is: Cx- [LIVMFQ] -x- [LIVMF] -x (2)-
[FY] -P-x-D-x (3) -C, where the two Cs are linked by a disulfide bond.

(PDZ domain (PDZ: Pfam accession number PF00595)) SEQ ID NO: 5
23 and 980 correspond to genes containing the PDZ domain (also known as the DHR domain or the GLGF domain). PDZ domain is 80 to 10
It contains 0 residue repeats, some of which are C-terminal tetrapeptide motifs X-Ser / Thr-X-Val-CO of ion channels and / or receptors.
It interacts with O-and is found in mammalian proteins and in bacteria, yeast and plants (Pontig et al., Protein Sci. (19).
97) 6 (2): 464-8). Proteins containing one or more PDZ domains are associated with diverse membrane-associated proteins (guanylate kinase homologs, some protein phosphatases and protein kinases, neuronal nitric oxide synthase,
And several dystrophin-related proteins (collectively syntrophin (s
(including members of the MAGUK family of proteins (known as yntropins)) (including members of the MAGUK membrane) (ponting).
Et al., Bioessays (1997) 19 (6): 469-79). Many PD
Z domain-containing proteins are localized to well-defined submembrane sites, suggesting that they are involved in cell junction formation, receptor or channel cluster formation, and intracellular signaling events. For example, some P of MAGUK
The DZ domain interacts with C-terminal polypeptides of a subset of NMDA receptor subunits and / or Shaker-type K + channels. Other PDZ
Domains have been shown to bind similar ligands for other transmembrane receptors. In cell-linkage related proteins, PDZs mediate clustering of membrane ion channels by binding to their C-termini. The X-ray crystallographic structures of several proteins containing PDZ domains have been solved (see, eg, Doyle et al., Cell (1996) 85 (7): 1067-76).

(Protein phosphatase 2A regulatory subunit PR55 signature (PR5
5; Pfam accession number PF01240)) SEQ ID NO: 1028 corresponds to the gene encoding the protein phosphatase 2A regulatory subunit. Protein phosphatase 2A (PP2A) is a serine / threonine phosphatase involved in many aspects of cellular function, including regulation of metabolic enzymes and proteins involved in signal transduction. PP2A is a 65 Kd regulatory subunit (PR65,
Is a trimeric enzyme consisting of a core composed of catalytic subunits associated with subunit A); this complex then forms a third variable subunit (
Subunit B), which confers different properties to the holoenzyme (M
ayer et al., Trends Cell Biol. (1994) 4: 287-2.
91). One form of variable subunit is the 55Kd protein (PR55), which is highly conserved in mammals, where it is known that there are three isoforms. This subunit may perform the substrate recognition function or may be responsible for targeting the enzyme complex to the appropriate subcellular compartment. Two perfectly conserved 15-residue sequences (one for N
Located in the terminal region, the other in the center of the protein) is given as a reference for the following consensus pattern: 1) E-F-D-Y-L-K-
S-LE-IE-E-K-I-N; 2) N- [AG] -H- [TA] -Y-
H-I-N-S-I-S- [LIVN] -N-S-D.

(Protein Kinase (protkinase; Pfam Accession No. PF000
69)) SEQ ID NOS: 635, 992 and 1078 represent polynucleotides encoding protein kinases that catalyze the phosphorylation of proteins in various pathways and are involved in cancer. Eukaryotic protein kinases (Hanks et al., F
ASEB J. (1995) 9: 576; Hunter, Meth. Enzym
ol. (1991) 200: 3: Hanks et al., Meth. Enzymol. (
1991) 200: 38; Hanks, Curr. Opin. Struct. B
iol. (1991) 1: 369; Hanks et al., Science (1988).
241: 42) belongs to a very broad family of proteins that share a common conserved catalytic core for 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 has been shown to be 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 (Knight).
on et al., Science (1991) 253: 407).

The protein kinase profile 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 the alignment (Han
Ks et al., FASEB J .; (1995) 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)
-[LIVMFYWCSTAR]-[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
] -D- [LIVMFY]-[RSTAC] -x (2) -N- [LIVMFYC
], Where D is an active site residue.

(Ras Family Protein (ras; Pfam Accession No. PF00071)
) Sequence field number 527 shows a polynucleotide encoding the ras family of small GTP / GDP binding proteins (Valencia et al., 1991, Bio).
Chemistry 30: 4637-4648). Ras family members generally require specific guanine nucleotide exchange factors (GEFs) and specific GTPase activating proteins (GAPs) as stimulators of overall GTPases. The highest degree of sequence conservation among ras-related proteins is found in the four regions directly involved in guanine nucleotide binding. First two
One constitutes most of the phosphate and Mg2 + binding sites (PM sites),
And it is located in the first half of the G-domain. The other two regions are involved in guanosine binding, which is located in the C-terminal half of the molecule. The ras-related protein motifs and conserved structural features are reviewed by Valencia et al.
1991, Biochemistry 30: 4637-4648. The major consensus patterns of ras protein are: DTA
-G-Q-E-K- [LF] -G-G-LR- [DE] -G-Y-Y.

(Src Homology Domain 3 (SH3; Pfam Accession No. PF00018)) SEQ ID NO: 450 corresponds to the gene containing the Src homology domain. Src homology 3 (
The SH3) domain is associated with several cytoplasmic protein tyrosine kinases (eg,
Src, Abl, Lck) is a small protein domain of approximately 60 amino acid residues that was first identified as a conserved sequence in the non-catalytic part of (Sca, Ack, Lck) (Mayer et al., N.
ature (1988) 332: 272-275). Since then, it has been found in a great variety of other intracellular or membrane-bound proteins (Mus.
accio et al., FEBS Lett. (1992) 307: 55-61; Pa
Wson et al., Curr. Biol. (1993) 3: 434-442; Maye.
r et al., Trends Cell Biol. (1993) 3: 8-13; Paw.
Son et al., Nature (1995) 373: 573-580). The SH3 domain has a characteristic fold consisting of 5 or 6 β-strands arranged as two closely packed anti-parallel β-sheets. This linker region may contain a short helix (Kuriyan et al., Curr. Op.
in. Struct. Biol. (1993) 3: 828-827). This SH
The 3 domain is thought to mediate the assembly of specific protein complexes via binding to proline-rich peptides (Morton et al., Curr. B.
iol. (1994) 4: 615-617). Generally, the SH3 domain is found as a single copy in a given protein, but there are significant numbers of proteins with two SH3 domains, and a few proteins with 3 or 4 copies. The profile for detecting the SH3 domain is based on a structural alignment consisting of 5 ungapped blocks and a total of 62 coincident positions of the 4 linker regions.

(Trypsin (trypsin; Pfam accession number PF00089)). SEQ ID NOs: 635, 995 and 984 correspond to novel serine proteases of the trypsin family. 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 flanking the active site serine and histidine residues are well conserved (Brenner, N.
ature (1988) 334: 528). The consensus pattern for this trypsin protein family is: 1) [LIVM]-[ST
] -A- [STAG] -HC (where H is an active site residue); 2) [D
NSTAGC]-[GSTAPIMVQH] -x (2) -G- [DE] -SG
-[GS]-[SAPHV]-[LIVMFYWH]-[LIVMFYSTAN
QH] (where S is an active site residue). All sequences known to belong to this family are detected by the consensus sequence above, except for 18 different proteases that lost the first conserved glycine. If the protein contains both serine and histidine active site features, then the probability that it is a trypsin family serine protease is 100%.

(WD domain, G-β repeat (WD domain; Pfam accession number PF0040
0)). SEQ ID NOs: 505, 721 and 1018 show members of the WD domain / G-β repeat family. β-transducin (G-β) is a three-subunit (α, β) subunit of guanine nucleotide-binding protein (G protein) that acts as a mediator in the transduction of signals produced by transmembrane receptors.
And γ) (Gliman, Annu. Rev. Biochem.
(1987) 56: 615). The α subunit binds and hydrolyzes GTP; the β and γ subunits require GDP displacement of GDP, as well as membrane anchoring and receptor recognition. In higher eukaryotes, G-
β exists as a small multigene family of highly conserved proteins of approximately 340 amino acid residues. Structurally, G-β has a central Trp-Asp, respectively.
8 tandem repeats of approximately 40 residues containing the motif (this type of repeat is sometimes referred to as WD
-40 repeats). The consensus pattern for the WD domain / G-β repeat family is: [LIVMSTAC]-[LIVMFYWST
AGC]-[LIMSTAG]-[LIVMSTAGC] -x (2)-[DN]
-X (2)-[LIVMWSTAC] -x- [LIVMFSTAG] -W- [D
EN]-[LIVMFSTAGCN].

(WW / rsp5 / WWP domain characteristics and profile (WW domain;
Pfam registration number PF00397)). SEQ ID NO: 606 corresponds to the gene encoding the protein containing the WW domain. This WW domain (Bork et al., T
rends Biochem. Sci. (1994) 19: 531-533; A
ndre et al., Biochem. Biophys. Res. Commun. (19
94) 205: 1201-1205; Hofmann et al., FEBS Lett.
(1995) 358: 153-157; Sudol et al., FEBS Lett. (
1995) 369: 67-71; http: // www. Bork. embl-
heidelberg. de / Modules / ww-gif. html) (r
(also known as sp5 or WWP) was discovered as a short conserved region of many unrelated proteins. Of those dystrophins, this gene is responsible for Duchenne muscular dystrophy. This domain, which spans approximately 35 residues, is repeated up to four times in some proteins. The protein binds to a specific proline motif, [AP] -PP- [AP] -Y, thus SH3
It has been shown to be somewhat similar to domains (Chen et al., Proc. Natl.
． Acad. Sci. USA (1995) 92: 7819-7823). This W
The W domain contains a β chain assembled around four conserved aromatic positions (generally tryptophan). The name WW or WWP derives from the presence of two tryptophans and the conserved one proline. This WW domain frequently binds to other domains that are typical of proteins in signal transduction processes. The consensus pattern for the WW domain is: W-
x (9,11)-[VFY]-[FYW] -x (6,7)-[GSTNE]-[
GSTQCR]-[FYW] -x- (2) -P.

(Zinc finger, C2H2 type (Zincfing C2H2; Pfam registration number PF00096)). Some sequences contain a zinc finger domain of the C2H2 type that contains a zinc finger domain that facilitates nucleic acid binding (
Klug et al., Trends Biochem. Sci. (1987) 12:46.
4; Evans et al., Cell (1988) 52: 1; Payre et al., FEBS.
Lett. (1988) 234: 245; Miller et al., EMBO J. et al. (1
985) 4: 1609; and Berg, Proc. Natl. Acad. Sc
i. USA (1988) 85:99). In addition to the conserved zinc ligand residue, many other positions also have C
Important for the structural integrity of the 2H2 zinc finger (Rosenfeld et al., J.
． Biomol. Struct. Dyn. (1993) 11: 557). The best conserved positions are generally aromatic or aliphatic residues, located four residues after the second cysteine. The consensus pattern for the C2H2 zinc finger is: Cx (2,4) -Cx (3)-[LIVMFYWC]-
x (8) -H-x (3,5) -H. Two Cs and two Hs are zinc ligands.

(Zinc finger, C3HC4 type (RING finger) characteristics (Zincf
ing C3H4; Pfam registration number PF00097)). SEQ ID NOs: 805 and 1078 set forth polynucleotides encoding polypeptides having C3HC4 type zinc finger characteristics. Many eukaryotic and viral proteins contain this feature, which is predominantly 40-6, which connects the two atoms of zinc.
It is a conserved cysteine-rich domain of 0 residues (Borden KL;
B. Curr. Opin. Struct. Biol. (1996) 6:39.
5), and possibly involved in mediating protein-protein interactions. The 3D structure of the zinc-linked system is unique to the RING domain, and the "cross prosthesis (cro).
ss-brace) motif. The spacing of cysteines in this domain is: Cx (2) -Cx (9-39) -Cx (1-3) -H.
-X (2-3) -Cx (2) -Cx (4-48) -Cx (2) -C. C3
The characteristic pattern for the HC4 fingers is based on the central region of the following domains: Cx-Hx- [LIVMFY] -Cx (2) -C- [LIVMYA].
.

(Zinc finger, CCHC type (Zincfing CCHC; Pfam registration number PF00098)). SEQ ID NOs: 693, 973 and 1078 are CCH
Corresponds to the gene encoding a member of the C zinc finger family. This domain is also referred to as the retroviral zinc finger domain because the original CCHC zinc finger structure is derived from the HIV protein. This family also includes proteins involved in eukaryotic gene regulation (eg, C. ele).
gans GLH-1). This structure is an 18-residue zinc finger; there are no examples of indels in the alignment. The motif defining the CCHC-type zinc finger domain is: C-X2-C-X4-H-X4-C.
(Summers J Cell Biochem 1991 Mu January; 45 (1
): 41-8). This domain includes, for example, the HIV-1 nucleocapsid protein, Moloney murine leukemia virus nucleocapsid protein NCp10 (
De Rocquigny et al., Nucleic Acids Res. (199
3) 21: 823-9), and myelin transcription factor 1 (Myt1) (Kim et al.,
J. Neurosci. Res. (1997) 59: 272-90).

Example 5 Differential Expression of Polynucleotides of the Invention: Library Description and Detection of Differential Expression Relative expression levels of the polynucleotides of the invention include cell lines and patient tissue samples. It was evaluated in several libraries prepared from different sources. Table 4 provides a summary of these libraries. This includes the abbreviated library name (used hereafter), the mRNA source used to prepare the cDNA library, the "nickname" of the library used in the table below (enclosed in quotation marks). ), And the approximate number of clones in the library.

[0196]

[Table 1]

The KM12L4, KM12C, and MDA-MB-231 cell lines are described in Example 1 above. The MCF7 cell line was derived from breast pleural effusion. And it is non-metastatic. The MV-522 cell line is derived from human lung carcinoma and is highly metastatic. The UCP-3 cell line is a low metastatic human lung carcinoma cell line; MV-522 is a highly metastatic variant of UCP-3. These cell lines are well recognized in the art as models for the study of human breast and lung cancers (eg Chandracekaran et al. Cancer Re.
s. (1979) 39: 870 (MDA-MB-231 and MCF-7); G.
aspar et al., J Med Chem (1998) 41: 4965 (MDA-
MB-231 and MCF-7); Ranson et al., Br J Cancer (
1998) 77: 1586 (MDA-MB-231 and MCF-7); Kua.
ng et al., Nucleic Acids Res (1998) 26: 1116 (M.
DA-MB-231 and MCF-7); Varki et al., Int J Canc.
er (1987) 40:46 (UCP-3); Varki et al., Tumour B.
iol. (1990) 11: 327; (MV-522 and UCP-3); Va
rki et al., Anticancer Res. (1990) 10: 637; (MV
-522); Kelner et al., Anticancer Res (1995) 15
: 867 (MV-522); and Zhang et al., Anticancer Dr.
ugs (1997) 8: 696 (MV522)). Library 1
The 5-20 samples are from two different patients (UC # 2 and UC # 3). bFGF-treated HMEVC was prepared by incubation with bFGF at 10 ng / ml for 2 hours; VEGF-treated HMEVC had 20
Prepared by incubation with ng / ml VEGF for 2 hours. After incubation with each growth factor, cells were washed and lysis buffer was added for RNA preparation. The GRRpz and WOca cell lines were Donn
aM. Dr. Peehl, Department of Medicine, S
tanford University School of Medicine
provided by e. GRRpz was derived from normal prostate epithelium. WOca
The cell line is a Gleason grade 4 cell line.

Each library contains the mR expressed in the indicated mRNA source.
It consists of a collection of cDNA clones representing NA in order. Sequences were assigned to clusters to facilitate analysis of millions of sequences in each library. The concept of "cluster of clones" is based on their hybridization pattern into a panel of approximately 300 7 bp oligonucleotide probes, cdD.
Derived from selection / grouping of NA clones (Drmanac et al., Genomi
cs (1996) 37 (1): 29). Random cDNA clones from a tissue library are hybridized to 300 7 bp oligonucleotides with moderate stringency. Each oligonucleotide has several measures of specific hybridization to its particular clone.
300 of these measures of hybridization for 300 probes
The individual combinations are the "hybridization characteristics" for specific clones.
be equivalent to. Clones with similar sequences have similar hybridization characteristics. By developing a selection / grouping algorithm to analyze these characteristics, groups of clones in the library can be identified and computationally assembled. These groups of clones are called "clusters". Stringency of choice in algorithms (classical library cd
Depending on the stringency of hybridization in the NA screening protocol), the "purity" of each cluster can be controlled. For example, even if the artificial result is the highest stringency, 400
"web-lab" of a cDNA library containing a bp cDNA fragment
As with screening, the artifacts of clustering can occur with computerized clustering. The stringency used in the implementation of the clusters herein generally provides a group of clones that are derived from the same or closely related cDNAs. Closely related clones can be the result of different length clones of the same cDNA, closely related clones from a highly related gene family, or splice variants of the same cDNA.

Differential expression for selected clusters first determined the number of cDNA clones (clones in the first round) corresponding to the selected cluster in the first library and selected in the second library. It was evaluated by determining the number of cDNA clones corresponding to the cluster (clones in the second round). The differential expression of the selected clusters in the first library as compared to the second library is the percentage of expression between the two libraries.
Expressed as "ratio". Generally, the "ratio" is: 1) in the first library, by dividing the number of clones corresponding to the selected cluster in the first library by the total number of clones analyzed from the first library. Calculating the percent expression of the selected cluster; 2) dividing the number of clones corresponding to the selected cluster in the second library by the total number of clones analyzed from the second library. Calculated by calculating the percent expression of the selected clusters in the library; 3) dividing the calculated percent expression from the first library by the calculated percent expression from the second library. If the "number of clones" corresponding to the selected cluster in the library is zero, the value is set to 1 to aid the calculation. The formula used in calculating ratios takes into account the “depth” of each library being compared, ie the total number of clones analyzed in each library.

Generally, a polynucleotide is significantly differentially expressed between two samples if the ratio value is at least greater than about 2, preferably greater than at least about 3, and more preferably greater than at least about 5. , Where the ratio values are calculated using the above method. The significance of the differential expression was determined by the z-score test (Za
r, Biostatistical Analysis, Prentice H
all, Inc. , USA, “Differences between Pr
pp. 296-298 (1974)).

Examples 6-11: Differential Expression of Polynucleotides of the Invention For example, between cells derived from cancerous tissue with high metastatic potential and cells derived from low metastatic cancerous tissue, and high. Many polynucleotide sequences have been identified that are differentially expressed between cells derived from metastatic cancer tissue and normal tissue. Assessing the level of expression of genes corresponding to these sequences can be used in diagnosis, prognosis, and / or treatment (eg, to facilitate rational design of therapy, monitoring during or after therapy, etc.). Can be worth it. In addition, genes corresponding to the differentially expressed sequences described herein may be targeted at therapeutic targets due to, for example, their involvement in the regulation (eg, inhibition or promotion) of development of the metastatic phenotype. possible. For example, sequences corresponding to genes whose expression is increased in cells with high metastatic potential compared to normal or non-metastatic tumor cells are involved in processes such as angiogenesis, differentiation, cell replication, and metastasis. Gene or regulatory sequence.

Detection of relative expression levels of differentially expressed polynucleotides, as described herein, can provide valuable information to guide the clinician in selecting a treatment. For example, a sample of a patient exhibiting expression levels of one or more of these polynucleotides corresponding to genes whose expression is increased in metastatic or highly metastatic cells is treated with a more aggressive treatment for that patient. Can be guaranteed. In contrast, detection of the expression levels of polynucleotide sequences corresponding to those associated with low metastatic potential cells may warrant a more positive prognosis than is suggested by gross pathology.

Many of the polynucleotide sequences of the present invention are found in naive human microvascular endothelial cells (H
Differentially expressed between growth factor treated HMVECs compared to MVECs).
Sequences differentially expressed between growth factor-treated and untreated HMVECs encode gene products involved in angiogenesis, metastasis (cell migration), and other developmental and oncogenic processes. The sequence can be shown. For example, sequences that are more highly expressed in HMVEC treated with growth factors (eg, bFGF or VEGF) as compared to untreated HMVEC may act as drug targets for chemotherapeutic agents, such as Decreasing the expression of a regulated gene or inhibiting the activity of its encoded gene product acts to inhibit tumor cell angiogenesis. Detection of the expression of these sequences in colon cancer tissues may be valuable in determining diagnostic, prognostic, and / or treatment information associated with preventing the achievement of malignant conditions in these tissues, and It may be important in a risk assessment for a patient. Therefore, a sample of patients exhibiting increased levels of one or more of these polynucleotides warrants more careful attention procedures or more frequent screening procedures to ascertain malignant status as early as possible. obtain.

Accordingly, the differential expression of the polynucleotides described herein can be used as a diagnostic, prognostic marker, eg, for risk assessment, patient treatment, etc. These polynucleotide sequences can also be used in combination with other known molecular and / or biochemical markers. The following examples provide the relative expression levels of polynucleotides from specific cell lines and patient tissue samples.

Example 6: High metastatic breast cancer cells versus low metastatic breast cancer cells The table below lists genes that are differentially expressed between high and low metastatic breast cancer cells. The data for the indicated polynucleotides are summarized.

Table 5. Breast cancer cells with high metastatic potential (lib3)> Breast cancer cells with low metastatic potential (lib4)
)

[0207]

[Table 2]

Table 6. Breast cancer cells with low metastatic potential (lib4)> Breast cancer cells with high metastatic potential (lib3)
)

[0209]

[Table 3]

Example 7 High Metastatic Potential Lung Cancer Cells vs. Low Metastatic Potential Lung Cancer Cells The following is a poly showing differentially expressed genes between highly metastatic and low metastatic lung cancer cells. Summarize the nucleotides.

Table 7. High metastatic lung cancer cells (lib8)> Low metastatic lung cancer cells (lib9)
)

[0212]

[Table 4]

Example 8 Highly Metastatic Metastatic Colon Cancer Cells vs Low Metastatic Metastatic Colon Cancer Cells Table 8 is differentially expressed between high and low metastatic colon cancer cells. The polynucleotides that represent the genes are summarized.

Table 8. Low metastatic metastatic colon cancer cells (lib2)> High metastatic metastatic colon cancer cells (li)
b1)

[0215]

[Table 5]

Example 9: High Tumor Probability Colon Tissues vs. Metastasized Colon Cancer Tissues The following table shows between high tumor potential colon cancer cells and cells from high metastatic potential colon cancer cells of patients. The polynucleotides that represent the differentially expressed genes are summarized.

Table 9. Highly tumor-prone colon tissue (lib16) versus highly metastatic colon tissue (lib1)
7)

[0218]

[Table 6]

Example 10: Differential Expression Across Multiple Libraries A number of polynucleotide sequences were identified that represent genes that are differentially expressed across multiple libraries. Expression of these sequences in tissues or any origin may be beneficial in determining diagnostic, prognostic and / or treatment information associated with prevention of achievement of malignant conditions in these tissues, and patient Can be important in assessing the risk of These polynucleotides may also serve as non-tissue specific markers of risk of tumor metastasis, for example. Table 10 below
Summarizes this data.

[0220]

[Table 7]

Some polynucleotides showed opposite differential expression trends in libraries of different origin (see, eg, SEQ ID NO: 316). These data suggest that the differential expression patterns of some genes involved in the development of metastases indicate a unique role for the tissue-specific genes of origin.

Those skilled in the art will be able to recognize or ascertain the many equivalents to the particular embodiments of the invention described herein using experiments that are at best routine.
Such particular embodiments and equivalents are intended to be covered by the following claims.

All publications and patent applications cited in this specification are described in this specification as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Incorporated as a reference in. The citation of any publication is for its disclosure prior to the filing date of the present application and is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Should not be.

While the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is understood that certain changes and modifications are within the spirit or scope of the appended claims. It will be readily apparent to one of ordinary skill in the art in view of the teachings of the present invention, which may be made without departing from the scope.

(Deposit Information) The following materials are American Type Culture Collection (CMCC = Chi)
ron Master Culture Collection).

[0226]

[Table 8]

In addition, the pool of selected clones, as well as the library containing the particular clones, were assigned an “ES” number (internal reference) and deposited with the ATCC. Table 21 below provides the ATCC Deposit Numbers for ES deposits. All of these were deposited on or before May 13, 1999. The names of the clones containing each of these deposits are provided in the table numbered 22 and above (inserted after the claims).

Table 12: Pools and libraries of clones deposited with the ATCC before September 23, 1999.

[0229]

[Table 9]

The deposits described herein are provided solely as a convenience to those of ordinary skill in the art and are not an admission that the deposit is required by US Pat. The sequences of the polynucleotides contained in the deposited material, as well as the amino acid sequences of the polypeptides encoded thereby, are incorporated herein by reference and are in the event of any conflict with the description of the sequences herein. Also controls. A permit may be required to make, use, or sell the deposited material, and such a permit is hereby granted.

Recovery of Individual Clones from Pooled Clone Deposits If the ATCC deposit consists of a pool of cDNA clones or a library of cDNA clones, this deposit separates each clone separately. Prepared by first transfecting bacterial cells. Clones in the pool or library were then deposited as a pool of equivalent mixtures in the composite deposit. Specific clones may be obtained from composite deposits using methods well known in the art. For example, a bacterial cell containing a particular clone,
Isolation of a single colony and oligonucleotide probe (s) designed to specifically hybridize to the sequence of the insert of that clone (eg, encoded with the sequence number shown) (A probe based on the unmasked sequence of the polynucleotide) can be used to identify colonies containing particular clones by standard colony hybridization techniques. This probe should be designed to have a T _m of about 80 ° C. (assuming 2 ° C. for each A or T and 4 ° C. for each G or C). Positive colonies can then be picked, cultured and propagated,
The recombinant clone can then be isolated. Alternatively, a probe designed in this manner can be used in PCR according to methods well known in the art (eg purifying cDNA from the deposited culture pool and amplifying with the corresponding desired polynucleotide sequence). Nucleic acid molecules can be isolated from pooled clones (by using this probe in a PCR reaction to generate a product).

[0232]

[Table 10]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[Table 11]

[0256]

[Table 12]

[0257]

[0258]

[0259]

[Table 13]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[Table 14]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[Table 15]

[0340]

[Table 16]

[0341]

[0342]

[0343]

[0344]

[0345]

[Table 17]

[0346]

[Sequence list]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12N 1/21 C12Q 1/68 Z 5/10 C12N 15/00 ZNAA C12Q 1/68 5/00 A (31 ) Priority claim number 60/102, 380 (32) Priority date September 29, 1998 (September 29, 1998) (33) Country of priority claim United States (US) (31) Priority claim number 60/103 , 815 (32) Priority date October 8, 1998 (Oct. 8, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 60/105, 877 (32) Priority date Heisei October 27, 2010 (October 27, 1998) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE), A (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, 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, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, N, YU, ZA, ZW (72) inventor Escobedo, Jaime United States California 94507, Alamo, Raboruna load 1470 (72) inventor Innis, Michael er. United States California 94556, Moraga, Constance Place 315 (72) Inventor Garcia, Pablo Dominga United States California 94131, San Francisco, Chenery Street 882 (72) Inventor Sados Klinger, Julie United States California 94707, Kensington, Lexington Road 280 ( 72) Inventor Linahart Christoff, California 94501, Alameda, Clinton Avenue 1633 (72) Inventor Gies, Klaus Germany Berlin D-10115, Chossetlass 92 (72) Inventor Randazo, Filippo United States California 94133, San Francisco, Chestnut Street 690 Apartment 403 (72) Inventor Kennedy, Giulian Sea. United States California 94116, San Francisco, Castenada Avenue 360 (72) Inventor Pot, David United States California 94112, San Francisco, Number 102, 5T Avenue 1565 (72) Inventor Cassam, Altaf United States California 94602, Oakland, Harold Street 2659 (72) Inventor Ramson, George United States California 94556, Moraga, Sandringham Drive 232 (72) Inventor Dolmanac, Ladge United States California 94303, Palo Alto, East Greenwich Place 850 (72) Inventor Kurk Benjacob, Radmir United States California 94068, Sunnyvale, Haverhill Drive 762 (72) Inventor Dickson, Mark United States California 95025, Hollister, Number B. , Gabiran Drive 1411 (72) Inventor Dormanac, Snezana United States California 94303, Palo Alto, East Greenwich Place 850 (72) Inventor Rabat, Iban United States California 94086, Sunnyvale, Akaranes Drive 140 (72) Inventor Reshko Witts, Dena United States California 94087, Sunnyvale, Darshire Way 678 (72) Inventor Kita, David United States California 94404, Foster City, Bounty Drive 899 (72) Inventor Garcia, Veronica United States California 94086, Sunnyvale, Anno Nuevo 396 , Apartment 412 (72) Inventor Jones, Lee William United States California 94086, Sunnyvale, number 412 Anon Bo 396 (72) Inventor Stacey Crane, Bargit United States California 94086, Sunnyvale, South Mary Avenue 345 F Term (reference) 4B024 AA01 AA12 AA19 BA80 CA02 CA04 CA09 CA12 CA20 DA01 DA02 DA05 DA11 DA12 GA11 HA11 4B063 QA01 QA20 QQ43Q QR56 QR84 QS34 QS39 QX10 4B065 AA01X AA58X AA72X AA87X AA93Y AB01 AC14 BA01 CA24 CA44 CA46 4H045 AA10 AA11 BA10 CA40 DA75 EA20 EA50 FA71 FA74

Claims (12)

[Claims] 1. A library of polynucleotides, SEQ ID NOs: 1 to 1.
A library comprising sequence information for at least one of 079. 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 computer readable format. 4. The library comprises a polynucleotide corresponding to a gene that is differentially expressed in a cell having a high metastatic potential relative to a control cell, the control cell being a normal cell or a cell having a low metastatic potential, And here, the sequences are SEQ ID NOS: 350, 571, 781, 778, 756, 779, 691,
The library of claim 1 selected from the group consisting of 686, 916, and 969. 5. The library comprises a polynucleotide corresponding to a gene that is differentially expressed in a cancer cell having a relatively low metastatic potential as compared with a control cell,
The control cells are normal cells or cells with high metastatic potential, and wherein the sequences are SEQ ID NOs: 34, 57, 103, 110, 113, 189, 214, 3
59, 521, 532, 533, 536, 547, 549, 554, 555, 5
58, 561, 562, 572, 582, 584, 587, 589, 590, 5
91, 592, 599, 603, 607, 609, 623, 624, 635, 6
36, 637, 641, 646, 647, 648, 650, 653, 654, 6
The library of claim 1, selected from the group consisting of 56, 657, and 661. 6. An isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to the identified sequences of SEQ ID NOs: 1-1079 or degenerate variants or fragments thereof. 7. A recombinant host cell comprising the polynucleotide of claim 6. 8. A polynucleotide encoded by the polynucleotide of claim 6,
Isolated polypeptide. 9. An antibody that specifically binds to the polypeptide of claim 8. 10. A vector comprising the polynucleotide according to claim 6. 11. A polynucleotide comprising the nucleotide sequence of interest contained in ATCC Deposit No. xx, or the clone deposited as xx. 12. A method for detecting a gene differentially expressed in correlation with the cancerous state of a mammalian cell, the method comprising the steps of: differentially in a test sample derived from cells suspected of being cancerous. Detecting at least one expressed gene product, wherein said gene product is SEQ ID NO: 34, 57, 100, 103, 110, 113, 189, 209, 214, 3,
16, 350, 359, 370, 521, 532, 533, 536, 547, 5
49, 554, 555, 558, 561, 562, 571, 572, 582, 5
84, 587, 589, 590, 591, 592, 599, 603, 607, 6
09, 623, 624, 635, 636, 637, 641, 645, 646, 6
47, 648, 650, 653, 654, 656, 657, 661, 781, 7
78, 756, 779, 691, 686, 854, 916, and 969 encoded by a gene corresponding to a sequence of the gene, wherein the detection of the differentially expressed gene product comprises: , Correlating with the cancerous state of the cells from which the test sample was derived.