WO2004033643A2 - Methods of use of a dna helicase in the diagnosis and treatment of colon and lung cancer - Google Patents

Abstract

The invention provides a DNA helicase that is differentially expressed in colon and lung cancer. It also provides for the use of the protein, a cDNA encoding the protein, and antibodies that specifically bind the protein in various methods to diagnose, stage, treat, or monitor the treatment of colon and lung cancer.

Description

METHODS OF USE OF A DNA HELICASE IN THE DIAGNOSIS AND TREATMENT

OF COLON AND LUNG CANCER

TECHNICAL FIELD This invention relates to a DNA Helicase, its encoding cDNA, and an antibody that specifically binds the protein and to their use to diagnose, to stage, to treat, or to monitor the progression or treatment of colon and lung cancer.

BACKGROUND OF THE INVENTION Cancers and malignant tumors are characterized by continuous cell proliferation and cell death and are related causally to both genetics and the environment. Genes whose expression are associated with cancer are of potentially great importance as cancer markers in the early diagnosis and prognosis of various cancers, as well as potential targets in cancer treatment.

Colorectal cancer is the fourth most common cancer and the second most common cause of cancer death in the United States with approximately 130,000 new cases and 55,000 deaths per year. Colon and rectal cancers share many environmental risk factors and both are found in individuals with specific genetic syndromes. (See Potter (1999) J Natl Cancer Institute 91:916-932 for a review of colorectal cancer.) Colon cancer is the only cancer that occurs with approximately equal frequency in men and women, and the five-year survival rate following diagnosis of colon cancer is around 55% in the United States (Ries et al. (1990) National Institutes of Health, DHHS Publ No. (NIH)90-2789).

Lung cancer is the leading cause of cancer death in the United States affecting more than 100,000 men and 50,000 women each year, and nearly 90% of those diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in exposed bronchial epithelium. In nearly 80% of those diagnosed, the lung cancer has metastasized to pleura, brain, bone, pericardium, and liver. Treatment with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to the identification, staging, and treatment of lung cancer could positively affect prognosis. RuvBLl/TIP49a DNA Helicase

DNA helicases are important components of nuclear complexes involved in DNA replication, repair, and transcription. The role of the DNA helicase in these complexes is to unwind duplex DNA into single-stranded DNA using the energy of ATP hydrolysis. Features common to all DNA helicases are the conserved Walker A and B motifs. The Walker A motif contains the sequence GxxxxGKT. The lysine residue directly contacts phosphate residues in ATP and the threonine binds to the coordinated Mg²⁺. The Walker B motif has the sequence DExx. The aspartic acid residue contacts Mg²⁺ and the glutamic acid is thought to be involved in ATP hydrolysis (Caruthers and McKay (2002) Curr Opin Struct Biol 12:123-133).

The RuvB DNA helicase is an E. coli protein that functions in homologous recombination and recombination repair of DNA damage (Mezard et al. (1997) J Mol Biol 271:704-717). Functional motifs associated with ATP binding and hydrolysis in RuvB have been identified by mutational analysis (Iwasaki et al. (2000) Molecular Microbiology 36:528-538). Eukaryotic helicases have been identified that share sequence similarity with E. coli RuvB. These include the Saccharomvces cerevisiae scRUVBLl, a gene that is essential for viability (Qiu et al. (1998) J Biol Chem 273:27786-27793), and the rat and human TIP49a. Rat TIP49a was originally identified by binding in a complex with TATA-binding protein (TBP) from rat liver nuclear extracts (Kanemaki et al. (1999) J Biol Chem 274:22437-22444). Since TBP is involved in the initiation of eukaryotic transcription, it has been suggested that TIP49a may play a role in transcriptional processes in addition to recombination repair. Human TIP49a is 99% identical to rat TIP49a and was isolated from a human liver cDNA library using rat TIP49a as a probe (Makino et al. (1998) Biochem Biophys Res Comm 245:819-823).

Human TIP49a has also been found to interact with other proteins involved in a variety of DNA-related functions. These include the 14 kDa subunit of replication protein A (hsRPA3, Qiu, supra), RuvBL2/TIP49b, another RuvB-like helicase, (Kanemaki, supra), c-myc (Wood et al. (2000) Molecular Cell 5:321-330), and β-catenin, a member of the Wnt signaling pathway (Bauer et al. (1998) Proc Natl Acad Sci USA 95:14787-14792). The Wnt Signaling Pathway

Wnt proteins are secreted, cysteine-rich factors that are involved in many developmental and biological processes (reviewed in Wodarz and Nusse (1998) A nu Rev Cell Dev Biol 14:59-88). Wnt signals are transduced into the cell upon binding of the Wnts to frizzled receptors. In cells that have not been stimulated by Wnt, intracellular components of the Wnt signaling pathway form a complex that includes β-catenin, Axin or conductin, glycogen synthase kinase 3 β (GSK3), and the APC tumor suppressor protein. Interactions between these components of the Wnt signaling pathway facilitate phosphorylation of β-catenin by GSK3 on serine and threonine residues. Phosphorylation of β- catenin results in ubiquitination and subsequent proteolytic degradation of β-catenin (reviewed in Bienz and Clevers (2000) Cell 103:311-320). However, when cells are stimulated with the appropriate Wnt factor, a protein known as dishevelled inhibits the complex containing β-catenin. This results in an increase in the cytoplasmic, unphosphorylated pool of β-catenin. β-catenin is then free to translocate into the nucleus, complex with members of the TCF/LEF family of transcription factors, and activate transcription of target genes in the Wnt signaling pathway. Thus, β-catenin is one of the primary effectors of the Wnt signaling pathway. Wnt Signaling Pathway and Colorectal Cancer

Mutations in the Wnt signaling pathway occur in the majority of colorectal cancers. Approximately 85% of colorectal cancers contain loss of function mutations in the APC gene (Kinzler and Vogelstein (1998) The Genetic Basis of Human Cancer, pp. 565-587, McGraw Hill, New York, NY). Many of these mutations result in a truncated protein lacking β-catenin interaction domains. In the 15% of colon cancers that do not have mutations in APC, mutations in β-catenin are found in the arrύno-terminal serines and threonines that are phosphorylated by GSK3 (Morin et al. (1997) Science 275: 1787-1790). These mutations result in a stablization of β-catenin protein and constitutive activation of β-catenin/TCF transcription. In addition, mutations in another member of the Wnt pathway, conductin, have also been found in colorectal cancers (Liu et al. (2000) Nature Genet 26:146-147).

The importance of β-catenin transcriptional activation in colon cancer has been further investigated using wild-type and mutant APC constructs. Introduction of wild-type APC into colon tumor cell lines results in the suppression of β-catenin-mediated transcriptional activation (Korinek et al. (1997) Science 275: 1784-1787), while mutant forms of APC that cannot bind to β-catenin are unable to elicit this effect (Morin, supra). The β-catenin interaction domains of APC are sufficient to induce apoptosis in colon tumor cell lines (Shih et al. (2000) Cancer Res 60: 1671-1676). Target genes of the β-catenin/TCF complex include c-myc, cyclin Dl, MMP-7, and VEGF to name a few (He et al. (1998) Science 281:1509-1512; Tetsu and McCormick (1999) Nature 398:422- 426; Brabletz et al. (1999) Am J Path 155:1033-1038; Crawford et al. (1999) Oncogene 18:2883- 2891; and Shtutman et al. (1999) Proc Natl Acad Sci 96:5522-5527; Zhang et al. (2001) Cancer Research 61:6050-6054). Many of the β-catenin target genes are involved in cell proliferation and the cell cycle, and thus play direct roles in the initiation and progression of cancer.

Array technologies and quantitative PCR provide the means to explore the expression profiles of a large number of related or unrelated genes. When an expression profile is examined, arrays provide a platform for examining which genes are tissue-specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder. The application of expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of the disease. For example, both the sequences and the amount of expression can be compared between tissues from subjects with different disorders. The discovery of a DNA helicase, its encoding cDNA, and the making of an antibody that specifically binds the protein satisfies a need in the art by providing compositions which are useful to diagnose, to stage, to treat, or to monitor the progression or treatment of colon and lung cancer.

SUMMARY OF THE INVENTION

The invention provides an isolated cDNA comprising a nucleic acid sequence encoding a protein having the amino acid sequence of SEQ ID NO: 1 (RUVBL1/TIP49). The invention also provides an isolated cDNA or the complement thereof comprising a nucleic acid seqeuence of SEQ ID NO:2. The invention further provides a probe consisting of the cDNA encoding RUVBLl, A cell transformed with the cDNA encoding RUVBLl, a composition comprising the cDNA encoding RUVBLl, and a labeling moiety, an array element comprising the cDNA encoding RUVBLl, and a substrate upon which the cDNA encoding RUVBLl, is immobilized.

The invention provides a vector containing the cDNA encoding RUVBLl, a host cell containing the vector and a method for using the cDNA to make the protein, the method comprising culturing the host cell containing the vector containing the cDNA encoding the protein under conditions for expression and recovering the protein from the host cell culture. The invention also provides a transgenic cell line or organism comprising the vector containing the cDNA encoding RUVBLl. The invention further provides a composition, a substrate or a probe comprising the cDNA, a fragment, a variant, or complements thereof, which can be used in methods of detection, screening, and purification. In one aspect, the probe is a single-stranded complementary RNA or DNA molecule.

The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose colon or lung cancer.

The invention provides a purified protein or a portion thereof comprising an amino acid sequence of SEQ ID NO: 1. The invention also provides a composition comprising the purified protein and a pharmaceutical carrier, a composition comprising the protein and a labeling moiety, a substrate upon which the protein is immobilized, and an array element comprising the protein. The invention further provides a method for detecting expression of a protein having the amino acid sequence of SEQ ID NO: 1 in a sample, the method comprising performing an assay to determine the amount of the protein in a sample; and comparing the amount of protein to standards, thereby detecting expression of the protein in the sample. The invention still further provides a method for diagnosing cancer comprising performing an assay to quantify the amount of the protein expressed in a sample and comparing the amount of protein expressed to standards, thereby diagnosing a cancer. In a one aspect, the assay is selected from antibody arrays, enzyme-linked immunosorbent assays, fluorescence-activated cell sorting, 2D-PAGE and scintillation counting, protein arrays, radioimmunoassays, and western analysis. In a second aspect, the sample is from colon or lung tissue. In a third aspect, the cancer is a colon or lung cancer.

The invention provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from agonists, antagonists, bispecific molecules, DNA molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules. In another aspect, the ligand is used to treat a subject with a cancer, in particular, a colon or lung cancer. The invention also provides an therapeutic antibody that specifically binds the protein having the amino acid sequence of SEQ ID NO: 1. The invention further provides an antagonist which specifically binds the protein having the amino acid sequence of SEQ ID NO:l. The invention yet further provides a small drug molecule which specifically binds the protein having the amino acid sequence of SEQ ID NO: 1. The invention also provides a method for testing a ligand for effectiveness as an agonist or antagonist comprising exposing a sample comprising the protein to the molecule or compound, and detecting agonist or antagonist activity in the sample.

The invention provides a method for using a protein to screen a plurality of antibodies to identify an antibody that specifically binds the protein comprising contacting a plurality of antibodies with the protein under conditions to form an antibody:protein complex, and dissociating the antibody from the antibodyφrotein complex, thereby obtaining antibody that specifically binds the protein. In one aspect the antibodies are selected from intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a bispecific molecule, a multispecific molecule, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an F(ab')₂ fragment, an Fv fragment, and an antibody-peptide fusion protein. The invention provides purified antibodies which bind specifically to a protein.

The invention also provides methods for using a protein to prepare and purify polyclonal and monoclonal antibodies which specifically bind the protein. The method for preparing a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies. The method for preparing a monoclonal antibodies comprises immunizing a animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating monoclonal antibodies from culture.

The invention also provides a method for using an antibody to detect expression of a protein in a sample, the method comprising combining the antibody with a sample under conditions for formation of antibody:protein complexes, and detecting complex formation, wherein complex formation indicates expression of the protein in the sample. In one aspect, the sample is selected from colon and lung tissue. In a second aspect, complex formation is compared to standards and is diagnostic of a colon or lung cancer. The invention provides a method for immunopurification of a protein comprising attaching an antibody to a substrate, exposing the antibody to a sample containing protein under conditions to allow antibody:protein complexes to form, dissociating the protein from the complex, and collecting purified protein. The invention also provides a composition comprising an antibody that specifically binds the protein and a labeling moiety or pharmaceutical agent; a kit comprising the composition; an array element comprising the antibody; a substrate upon which the antibody is immobilized. The invention further provides a method for using a antibody to assess efficacy of a molecule or compound, the method comprising treating a sample containing protein with a molecule or compound; contacting the protein in the sample with the antibody under conditions for complex formation; determining the amount of complex formation; and comparing the amount of complex formation in the treated sample with the amount of complex formation in an untreated sample, wherein a difference in complex formation indicates efficacy of the molecule or compound.

The invention provides a method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention an antibody that specifically binds the protein, a bispecific molecule that specifically binds the protein, a multispecific molecule that specifically binds the protein, or a composition comprising an antibody and a pharmaceutical agent. The invention also provides a method for delivering a pharmaceutical or therapeutic agent to a cell comprising attaching the pharmaceutical or therapeutic agent to a bispecific molecule that specifically binds the protein and administering the bispecific molecule to a subject in need of therapeutic intervention, wherein the bispecific molecule delivers the pharmaceutical or therapeutic agent to the cell.

The invention provides an agonist that specifically binds the protein, and a composition comprising the agonist and a pharmaceutical carrier. The invention also provides an antagonist that specifically binds the protein, and a composition comprising the antagonist and a pharmaceutical carrier. The invention further provides a pharmaceutical agent or a small drug molecule that specifically binds the protein.

The invention provides a method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention pharmaceutical agent or a small drug molecule that specifically binds the protein. The invention provides an antisense molecule of 18 to 30 nucleotides in length that specifically binds a portion of a polynucleotide having a nucleic acid sequence of SEQ ID NO: 2 or the complement thereof wherein the antisense molecule inhibits expression of the protein encoded by the polynucleotide.

The invention also provides an antisense molecule with at least one modified intemucleoside linkage or at least one nucleotide analog. The invention further provides that the modified intemucleoside linkage is a phosphorothioate linkage and that the modified nucleobase is a 5-methylcytosine.

The invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA of SEQ ID NO:3-8 transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby foπriing a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES Figure 1 shows the relative expression of RuvBLl in cancerous and non-cancerous colon tissue from donor-matched samples obtained from patients with colon cancer. The X axis identifies individual donors and the Y axis the expression of RuvBLl in each sample relative to a pool of normal colon tissue. Tissue samples were obtained from Huntsman Cancer Institute (HCl; Salt Lake City UT). The analysis was performed by QPCR using the TAQMAN protocol (Applied Biosystems (ABI), Foster City CA) and an oligonucleotide probe extending from about nucleotide C1022 to about nucleotide C1075 of SEQ ID NO:2.

Figure 2 shows the relative expression of RuvBLl in cancerous and non-cancerous lung tissue from donor-matched samples obtained from patients with lung cancer. The X axis identifies individual donors and the Y axis the expression of RuvBLl in each sample relative to normal lung tissue from Donor 9752. Tissue samples were obtained from the Roy Castle Institute for Lung Cancer Research (RCI; Liveφool UK). The analysis was performed by QPCR using the TAQMAN protocol(ABI) and an oligonucleotide probe extending from about nucleotide C1022 to about nucleotide C1075 of SEQ ID NO:2.

Figure 3 shows the relative expression of RuvBLl in various tumorigenic and non-tumorigenic cell lines derived from colon, lung, prostate, breast and ovary tissue using QPCR (ABI) and an oligonucleotide probe extending from about nucleotide C1022 to about nucleotide C1075 of SEQ ID NO:2. The Y axis shows the expression in each cell line relative to a mixed pool of tissues and cell lines. The X axis indicates each cell line grouped according to the tissue origin. Cell lines were obtained from the ATCC (Manassas VA) and Clonetics and are described in Example VII.

Figure 4 shows the relative expression of RuvBLl in a panel of normal tissues using QPCR (ABI) and an oligonucleotide probe extending from about nucleotide C1022 to about nucleotide C1075 of SEQ ID NO:2. The Y axis shows the expression in each tissue relative to that in a pool of mixed tissues (positive control). The Y axis indicates the tissue type.

Figure 5 shows the expression of RuvBLl and various mutant genes of RuvBLl in HCT116 colon tumor cells using western analysis. Cells were transfected with a pTRIEX-3 NEO vector alone (ptx) or the vector containing the cDNA encoding RuvBLl (WT=wild type) or various single amino acid mutants of RuvBLl (DN, KR, TA or KA) and a C-teirninal HIS tag. The mutant genes are described in Table at p. 16. The X axis shows the time course of expression for each sample at 24, 48, and 72 hours post transfection.

Figure 6 shows the results of preparation and purification of an RuvBLl antibody using a peptide from amino acid residue R118 to E132 of RuvBLl. Panel A shows the results of an analysis for RuvBLl expression using the crude serum from an immunized rabbit, and Panel B, results using the affinity purified antibody. The analyses were conducted with (+ peptide) and without the Rl 18 to E132 peptide to block antibody binding. The individual sample lanes are cell lysates from 1=HCT116 cells transfected with RuvBLl; 2 =HCT116 cells + empty vector, and 3 =HCT116 cells alone.

Table 1 shows the differential expression of RuvBLl in cancerous colon tissue relative to normal colon tissue as deteπrύned by microarray analysis. Column 1 shows the differential expression of RuvBLl in terms of the ratio of the signal intensity for the flourescent dye Cy5 in labeled tumor tissue relative to that for flourescent dye Cy3 in labeled normal colon tissue. Column 2 shows the source of the normal colon tissue as the individual donor (Dn), or pooled tissue from more than one donor (pool), column 3 is a description of the colon tumor sample, and column 4, the source of the tumor sample. Tissue samples were obtained from HCl and Asterand Bioresources.

Table 2 shows the differential expression of RuvBLl in cancerous lung tissue relative to normal lung tissue as determined by microarray analysis. Column headings are the same a those described above for Table 1. Tissue samples were obtained from RCI Table 3 shows the differential expression of RuvBLl in cancerous ovary tissue relative to normal ovary tissue from a patient with ovarian cancer as determined by microarray analysis. Column headings are the same a those described above for Table 1. Tissue samples were obtained from HCL

DESCRIPTION OF THE INVENTION It is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the p pose of describing particular embodiments and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms "a", "an", and "the" may include plural reference unless the context clearly dictates otherwise. For example, a reference to "a host cell" includes a plurality of such host cells known to those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are cited for the prupose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and whiςh might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Definitions

"Antibody" refers to intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an Fføb'^ fragment, an Fv fragment, and an antibody-peptide fusion protein. "Antigenic deteπ inant" refers to an antigenic or immunogenic epitope, structural feature, or region of an oligopeptide, peptide, or protein which is capable of inducing formation of an antibody that specifically binds the protein. Biological activity is not a prerequisite for immunogenicity.

"Array" refers to an ordered arrangement of at least two cDNAs, proteins, or antibodies on a substrate. At least one of the cDNAs, proteins, or antibodies represents a control or standard, and the other cDNA, protein, or antibody is of diagnostic or therapeutic interest. The arrangement of at least two and up to about 40,000 cDNAs, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each cDNA and at least one nucleic acid, each protein and at least one ligand or antibody, or each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable. A "cancer" refers to an adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and tumors of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, esophagus, gall bladder, ganglia, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, pituitary gland, prostate, salivary glands, skin, small intestine, spleen, stomach, testis, thymus, thyroid, and uterus.

"RuvBLl" refers to a DNA helicase that is exactly or highly homologous (>85%) to the amino acid sequence of SEQ ID NO: 1 obtained from any species including bovine, ovine, porcine, murine, equine, and preferably the human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant. The "complement" of a cDNA of the Sequence Listing refers to a nucleic acid molecule which is completely complementary over its full length and which will hybridize to the cDNA or an mRNA under conditions of high stringency.

"cDNA" refers to an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, may be double-stranded or single-stranded, represents coding and noncoding 3' or 5' sequence, and lacks introns.

The phrase "cDNA encoding a protein" refers to a nucleotide sequence that closely aligns with sequences which encode conserved regions, motifs or domains that were identified by employing analyses well known in the art. These analyses include BLAST (Basic Local Alignment Search Tool) which provides identity within the conserved region (Altschul (1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403-410).

A "composition" refers to the polynucleotide and a labeling moiety; a purified protein and a pharmaceutical carrier or a heterologous, labeling or purification moiety; an antibody and a labeling moiety or pharmaceutical agent; and the like.

"Derivative" refers to a cDNA or a protein that has been subjected to a chemical modification. Derivatization of a cDNA can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. These substitutions are well known in the art. Derivatization of a protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or moφholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer advantages such as longer lifespan or enhanced activity. "Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.

"Disorder" refers to conditions, diseases or syndromes in which RuvBLl or the mRNA encoding RuvBLl are differentially expressed; these include neoplastic disorders such as cancers of the colon, lung, ovary, and prostate.

An "expression profile" is a representation of gene expression in a sample. A nucleic acid expression profile is produced using sequencing, hybridization, or amplification technologies and mRNAs or cDNAs from a sample. A protein expression profile mirrors the nucleic acid expression profile and uses labeling moieties or antibodies to quantify the protein expression in a sample. The nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art.

"Fragment" refers to a chain of consecutive nucleotides from about 50 to about 5000 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Such ligands are useful as therapeutics to regulate replication, transcription or translation.

"Guilt-by-association" (GBA) is a method for identifying cDNAs or proteins that are associated with a specific disease, regulatory pathway, subcellular compartment, cell type, tissue type, or species by their highly significant co-expression with known markers or therapeutics.

A "hybridization complex" is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5'-A- G-T-C-3' base pairs with 3'-T-C-A-G-5'. Hybridization conditions, degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions. "Identity" as applied to nucleic acid or protein sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. Similarity is an analogous score, but it is calculated with conservative substitutions taken into account; for example, substitution of a valine for a isoleucine or leucine

"Labeling moiety" refers to any reporter molecule including radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cofactors, inhibitors, or magnetic particles than can be attached to or incoφorated into a polynucleotide, protein, or antibody. Visible labels and dyes include but are not limited to anthocyanins, β glucuronidase, biotin, BIODIPY, Coomassie blue, Cy3 and Cy5, 4,6-diamidino-2-phenylindole (DAPI), digoxigenin, fluorescein, FITC, gold, green fluorescent protein, lissamine, luciferase, phycoerythrin, rhodamine, spyro red, silver, streptavidin, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like. "Ligand" refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats, and lipids. A "multispecific molecule" can bind with at least two different binding specificities to at least two different molecules or two different sites on a molecule. Antibodies can perform as multispecific molecules in that they can bind to both a target protein and a pharmaceutical agent.

"Oligonucleotide" refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplicon, amplimer, primer, and oligomer.

An "oligopeptide" is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.

"Portion" refers to any part of a protein used for any puφose; but especially, to an epitope for the screening of ligands or for the production of antibodies. A "pharmaceutical agent" or "therapeutic agent" may be an antibody, an antisense or RNAi molecule, a multispecific molecule, a peptide, a protein, a radionuclide, a small drug molecule, a cytospecific or cytotoxic drug such as abrin, actinomyosin D, cisplatin, crotin, doxorubicin, 5-fluorouracil, methotrexate, ricin, vincristine, vinblastine, or any combination of these elements.

"Post-translational modification" of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.

"Probe" refers to a cDNA that hybridizes to at least one nucleic acid in a sample. Where targets are single-stranded, probes are complementary single strands. Probes can be labeled with reporter molecules for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.

"Protein" refers to a polypeptide or any portion thereof. A "portion" of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic determinant of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR, Madison WI).

"Purified" refers to any molecule or compound that is separated from its natural environment and is from about 60% free to about 90% free from other components with which it is naturally associated.

"Sample" is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like. A sample may comprise a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingeφrint, buccal cells, skin, or hair; and the like.

"Similarity" as applied to sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith- Waterman alignment (Smith and Waterman (1981) J Mol Biol 147: 195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. Particularly in proteins, similarity is greater than identity in that conservative substitutions, for example, valine for leucine or isoleucine, are counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art. "Specific binding" refers to a precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule or the binding between an epitope of a protein and an agonist, antagonist, or antibody.

"Substrate" refers to any rigid or semi-rigid support to which polynucleotides, proteins, or antibodies are bound and includes magnetic or nonmagnetic beads, capillaries or other tubing, chips, fibers, filters, gels, membranes, plates, polymers, slides, wafers, and microparticles with a variety of surface forms including channels, columns, pins, pores, trenches, and wells. A "transcript image" (TI) is a profile of gene transcription activity in a particular tissue at a particular time. TI provides assessment of the relative abundance of expressed polynucleotides in the cDNA libraries of an EST database as described in USPN 5,840,484, incoφorated herein by reference. "Variant" refers to molecules that are recognized variations of a protein or the polynucleotides that encode it. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. AUelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. "Single nucleotide polymoφhism" (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure. THE INVENTION

The invention is based on the discovery of a DNA helicase (RuvBLl), its encoding cDNA, and an antibody that specifically binds the protein and on their use in the characterization, diagnosis, prognosis, treatment and evaluation of treatment of colon and lung cancer.

RuvBLl was identified as a potential gene associated with colon cancer by its coexpression with known β-catenin target genes in the Wnt pathway. In particular RuvBLl was highly significantly coexpressed with VEGF, cyclin Dl and c-myc in colon tumor samples from a microarray analysis comparing colon tumors with normal colon tissue. The Table below shows the results of the GB A analysis in which two clones representing partial transcripts of RuvBLl were found to be highly significantly coexpressed (Correlation Coefficient > 0.50) with each of the known β-catenin target genes, VEGF, cyclin Dl and c-myc in colon tumor samples. The first column shows the Incyte clone number, and columns 2-4, the correlation coefficient of expression of the clone with each of the genes encoding VEGF, cyclin Dl and c-myc, respectively.

Clone ID Correlation Coefficient

RuvBLl was furthermore found to be overexpressed in several of the colon tumors profiled on microarrays compared to normal colon tissue. Table 1 shows that RuvBLl was expressed at least 1.5 fold higher in 7 of 16 colon tumor samples compared to normal colon tissue.

Table 2 shows the results of a similar microarray analysis using lung tumor tissue. RuvBLl was significantly overexpressed (>1.5 fold) in a majority of lung tumor samples relative to donor-matched normal lung tissue (22 of 35 samples). RuvBLl was also consistently over expressed over 2 fold in an ovarian tumor sample compared with donor-matched normal tissue (Table 3).

QPCR analysis of tumor samples confirmed the overexpression of RuvBLl in both colon and lung tumors. Figure 1 shows that RuvBLl was overexpressed in 8 of 9 colon tumor samples relative to either a pool of normal tissue or to donor-matched normal colon tissue. Likewise, Figure 2 shows that RuvBLl was overexpressed in all (9 of 9) lung tumor samples analyzed by QPCR. Differences in results obtained from microarry compared to QPCR analysis may be attributable to the greater sensitivity and larger dynamic range of the latter method.

Figure 3 shows the results of QPCR analysis of RuvBLl expression in various normal and tumorigenic human cell lines from colon, lung, prostate, breast and ovary tissues relative to a mixed pool of normal tissues. In colon cell lines, RuvBLl was expressed at high levels in the colon tumor cell lines

Caco-2, HCT116, SW480, and SW620. RuvBLl was expressed at higher levels in virtually all of the lung tumor cell lines examined relative to both the normal tissue pool control and the non-tumorigenic ell lines

NHBE and BEAS. RuvBLl was expressed at higher levels in the prostate tumor cell lines LNCaP and

22Rvl relative to both the normal tissue pool control and the non-tumorigenic cell lines PrEC and RWPE- 1. RuvBLl was also highly expressed in the metastatic breast adenocarcinoma cell line, MDA-MB-361. An analysis of a panel of normal tissues for RuvBLl expression by QPCR (Figure 4) shows that the gene is most highly expressed in testis and brain tissue. Of particular note is the low expression of

RuvBLl in normal colon (large intestine), lung, and prostate. 5 Figure 5 shows the effect of various mutations in RuvBLl on the expression of the gene product in HCTl 16 cells transfected with the gene. The various mutations were engineered to mirror mutations previously shown to affect ATP binding and/or hydrolysis in E. coli RuvB (Iwasaki et al. supra). The

Table below indicates the location of each mutation in RuvBLl, the comparable mutation in E. coli RuvB, and its known effect in that gene product (Iwasaki et al. supra). 10

15

The results clearly show that the mutations K76R and K76A, the replacement of lysine at position 76 in RuvBLl by either a conservative (arginine) or nonconservative (alanine) mutation, results in the 20 inhibition of gene expression over time. These mutations have been shown to result in defective ATP binding in E. coli RuvB and may therefore provide a means of inhibiting RuvBLl expression and tumor progression, perhaps by induction of apoptosis in tumor cells.

Figure 6 shows the results of purification an analysis of an antibody for RuvBLl derived from immunization of a rabbit with the polypeptide from amino acid residue Rl 18 to E152 of RuvBLl. In 25 Panel B of Figure 6, the highest expression of the protein is evident in HCTl 16 cells transfected with the RuvBLl plasmid compared with either mock transfected cells or untransfected HCTl 16 cells (which express and endogenous level of HCTl 16).

Mammalian variants of the cDNA encoding RuvBLl were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics). These highly homologous cDNAs have about 30 87% identity to all or part of the coding region of the human cDNA as shown in the, table below. The first column represents the SEQ ID NO: for homologous cDNAs (SEQ ΪD_V ; the second column, the Incyte ID for the homologous cDNAs (Incyte ID_Var); the third column, the species; the fourth column, the percent identity to the human cDNA; and the fifth column, the nucleotide alignment of the homologous cDNA to the human cDNA.

SEQ ID_Var Incyte ID_Var Species Identity Nt_H Alignment

3 005137_Mm.l Mouse 89% 69-1442

4 703879024 Jl Rat 89% 338-957

5 703094507T1 Rat 88% 853-1442

6 205377_Rn.l Rat 90% 70-1442

7 704087505J1 Dog 95% 36-793

8 703558404J1 Dog 92% 460-1138

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of cDNAs encoding RuvBLl, some bearing minimal similarity to the cDNAs of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of cDNA that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide encoding naturally occurring RuvBLl, and all such variations are to be considered as being specifically disclosed.

The mammalian cDNAs, SEQ ID NOs:3-8 may be used to produce transgenic cell lines or organisms which are model systems for human colon and lung cancer and upon which the toxicity and efficacy of therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the cDNAs, proteins, antibodies and molecules and compounds identified using the cDNAs and proteins of the present invention. Characterization and Use of the Invention cDNA libraries

In a particular embodiment disclosed herein, mRNA is isolated from mammalian cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries. The Incyte cDNAs were isolated from mammalian cDNA libraries prepared as described in the EXAMPLES. The consensus sequence is present in a single clone insert ,or chemically assembled, based on the electronic assembly from sequenced fragments including Incyte cDNAs and extension and/or shotgun sequences. Computer programs, such as PHRAP (P Green, University of Washington, Seattle WA) and the AUTO ASSEMBLER application (ABI), are used in sequence assembly and are described in EXAMPLE V. After verification of the 5' and 3' sequence, at least one representative cDNA which encodes RuvBLl is designated a reagent for research and development. Sequencing

Methods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Biosciences (APB), Piscataway NJ), or combinations of polymerases and proofreading exonucleases (Invitrogen, Carlsbad CA). Sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton, Reno NV) and the DNA ENGINE thermal cycler (MJ Research, Watertown MA) and sequencing, with the PRISM 3700, 377 or 373 DNA sequencing systems (ABI) or the MEGABACE 1000 DNA sequencing system (APB).

The nucleic acid sequences of the cDNAs presented in the Sequence Listing were prepared by such automated methods and may contain occasional sequencing errors and unidentified nucleotides, designated with an N, that reflect state-of-the-art technology at the time the cDNA was sequenced. Vector, linker, and polyA sequences were masked using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. Ns and SNPs can be verified either by resequencing the cDNA or using algorithms to compare multiple sequences that overlap the area in which the Ns or SNP occur. Both of these techniques are well known to and used by those skilled in the art. The sequences may be analyzed using a variety of algorithms described in Ausubel et al. (1997; Short Protocols in Molecular Biology. John Wiley & Sons, New York NY, unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853).

Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the cDNAs of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8:195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, can be removed, and deleted sequences can be restored to complete the assembled, finished sequences. Extension of a Nucleic Acid Sequence

The sequences of the invention may be extended using various PCR-based methods known in the art. For example, the XL-PCR kit (ABI), nested primers, and cDNA or genomic DNA libraries may be used to extend the nucleic acid sequence. For all PCR-based methods, primers may be designed using software, such as OLIGO primer analysis software (Molecular Biology Insights, Cascade CO) to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C to about 68C. When extending a sequence to recover regulatory elements, genomic, rather than cDNA libraries are used. Hybridization

The cDNA and fragments thereof can be used in hybridization technologies for various puφoses. A probe may be designed or derived from unique regions such as the 5' regulatory region or from a nonconserved region (i.e., 5' or 3' of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding the RuvBLl, allelic variants, or related molecules. The probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to any of the nucleic acid sequences, SEQ JD NOs:2-8. Hybridization probes may be produced using oligolabeling, nick-translation, end-labeling, or PCR amplification in the presence of a reporter molecule. A vector containing the cDNA or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using kits such as those provided by APB.

The stringency of hybridization is determined by G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. Hybridization can be performed at low stringency with buffers, such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60C, which permits the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45C (medium stringency) or 68C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, from about 35% to about 50% formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals can be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Plainview NY.

Arrays may be prepared and analyzed using methods well known in the art. Oligonucleotides or cDNAs may be used as hybridization probes or targets to monitor the expression level of large numbers of genes simultaneously or to identify genetic variants, mutations, and single nucleotide polymoφhisms. Arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., USPN 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; USPN 5,605,662.)

Hybridization probes are also useful in mapping the naturally occurring genomic sequence. The probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction. Such constructions include human artificial chromosomes , yeast artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions, or the cDNAs of libraries made from single chromosomes. QPCR QPCR is a method for quantifying a nucleic acid molecule based on detection of a fluorescent signal produced during PCR amplification (Gibson et al. (1996) Genome Res 6:995-1001; Heid et al. (1996) Genome Res 6:986-994). Amplification is carried out on machines such as the PRISM 7700 detection system (ABI) which consists of a 96-well thermal cycler connected to a laser and charge-coupled device (CCD) optics system. To perform QPCR, a PCR reaction is carried out in the presence of a doubly labeled probe. The probe, which is designed to anneal between the standard forward and reverse PCR primers, is labeled at the 5' end by a flourogenic reporter dye such as 6-carboxyfluorescein (6-FAM) and at the 3' end by a quencher molecule such as

6-carboxy-tetramethyl-rhodamine (TAMRA). As long as the probe is intact, the 3' quencher extinguishes fluorescence by the 5' reporter. However, during each primer extension cycle, the annealed probe is degraded as a result of the intrinsic 5' to 3' nuclease activity of Taq polymerase (HoUandet al. (1991) Proc Natl Acad Sci 88:7276-7280). This degradation separates the reporter from the quencher, and fluorescence is detected every few seconds by the CCD. The higher the starting copy number of the nucleic acid, the sooner an increase in fluorescence is observed. A cycle threshold (C_τ ) value, representing the cycle number at which the PCR product crosses a fixed threshold of detection is determined by the instrument software. The C_τ is inversely proportional to the copy number of the template and can therefore be used to calculate either the relative or absolute initial concentration of me nucleic acid molecule in the sample. The relative concentration of two different molecules can be calculated by determining their respective C_τ values (comparative C_τ method). Alternatively, the absolute concentration of the nucleic acid molecule can be calculated by constructing a standard curve using a housekeeping molecule of known concentration. The process of calculating C_τ values, preparing a standard curve, and determining starting copy number is performed using SEQUENCE DETECTOR 1.7 software (ABI).

Expression

Any one of a multitude of cDNAs encoding RuvBLl may be cloned into a vector and used to express the protein, or portions thereof, in host cells. The nucleic acid sequence can be engineered by such methods as DNA shuffling (USPN 5,830,721) and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life, and the like. The expression vector may contain transcriptional and translational control elements (promoters, enhancers, specific initiation signals, and polyadenylated 3' sequence) from various sources which have been selected for their efficiency in a particular host. The vector, cDNA, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17). A variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors or plant cell systems transformed with expression vectors containing viral and/or bacterial elements (Ausubel supra, unit 16). In mammalian cell systems, an adenovirus transcriptional/ translational complex may be utilized. After sequences are ligated into the El or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells. The Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.

Routine cloning, subcloning, and propagation of nucleic acid sequences can be achieved using the multifunctional pBLUESCRIPT vector (Stratagene, La Jolla CA) or pSPORTl plasmid (Invitrogen). Introduction of a nucleic acid sequence into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.

For long term production of recombinant proteins, the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media and then are transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired cDNA is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification. The host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a "prepro" form may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein. Recovery of Proteins from Cell Culture Heterologous moieties engineered into a vector for ease of purification include glutathione S- transferase (GST), 6xHis, FLAG, MYC, and the like. GST and 6-His are purified using affinity matrices such as immobilized glutathione and metal-chelate resins, respectively. FLAG and MYC are purified using monoclonal and polyclonal antibodies. For ease of separation foUowing purification, a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16). Protein Identification

Several techniques have been developed which permit rapid identification of proteins using HPLC and MS. Beginning with a sample containing proteins, the method is: 1) proteins are separated using two-dimensional gel electrophoresis (2-DE), 2) selected proteins are excised from the gel and digested with a protease to produce a set of peptides; and 3) the peptides are subjected to mass spectral analysis to derive peptide ion mass and spectral pattern information. The MS information is used to identify the protein by comparing it with information in a protein database (Shevenko et al. (1996) Proc Natl Acad Sci 93:14440-14445). Proteins are separated by 2DE employing isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in the second dimension. For IEF, an immobilized pH gradient strip is useful to increase reproducibility and resolution of the separation. Alternative techniques may be used to improve resolution of very basic, hydrophobic, or high molecular weight proteins. The separated proteins are detected using a stain or dye such as silver stain, Coomassie blue, or spyro red (Molecular Probes, Eugene OR) that is compatible with MS. Gels may be blotted onto a PVDF membrane for western analysis and optically scanned using a STORM scanner (APB) to produce a computer-readable output which is analyzed by pattern recognition software such as MELANIE (GeneBio, Geneva, Switzerland). The software annotates individual spots by assigning a unique identifier and calculating their respective x,y coordinates, molecular masses, isoelectric points, and signal intensity. Individual spots of interest, such as those representing differentially expressed proteins, are excised and proteolytically digested with a site-specific protease such as trypsin or chymotrypsin, singly or in combination, to generate a set of small peptides, preferably in the range of 1-2 kDa. Prior to digestion, samples may be treated with reducing and alkylating agents, and following digestion, the peptides are then separated by liquid chromatography or capillary electrophoresis and analyzed using MS. MS converts components of a sample into gaseous ions, separates the ions based on their mass-to-charge ratio, and determines relative abundance. For peptide mass fingeφrinting analysis, a MALDI-TOF (Matrix Assisted Laser Desoφtion/Ionization-Time of Flight), ESI (Electrospray Ionization), and TOF-TOF (Time of Flight/Time of Flight) machines are used to determine a set of highly accurate peptide masses. Using analytical programs, such as TURBOSEQUEST software (Finnigan, San Jose CA), the MS data is compared against a database of theoretical MS data derived from known or predicted proteins. A minimum match of three peptide masses is used for reliable protein identification. If additional information is needed for identification, Tandem-MS may be used to derive information about individual peptides. In tandem-MS, a first stage of MS is performed to determine individual peptide masses. Then selected peptide ions are subjected to fragmentation using a technique such as collision induced dissociation (CID) to produce an ion series. The resulting fragmentation ions are analyzed in a second round of MS, and their spectral pattern may be used to determine a short stretch of amino acid sequence (Dancik et al. (1999) J Comput Biol 6:327-342).

Assuming the protein is represented in the database, a combination of peptide mass and fragmentation data, together with the calculated MW and pi of the protein, will usually yield an unambiguous identification. If no match is found, protein sequence can be obtained using direct chemical sequencing procedures well known in the art (cf. Creighton (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY). Chemical Synthesis of Peptides Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds -amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylamine- derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N-α-protected by acid labile Boc (t-butyloxy carbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego CA pp. S1-S20). Automated synthesis may also be carried out on machines such as the 431A peptide synthesizer (ABI). A protein or portion thereof may be purified by preparative HPLC and its composition confirmed by amino acid analysis or by sequencing (Creighton (1984) Proteins, Structures and Molecular Properties , WH Freeman,

New York NY).

Antibodies

Antibodies, or immunoglobulins (Ig), are components of immune response expressed on the surface of or secreted into the circulation by B cells. The prototypical antibody is a tetramer composed of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds which binds and neutralizes foreign antigens. Based on their H-chain, antibodies are classified as IgA, IgD, IgE, IgG or IgM. The most common class, IgG, is tetrameric while other classes are variants or multimers of the basic structure.

Antibodies are described in terms of their two functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. The binding of antibody to antigen triggers destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface Fc receptors that specifically bind to the Fc region of the antibody and allow the phagocytic cells to destroy antibody-bound antigen. Fc receptors are single-pass transmembrane glycoproteins containing about 350 amino acids whose extracellular portion typically contains two or three Ig domains (Sears et al. (1990) J Immunol 144:371-378). Preparation and Screening of Antibodies Various hosts including mice, rats, rabbits, goats, llamas, camels, and human cell lines may be immunized by injection with an antigenic determinant. Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH; Sigma-Aldrich), and dinitrophenol may be used to increase immunological response. In humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum increase response. The antigenic determinant may be an oligopeptide, peptide, or protein. When the amount of antigenic determinant allows immunization to be repeated, specific polyclonal antibody with high affinity can be obtained (Klinman and Press (1975) Transplant Rev 24:41-83). Oligopepetides which may contain between about five and about fifteen amino acids identical to a portion of the endogenous protein may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule. Monoclonal antibodies may be prepared using any teclrnique which provides for the production of antibodies by continuous cell lines in culture. These include the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) Mol Cell Biol 62: 109-120). Chimeric antibodies may be produced by techniques such as splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; and Takeda et al. (1985) Nature 314:452-454). Alternatively, techniques described for antibody production may be adapted, using methods known in the art, to produce specific, single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci 88:10134-10137). Antibody fragments which contain specific binding sites for an antigenic determinant may also be produced. For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 246:1275-1281).

Antibodies may also be produced by inducing production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299). A protein may be used in screening assays of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies having a desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Antibody Specificity

Various methods such as Scatchard analysis combined with radioi munoassay techniques may be used to assess the affinity of particular antibodies for a protein. Affinity is expressed as an association constant, K_a, which is defined as the molar concentration of protein-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The K_a determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple antigenic determinants, represents the average affinity, or avidity, of the antibodies. The K_a determined for a preparation of monoclonal antibodies, which are specific for a particular antigenic determinant, represents a true measure of affinity. High-affinity antibody preparations with K_a ranging from about 10⁹ to 10¹² L/mole are commonly used in immunoassays in which the protein-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_a ranging from about 10⁶ to 10⁷ L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of the protein, preferably in active form, from the antibody (Catty (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Wasliington DC; Liddell and Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY). The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing about 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of protein-antibody complexes. Procedures for making antibodies, evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are discussed in Catty (supra) and Ausubel (supra) pp. 11.1-11.31. Cell Transformation Assays

Cell transformation, the conversion of a normal cell to a cancerous cell, is a highly complex and genetically diverse process. However, certain alterations in cell physiology that are associated with this process can be assayed using either in vitro cell-based systems or in vivo animal models. Known alterations include acquired self-sufficiency relative to growth signals, an insensitivity to growth- inhibitory signals, unlimited replicative potential, evasion of apoptosis, sustained angiogenesis, and cellular invasion and metastasis. See Hanahan and Weinberg (2000) Cell 100:57-70. Such assays can be used, for example, to assess the effect of transfecting a cell with a gene such as RuvBLl, on transformation of the cell to a neoplastic state. DIAGNOSTICS

Differential expression of RuvBLl, as detected using RuvBLl, cDNA encoding RuvBLl, or an antibody that specifically binds RuvBLl, and at least one of the assays below can be used to diagnose colon and lung cancer. Labeling of Molecules for Assay

A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using kits such as^" those supplied by Promega (Madison WI) or APB for incoφoration of a labeled nucleotide such as ³²P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Qiagen-Operon, Alameda CA), or amino acid such as ³⁵S-methionine (APB). Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chermlummescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes). Nucleic Acid Assays The cDNAs, fragments, oligonucleotides, complementary RNAs, and peptide nucleic acids (PNA) may be used to detect and quantify differential gene expression for diagnosis of a disorder. Similarly antibodies which specifically bind RuvBLl may be used to quantitate the protein. Disorders associated with such differential expression include colon and lung cancer. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect differential gene expression. Qualitative or quantitative methods for this comparison are well known in the art. Expression Profiles

An expression profile comprises the expression of a plurality of cDNAs or protein as measured using standard assays with a sample. The cDNAs, proteins or antibodies of the invention may be used as elements on a array to produce an expression profile. In one embodiment, the array is used to diagnose or monitor the progression of disease.

For example, the cDNA or probe may be labeled by standard methods and added to a biological sample from a patient under conditions for the formation of hybridization complexes. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. If complex formation in the patient sample is altered in comparison to either a normal or disease standard, then differential expression indicates the presence of a disorder.

In order to provide standards for establishing differential expression, normal and disease expression profiles are established. This is accomplished by combining a sample taken from normal subjects, either animal or human, with a cDNA under conditions for hybridization to occur. Standard hybridization complexes may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose or stage that disorder.

By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic. The invention can be used to formulate a prognosis and to design a treatment regimen. The invention can also be used to monitor the efficacy of treatment. For treatments with known side effects, the array is employed to improve the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment. In another embodiment, animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disease, or disorder; or treatment of the condition, disease, or disorder. Novel treatment regimens may be tested in these animal models using arrays to establish and then follow expression profiles over time. In addition, arrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug.

Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to years. Protein Assays

Methods for detecting and measuring complex formation as a measure of protein expression are known in the art. Examples of such techniques include affinity chromatography, antibody arrays, ELISAs, FACS, protein arrays, RIAs, and 2D-PAGE in conjunction with scintillation counting, HPLC, MS, or western analysis. These assays and their quantitation against purified, labeled standards are well known in the art (Ausubel, supra, unit 10.1-10.6). Such assays may involve the measurement of complex formation between the protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed (Pound (1998) hnmunochemical Protocols, Humana Press, Totowa NJ). These methods are also useful for diagnosing diseases that show differential protein expression.

Normal or standard values for protein expression are established by combining body fluids or cell extracts taken from a normal mammalian or human subject with specific antibodies to a protein under conditions for complex formation. Standard values for complex formation in normal and diseased tissues are established by various methods, often photometric means. Then complex formation as it is expressed in a subject sample is compared with the standard values. Deviation'from the normal standard and toward the diseased standard provides parameters for disease diagnosis or prognosis while deviation away from the diseased and toward the normal standard may be used to evaluate treatment efficacy.

Recently, antibody arrays have allowed the development of techniques for high-throughput screening of recombinant antibodies. Such methods use robots to pick and grid bacteria containing antibody genes, and a filter-based ELISA to screen and identify clones that express antibody fragments. Because liquid handling is eliminated and the clones are arrayed from master stocks, the same antibodies can be spotted multiple times and screened against multiple antigens simultaneously. Antibody arrays are highly useful in the identification of differentially expressed proteins. (See de Wildt et al. (2000) Nature Biotechnol 18:989-94.) THERAPEUTICS

Differential expression of RuvBLl is significantly associated with the cells and/or tissues of colon and lung cancers. Therefore, RuvBLl clearly plays a role in colon and lung cancer.

In one embodiment, when decreased expression or activity of the protein is desired, an antibody , antagonist, inhibitor, a pharmaceutical agent or a composition containing one or more of these molecules may be delivered to a subject in need of such treatment. Such delivery may be effected by methods well known in the art and may include delivery by an antibody that specifically binds the protein. For therapeutic use, monoclonal antibodies are used to block an active site, inhibit dimer formation, trigger apoptosis and the like. In another embodiment, when increased expression or activity of the protein is desired, the protein, an agonist, an enhancer, a pharmaceutical agent or a composition containing one or more of these molecules may be delivered to a subject in need of such treatment. Such delivery may be effected by methods well known in the art and may include delivery of a pharmaceutical agent by an antibody specifically targeted to the protein. Any of the cDNAs, complementary molecules, or fragments thereof, proteins or portions thereof, vectors delivering these nucleic acid molecules or expressing the proteins, therapeutic antibodies, and ligands binding the cDNA or protein may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular disorder at a lower dosage of each agent. Modification of Gene Expression Using Nucleic Acids

Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5', 3', or other regulatory regions of the gene encoding RuvBLl. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA. In one alternative, a library or plurality of cDNAs may be screened to identify those which specifically bind a regulatory, nontranslated sequence.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.

Complementary nucleic acids and ribozymes of the invention may be prepared via recombinant expression, in vitro or in vivo, or using solid phase phosphoramidite chemical synthesis. In addition, RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5 ' and/or 3 ' ends of the molecule or by the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules. Either the inclusion of

5 nontraditional bases such as inosine, queosine, and wybutosine, or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio- groups renders the molecule more resistant to endogenous endonucleases. RNA Interference

RNA interference (RNAi), also known as double-stranded RNA (dsRNA)-induced gene silencing,

10 is a method of interfering with the transcription of specific mRNAs through the production of small RNAs (siRNAs) and short haiφin RNAs (sliRNAs). These RNAs are naturally formed in a multicomponent nuclease complex (RISC) in the presence of an RNAse III family nuclease (Dicer), and they serve as a guide to identify and destroy complementary transcripts. Transient infection of cells with RNAs capable of interference can bypass the need for Dicer and result in silencing of a gene for the lifespan of the

15 introduced RNAs, usually from about 2 to about 7 days. See Paddison and Hannon (2002) Cancer Cell 2:17-23.

The RNAi pathway is believed to have evolved in early eukaryotes as a cell-based immunity against viral and genetic parasites. It is considered, however, to have great potential as a method of identifying gene function particularly in diseases such as cancer, as well as providing a highly specific

20 means for nucleic acid-based therapies for cancer and other disorders. cDNA Therapeutics

The cDNAs of the invention can be used in gene therapy. cDNAs can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow may be reintroduced into the subject. Expression of the protein encoded by

25 the cDNA may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein. Alternatively, cDNAs may be delivered in vivo using vectors such as retrovirus, adenovirus, adeno-associated virus, heφes simplex virus, and bacterial plasmids. Non-viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature

30 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(3-4): 184-192; Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics. Humana Press, Totowa NJ; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego CA). Monoclonal Antibody Therapeutics Antibodies, and in particular monoclonal antibodies, that specifically bind a particular protein, enzyme, or receptor and block its overexpression are now being used therapeutically. The first widely accepted therapeutic antibody was HERCEPTIN (Trastuzumab, Genentech, S. San Francisco CA). HERCEPTLN is a humanized antibody approved for the treatment of HER2 positive metastatic breast cancer. It is designed to bind and block the function of overexpressed HER2 protein. Other monoclonal antibodies are in various stages of clinical trials for indications such as prostate cancer, lymphoma, melanoma, pneumococcal infections, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and the like. Screening and Purification Assays The cDNA encoding RuvBLl may be used to screen a library or a plurality of molecules or compounds for specific binding affinity. The libraries may be antisense molecules, artificial chromosome constructions, branched nucleic acid molecules, DNA molecules, peptides, peptide nucleic acid, proteins such as transcription factors, enhancers, or repressors, RNA molecules, ribozymes, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene. The assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the cDNA.

In one embodiment, the cDNA of the invention may be incubated with a plurality of purified molecules or compounds and binding activity deteπnined by methods well known in the art, e.g., a gel- retardation assay (USPN 6,010,849) or a reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel- retardation assay.

In another embodiment, the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.

In a further embodiment, the protein or a portion thereof may be used to purify a ligand from a sample. A method for using a protein to purify a ligand would involve combining the protein with a sample under conditions to aUow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.

In a preferred embodiment, RuvBLl may be used to screen a plurality of molecules or compounds in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. For example, in one method, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured. Depending on the particular kind of molecules or compounds being screened, the assay may be used to identify agonists, antagonists, antibodies, DNA molecules, small drug molecules, immunoglobulins, inhibitors, mimetics, peptides, peptide nucleic acids, proteins, and RNA molecules or any other ligand, which specifically binds the protein.

In one aspect, this invention contemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in USPN 5,876,946, incoφorated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding. In another aspect, this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a mammalian model system to evaluate their toxicity or therapeutic potential.

Pharmaceutical Compositions

Pharmaceutical compositions may be formulated and administered, to a subject in need of such treatment, to attain a therapeutic effect. Such compositions contain the instant protein, agonists, antagonists, small drug molecules, immunoglobulins, inhibitors, mimetics, multispecific molecules, peptides, peptide nucleic acids, pharmaceutical agent, proteins, and RNA molecules. Compositions may be manufactured by conventional means such as mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing. The composition may be provided as a salt, formed with acids such as hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic, or as a lyophilized powder which may be combined with a sterile buffer such as saline, dextrose, or water. These compositions may include auxiliaries or excipients which facilitate processing of the active compounds. Auxiliaries and excipients may include coatings, fillers or binders including sugars such as lactose, sucrose, mannitol, glycerol, or sorbitol; starches from corn, wheat, rice, or potato; proteins such as albumin, gelatin and collagen; cellulose in the form of hydroxypropylmethyl-cellulose, methyl cellulose, or sodium carboxymethylceUulose; gums including arabic and tragacanth; lubricants such as magnesium stearate or talc; disintegrating or solubilizing agents such as the, agar, alginic acid, sodium alginate or cross-linked polyvinyl pyrrolidone; stabilizers such as carbopol gel, polyethylene glycol, or titanium dioxide; and dyestuffs or pigments added for identify the product or to characterize the quantity of active compound or dosage. These compositions may be administered by any number of routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

The route of administration and dosage will determine formulation; for example, oral administration may be accomplished using tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, or suspensions; parenteral administration may be formulated in aqueous, physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Suspensions for injection may be aqueous, containing viscous additives such as sodium carboxymethyl cellulose or dextran to increase the viscosity, or oily, containing hpophilic solvents such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Penetrants well known in the art are used for topical or nasal administration. Toxicity and Therapeutic Efficacy

A therapeutically effective dose refers to the amount of active ingredient which ameliorates symptoms or condition. For any compound, a therapeutically effective dose can be estimated from cell culture assays using normal and neoplastic cells or in animal models. Therapeutic efficacy, toxicity, concentration range, and route of administration may be determined by standard pharmaceutical procedures using experimental animals.

The therapeutic index is the dose ratio between therapeutic and toxic effects~LD50 (the dose lethal to 50% of the population)/ED50 (the dose therapeutically effective in 50% of the population)~and large therapeutic indices are preferred. Dosage is within a range of circulating concentrations, includes an ED50 with little or no toxicity, and varies depending upon the composition, method of delivery, sensitivity of the patient, and route of administration. Exact dosage will be determined by the practitioner in light of factors related to the subject in need of the treatment.

Dosage and administration are adjusted to provide active moiety that maintains therapeutic effect. Factors for adjustment include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition.

Normal dosage amounts may vary from 0.1 μg, up to a total dose of about 1 g, depending upon the route of administration. The dosage of a particular composition may be lower when administered to a patient in combination with other agents, drugs, or hormones. Guidance as to particular dosages and methods of delivery is provided in the pharmaceutical literature. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton PA). Model Systems

Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, gestation period, numbers of progeny, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under- or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene. Toxicology

Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess consequences on human health following exposure to the agent.

Genetic toxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations. Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny. Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy, or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.

Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range- finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.

Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals.

Chronic toxicity tests, with a duration of a year or more, are used to test whether long term administration may elicit toxicity, teratogenesis, or carcinogenesis. When studies are conducted on rats, a minimum of tliree test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment. Transgenic Animal Models

Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., USPN 5,175,383 and USPN 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies. Embryonic Stem Cells Embryonic (ES) stem cells isolated from rodent embryos retain the ability to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene. The vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.

ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes. Knockout Analysis

In gene knockout analysis, a region of a gene is enzymatically modified to include a non- mammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288- 1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene. Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene. In one example, the mammalian gene is a human gene. Knockin Analysis ES cells can be used to create knockin humanized animals (pigs) or transgenic animal models

(mice or rats) of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastulae and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases. Non-Human Primate Model

The field of animal testing deals with data and methodology from basic sciences such as physiology, genetics, chemistry, pharmacology and statistics. These data are paramount in evaluating the effects of therapeutic agents on non-human primates as they can be related to human health. Monkeys are used as human surrogates in vaccine and drug evaluations, and their responses are relevant to human exposures under similar conditions. Cynomolgus and Rliesus monkeys (Macaca fascicularis and Macaca mulatta, respectively) and Common Marmosets (Callithrix iacchus) are the most common non-human primates (NHPs) used in these investigations. Since great cost is associated with developing and maintaining a colony of NHPs, early research and toxicological studies are usually carried out in rodent models. In studies using behavioral measures such as drug addiction, NHPs are the first choice test animal. In addition, NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from "extensive metabolizers" to "poor metabolizers" of these agents.

In additional embodiments, the cDNAs which encode the protein may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of cDNAs that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

EXAMPLES I cDNA Library Construction Cells or tissues were homogenized and lysed in guanidinium isothiocyanate, in phenol or in a suitable mixture of denaturants such as TRIZOL reagent (Invitrogen) and guanidine isothiocyanate. The lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNAse. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (Qiagen, Chatsworth CA), or an OLIGOTEX mRNA purification kit (Qiagen). Alternatively, RNA was isolated directly from tissue lysates using RNA isolation kits such as the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).

In some cases, Stratagene was provided with RNA and constructed the cDNA libraries. cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel, supra, units 5.1-6.6). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with appropriate restriction enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (APB) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid such as pBLUESCRIPT plasmid or pBK- CMV plasmid (both Stratagene), pSPORTl plasmid or PCDNA2.1 plasmid (both Invitrogen), pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR (Stratagene) or DH5α, DH10B, or ElectroMAX DHlOB (Invitrogen). II Isolation, Preparation, and Sequencing of cDNAs Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system

(Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or REAL PREP 96 plasmid purification kit from Qiagen. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4C.

Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high- throughput format (Rao (1994) Anal Biochem 216: 1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye

(Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the CATALYST 800 (ABI) thermal cycler or the DNA ENGINE thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents obtained from APB or supplied in sequencing kits such as the PRISM BIGDYE Terminator cycle sequencing ready reaction kit (ABI). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (APB) or PRISM 373 or 377 sequencing systems (ABI) in conjunction with standard protocols and base calling software. Reading frames within the cDNA sequences were identified using standard methods (Ausubel, supra, unit 7.7). Ill Extension of cDNAs

The cDNAs were extended using the cDNA clone and oligonucleotide primers. One primer was synthesized to initiate 5' extension of the known fragment, and the other, to initiate 3' extension of the known fragment. The initial primers were designed using primer analysis software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68C to about 72C. Any stretch of nucleotides that would result in haiφin structures and primer-primer dimerizations was avoided. Selected cDNA libraries were used as templates to extend the sequence. If extension was performed than one time, additional or nested sets of primers were designed. Preferred libraries have been size-selected to include larger cDNAs and random primed to contain more sequences with 5' or upstream regions of genes. Genomic libraries can be used to obtain regulatory elements extending into the 5' promoter binding region. High fidelity amplification was obtained by PCR using methods such as that taught in USPN

5,932,451. PCR was performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg ²⁺, (NH₄)₂S0₄, and β-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): The parameters for the cycles are 1: 94C, three min; 2: 94C, 15 sec; 3: 60C, one min; 4: 68C, two min; 5: 2, 3, and 4 repeated 20 times; 6: 68C, five min; and 7: storage at 4C. In the alternative, the parameters for primer pair T7 and SK+ (Stratagene) were as follows: 1: 94C, three min; 2: 94C, 15 sec; 3: 57C, one min; 4: 68C, two min; 5: 2, 3, and 4 repeated 20 times; 6: 68C, five min; and 7: storage at 4C. The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% reagent in lx TE, v/v; Molecular Probes) and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Coming Life Sciences, Acton MA) and allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose minigel to deteπxiine which reactions were successful in extending the sequence.

The extended clones were desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC18 vector (APB). For shotgun sequences, the digested nucleotide sequences were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and the agar was digested with AGARACE enzyme (Promega). Extended clones were religated using T4 DNA ligase (New England Biolabs) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into E. coli competent cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37C in 384- well plates in LB/2x carbenicillin liquid media.

The cells were lysed, and DNA was amplified using primers, Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: 1: 94C, three min; 2: 94C, 15 sec; 3: 60C, one min; 4: 72C, two min; 5: 2, 3, and 4 repeated 29 times; 6: 72C, five min; and 7: storage at 4C. DNA was quantified using PICOGREEN quantitation reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the conditions described above. Samples were diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the PRISM BIGDYE terminator cycle sequencing kit (ABI). IV Homology Searching of cDNA Clones and Their Deduced Proteins

The cDNAs of the Sequence Listing or their deduced amino acid sequences were used to query databases such as GenBank, SwissProt, BLOCKS, and the like. These databases that contain previously identified and annotated sequences or domains were searched using BLAST or BLAST2 to produce alignments and to detennine which sequences were exact matches or homologs. The alignments were to sequences of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively, algorithms such as the one described in Smith and Smith (1992, Protein Engineering 5:35-51) could have been used to deal with primary sequence patterns and secondary structure gap penalties. All of the sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T). As detailed in Karlin and Altschul (1993; Proc Natl Acad Sci 90:5873-5877), BLAST matches between a query sequence and a database sequence were evaluated statistically and only reported when they satisfied the threshold of 10^"25 for nucleotides and 10^"14 for peptides. Homology was also evaluated by product score calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. In comparison with hybridization procedures used in the laboratory, the stringency for an exact match was set from a lower limit of about 40 (with 1-2% error due to uncalled bases) to a 100% match of about 70.

The BLAST software suite (NCBI, Bethesda MD), includes various sequence analysis programs including "blastn" that is used to align nucleotide sequences and BLAST2 that is used for direct pairwise comparison of either nucleotide or amino acid sequences. BLAST programs are commonly used with gap and other parameters set to default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; and Filter: on. Identity is measured over the entire length of a sequence. Brenner (supra) analyzed BLAST for its ability to identify structural homologs by sequence identity and found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40%, for alignments of at least 70 residues.

The cDNAs of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database (Incyte Genomics). Component sequences from cDNA, extension, full length, and shotgun sequencing projects were subjected to PHRED analysis and assigned a quality score. All sequences with an acceptable quality score were subjected to various pre-processing and editing pathways to remove low quality 3' ends, vector and linker sequences, polyA tails, Alu repeats, mitochondrial and ribosomal sequences, and bacterial contamination sequences. Edited sequences had to be at least 50 bp in length, and low-information sequences and repetitive elements such as dinucleotide repeats, Alu repeats, and the like, were replaced by "Ns" or masked.

Edited sequences were subjected to assembly procedures in which the sequences were assigned to gene bins. Each sequence could only belong to one bin, and sequences in each bin were assembled to produce a template. Newly sequenced components were added to existing bins using BLAST and CROSSMATCH. To be added to a bin, the component sequences had to have a BLAST quality score greater than or equal to 150 and an alignment of at least 82% local identity. The sequences in each bin were assembled using PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation of each template was deteπrrined based on the number and orientation of its component sequences.

Bins were compared to one another, and those having local similarity of at least 82% were combined and reassembled. Bins having templates with less than 95% local identity were split.

Templates were subjected to analysis by STIT CHER/EXON MAPPER algorithms that determine the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, and the like. Assembly procedures were repeated periodically, and templates were annotated using BLAST against GenBank databases such as GBpri. An exact match was defined as having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs and a homology match as having an E-value (or probability score) of <1 x 10^"8. The templates were also subjected to frameshift FASTx against GENPEPT, and homology match was defined as having an E-value of <1 x 10^"8. Template analysis and assembly was described in USSN 09/276,534, filed March 25, 1999.

Following assembly, templates were subjected to BLAST, motif, and other functional analyses and categorized in protein hierarchies using methods described in USSN 08/812,290 and USSN 08/811,758, both filed March 6, 1997; in USSN 08/947,845, filed October 9, 1997; and in USSN 09/034,807, filed March 4, 1998. Then templates were analyzed by translating each template in all three forward reading frames and searching each translation against the PFAM database of hidden Markov model-based protein families and domains using the EIIvπViER software package (Washington University School of Medicine, St. Louis MO). The cDNA was further analyzed using MACDNASIS PRO software (Hitachi Software Engineering), and LASERGENE software (DNASTAR) and queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.

V Northern Analysis, Transcript Imaging, and Guilt-By- Association

Northern analysis

Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. The technique is described in EXAMPLE VII below and in Ausubel, supra, units 4.1-4.9)

Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQ database (Incyte Genomics). This analysis is faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to deteπriine whether any particular match is categorized as exact or homologous. The basis of the search is the product score which was described above in EXAMPLE IV. The results of northern analysis are reported as a list of libraries in which the transcript encoding RuvBLl occurs. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library. Transcript Imaging

A transcript image is performed using the LIFESEQ GOLD database (Incyte Genomics). This process allows assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries and was described in USPN 5,840,484, incoφorated herein by reference. All sequences and cDNA libraries in the LIFESEQ database are categorized by system, organ/tissue and cell type. The categories include cardiovascular system, connective tissue, digestive system, embryonic structures, endocrine system, exocrine glands, female and male genitalia, germ cells, hemic/immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, sense organs, skin, stomatognathic system, unclassified/mixed, and the urinary tract. Criteria for transcript imaging are selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like.

For each category, the number of libraries in which the sequence was expressed are counted and shown over the total number of libraries in that category. For each library, the number of cDNAs are counted and shown over the total number of cDNAs in that library. In some transcript images, all enriched, normalized or subtracted libraries, which have high copy number sequences can be removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject' s tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be excluded where clinical relevance is emphasized. Conversely, fetal tissue can be emphasized wherever elucidation of inherited disorders or differentiation of particular adult or embryonic stem cells into tissues or organs (such as heart, kidney, nerves or pancreas) would be aided by removing clinical samples from the analysis. Transcript imaging can also be used to support data from other methodologies such as hybridization, guilt-by-association and array technologies. Guilt-Bv-Association

GB A identifies cDNAs that are expressed in a plurality of cDNA libraries relating to a specific disease process, subcellular compartment, cell type, tissue type, or species. The expression patterns of cDNAs with unknown function are compared with the expression patterns of genes having well documented function to determine whether a specified co-expression probability threshold is met. Through this comparison, a subset of the cDNAs having a highly significant co-expression probability with the known genes are identified.

The cDNAs originate from human cDNA libraries from any cell or cell line, tissue, or organ and may be selected from a variety of sequence types including, but not limited to, expressed sequence tags (ESTs), assembled polynucleotides, full length gene coding regions, promoters, introns, enhancers, 5' untranslated regions, and 3' untranslated regions. To have statistically significant analytical results, the cDNAs need to be expressed in at least five cDNA libraries. The number of cDNA libraries whose sequences are analyzed can range from as few as 500 to greater than 10,000.

The method for identifying cDNAs that exhibit a statistically significant co-expression pattern is as follows. First, the presence or absence of a gene in a cDNA library is defined: a gene is present in a library when at least one fragment of its sequence is detected in a sample taken from the library, and a gene is absent from a library when no corresponding fragment is detected in the sample.

Second, the significance of co-expression is evaluated using a probability method to measure a due-to-chance probability of the co-expression. The probability method can be the Fisher exact test, the chi-squared test, or the kappa test. These tests and examples of their applications are well known in the art and can be found in standard statistics texts (Agresti (1990) Categorical Data Analysis, John Wiley & Sons, New York NY; Rice (1988) Mathematical Statistics and Data Analysis, Duxbury Press, Pacific Grove CA). A Bonferroni correction (Rice, supra, p. 384) can also be applied in combination with one of the probability methods for correcting statistical results of one gene versus multiple other genes. In a preferred embodiment, the due-to-chance probability is measured by a Fisher exact test, and the threshold of the due-to-chance probability is set preferably to less than 0.001.

This method of estimating the probability for co-expression of two genes assumes that the libraries are independent and are identically sampled. However, in practical situations, the selected cDNA libraries are not entirely independent because: 1) more than one library may be obtained from a single subject or tissue, and 2) different numbers of cDNAs, typically ranging from 5,000 to 10,000, may be sequenced from each library. In addition, since a Fisher exact co-expression probability is calculated for each gene versus every other gene that occurs in at least five libraries, a Bonferroni correction for multiple statistical tests is used (See Walker et al. (1999; Genome Res 9:1198-203; expressly incoφorated herein by reference). VI Chromosome Mapping

Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any of the cDNAs presented in the Sequence Listing have been mapped. Any of the fragments of the cDNA encoding RuvBLl that have been mapped result in the assignment of all related regulatory and coding sequences to the same location. The genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cM (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the terminus of the chromosomal p-arm. VII Hybridization and Amplication Technologies and Analyses

The HUMAN GENOME GEM series 1-5 microarrays (Incyte Genomics) contain 45,320 array elements which represent 22,632 annotated clusters and 22,688 unannotated clusters. For the UNIGEM series microarrays (Incyte Genomics), Incyte clones were mapped to non-redundant Unigene clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med 75:694-698), and the 5' clone with the strongest BLAST alignment (at least 90% identity and 100 bp overlap) was chosen, verified, and used in the construction of the microarray. The UNIGEM V 2.0 microarray (Incyte Genomics) contains 8,502 array elements which represent 8,372 annotated genes and 130 unannotated clusters. Tissue Sample Preparation

Normal and cancerous tissue samples are described by donor identification number in the table below. The first column shows the donor ID; the second, donor age/sex; the third column, a description of 5 the disorder, the fourth column, classification of the tumor; and the fifth column, the source.

Donor Age/Sex Tissue and Descriptions- Stage Source

3579 55/M colon; well differentiated adenocarcinoma Dukes' C TMN T2N1 HCl

3580 38/M colon; poorly differentiated, metastatic adenoCA T3N1MX HCl

3581 U/M rectal; tumor NA HCl 10 3582 78/M colon; moderately differentiated adenocarcinoma TMN T4N2MX HCl

3583 58/M colon; tubulovillous adenoma (hypeφlastic polyp) NA HCl

3647 83U colon; invasive moderately differentiated TMN T3N1MX HCl adenocarcinoma (tubular adenoma)

3649 86/U colon; invasive well-differentiated adenoCA NA HCl 3479 68/M colon; adenocarcinoma NA HCl

15 4614 67/U colon; moderately differentiated adenocarcinoma Dukes' B , TMN T3N0 HCl 3579 55/M colon; well differentiated adenoCA Dukes' C , TMN T2N1 HCl 3839 59/M colon tumor NA HCl 8403 54/F colon; adenoCA Grade II Asterand 8309 40/M colon tumor, adenoCA Grade II Asterand

20 9573 60/F colon; moderately differentiated adenocarcinoma Dukes C; T2N2M0 Asterand 9574 34/F colon; well differentiated metastatic adenoCA Dukes C; T2N1M0 Asterand 9575 60/M colon; moderately differentiated metastatic adenoCA Dukes C; TXN1-2M0 Asterand 9576 65/M colon; well differentiated adenocarcinoma Dukes C; T3N2M0 Asterand 9577 46/F colon; well differentiated adenocarcinoma Dukes C; TXN1-2M0 Asterand

25 7164 79/M lung; pulmonary carcinoid LA RCI 7162 73/M lung; poorly differentiated,large cell endocrine IJJB RCI 7168 75/M lung; poorly differentiated adenoCA JJ3 RCI 7173 70/M lung; moderately differentiated squamous cell CA JJB RCI 7175 67/M lung; moderately differentiated adenoCA JJ3 RCI

30 7176 72/M lung; poorly differentiated, adenosquamous IB RCI 7178 68/F moderately differentiated squamous cell carcinoma ΠIA RCI 7188 54/M lung; poorly differentiated adenoCA ΠIA RCI 7189 78/M lung; poorly differentiated adenoCA IB RCI 7190 50/F lung; moderately differentiated squamous cell CA IB RCI

35 7191 43/M lung; poorly differentiated, squamous cell CA ΠB RCI 7963 71/M lung; poorly differentiated adenoCA ΠIA RCI 7196 71/M well differentiated squamous cell carcinoma IB RCI 7197 53/M lung poorly differentiated adenoCA IA RCI 7179 62/M lung poorly differentiated adenoCA JJ3 RCI

40 7194 60/F lung moderately differentiated squamous cell CA IIB RCI 7962 71/M lung: moderately differentiated adenoCA ΠIA RCI 7964 50/M lung moderately differentiated adenoCA ΠIA RCI 7965 54/F lung moderately differentiated adenoCA ΠIA RCI 7966 39/F lung moderately differentiated adenoCA ΠIA RCI

45 7967 57/F lung moderately differentiated adenoCA ΠIA RCI 7968 58/M lung moderately differentiated squamous cell CA ΠIA RCI 7969 62/M lung poorly differentiated adenoCA ΠIA RCI 7971 54/M poorly differentiated large cell CA IIIA RCI

7972 62/M moderately differentiated squamous cell CA IIIA RCI

7973 54/M poorly differentiated adenoCA IIIA RCI 9760 57/M , moderately differentiated squamous cell CA T3N2Mx RCI

5 9762 67/M poorly differentiated adenoCA T2NlMx RCI

9763 74/M moderately differentiated squamous cell CA T2N0Mx RCI

9765 54/F moderately differentiated squamous cell CA NA RCI

3969 60/U moderately differentiated adenoCA II RCI

3837 U/U tumor NA RCI

10 3838 U/U tumor NA RCI

5792 68/F poorly differentiated squamous cell CA T2N0Mx RCI

3840 78/F ovary; tumor TV HCl ^Abbreviations: CA=carcinoma, U=unknown, NA=not available

15 The table below describes the tissues used in Figure 4 for the analysis of RuvBLl expression in normal human tissues. The first column provides the name of the tissue, the second column, a description of the tissue, and the third column, the provider of the tissue.

Tissue Description Source* 20 Heart LV Normal heart, left ventricle, from 33yr-old male Caucasian; cause of death: NDRI ghoblastoma

Heart RA Normal heart, right atrium, from 33yr-old male Caucasian; cause of death: NDRI ghoblastoma

Striated Normal striated muscle from 31yr-old male Caucasian Clinomics Muscle Spleen Normal spleens, pooled from 5yr-old male Caucasian (self-inflicted gunshot NDRI wound to head) + 57yr-old female Caucasian (IC bleed) + 33yr-old male

Caucasian (ghoblastoma)

25 Lymph Node Normal lymph node from 65yr-old female Caucasian Clinomics

Tonsil Normal left tonsil from 3yr-old female Caucasian Clinomocs

Thymus Normal thymus pooled from 9 male/female Caucasians, ages 15-25 Clontech

PBMC Untreated peripheral blood mononuclear cells from 29yr-old female Caucasian Stanford University

Bone Marrow Normal bone marrow from 17yr-old female Caucasian; cause of death: MVA NDRI 30 Pancreas Normal pancreas from 33yr-old male Caucasian; cause of death: ghoblastoma NDRI

Adipose Adipose tissue from 41yr-old female Caucasian with type II diabetes; cause of NDRI death: self-inflicted gunshot wound to head

Pituitary Normal pituitary, pooled from 56yr-old female Caucasian (MVA, multiple Clinomics traumas) + 39yr-old female Caucasian (opiate intoxication, heroin overdose)

Thyroid Thyroid papillary carcinoma from 78yr-old female HCl Adrenal Normal adrenal gland from 8yr-old black male; cause of death: anoxia HAM 35 Salivary Normal salivary glands, pooled from 24 male/female Caucasians, ages 15-60 Clontech Gland years Uterus Endometrium from hysterectomy of 35yr-old female Caucasian Mayo Clinic (endometrium

) 40 Uterus Myometrium from hysterectomy of 57yr-old female HCl

(myometrium) Testis Normal testes, pooled from 45 male Caucasians, ages 14-64 Clontech Brain Normal cerebellum from 39yr-old female Caucasian; cause of death: opiate Clinomics (cerebellum) intoxication, heroin overdose Brain Normal Brain, Caudate, Putamen, Nucleus Accumbens from 23yr-old Clinomics (Various) Caucasian male (MVA, multiple traumas) Spinal Cord Normal spinal cord, pooled from 49 male/female Caucasians, ages 15-66 Clontech Liver Liver from 64yr-old male; cause of death: Incarcerated umbilical hernia with HCl small intestinal perforation and sepsis Lung Normal lung from 37yr-old Caucasian male; cause of death: self-inflicted gun IIAM shot wound to head

Breast Grossly uninvolved breast tissue from 63yr-old female with invasive ductal HCl carcinoma

Small intestine Normal small intestine, pooled from 5yr-old male Caucasian (self-inflicted NDRI gunshot wound to head) + 57yr-old female Caucasian (IC bleed)

Large intestine Normal colon (ascending + descending), pooled from 40yr-old Caucasian Clinomics

(pneumonia) + 50yr-old male Caucasian (sudden cardiac death)

Ovary Normal ovary from 47yr-old female Caucasian Ambion Kidney Normal kidney, pooled from 14 male/female Caucasians, ages 18-59 Clontech Prostate Non-tumorous prostate from 65yr-old male Caucasian Mayo Clinic Positive Pool of normal placenta (Clinomics) + pooled normal testes (Clontech) + Mixed Control normal cerebellum (Clinomics) + Raji B-cell Burkitt's lymphoma cell line

(Ambion)

* NDRI=National Disease Reserch Interchange (Philadelphia, PA); IIAM=International Institute for the

Advancement of Medicine (Jessup, PA); Clinomics (Pittsfield MA); Stanford University (Palo Alto, CA); Mayo Clinic (Rochester, NY)

The table below describes cancerous and non-cancerous human cell lines analyzed in Figure 3 for RuvBLl expression. The first column lists the name of the cell line, the second column, the tissue source, the third column, a description of the cell line, the fourth and fifth columns whether the cell line is tumorigenic and/or metastatic in mice, and the sixth column, the source of the cell line.

Immobilization of cDNAs on a Substrate

The cDNAs are applied to a substrate by one of the following methods. A mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library. The cDNAs are then arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37C for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH ), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2xSSC for 10 min each. The membrane is then UN irradiated in a STRATALIΝKER UN-crosslinker (Stratagene).

In the second method, cDΝAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UN irradiation as described above. Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in USPΝ 5,807,522. Polymer-coated slides are prepared by cleaning glass microscope slides (Corning Life Sciences) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (NWR Scientific Products, West Chester PA), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a HOC oven. The slides are washed extensively with distilled water between and after treatments. The nucleic acids are arranged on the slide and then immobilized by exposing the array to UN irradiation using a STRATALINKER UV-crosslinker (Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford MA) for 30 min at 60C; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before. Probe Preparation for Membrane Hybridization

Hybridization probes derived from the cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 μl TE buffer, denaturing by heating to 100C for five min, and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five μl of [³²P]dCTP is added to the tube, and the contents are incubated at 37C for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100C for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below. Probe Preparation for QPCR

Probes for the QPCR were prepared according to the ABI protocol. Probe Preparation for Polymer Coated Slide Hybridization

The following method was used for the preparation of probes for the microarray analysis presented in Fig. 3. Hybridization probes derived from mRNA isolated from samples are employed for screening cDNAs of the Sequence Listing in array-based hybridizations. Probe is prepared using the GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 μl TE buffer and adding 5 μl 5x buffer, 1 μl 0.1 M DTT, 3 μl Cy3 or Cy5 labeling mix, 1 μl RNAse inhibitor, 1 μl reverse transcriptase, and 5 μl lx yeast control mRNAs. Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1 : 100,000, 1 : 10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine mRNA differential expression patterns, a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w). The reaction mixture is mixed and incubated at 37C for two hr. The reaction mixture is then incubated for 20 min at 85C, and probes are purified using two successive CHROMA SPIN+TE 30 columns (Clontech, Palo Alto CA). Purified probe is ethanol precipitated by diluting probe to 90 μl in DEPC-treated water, adding 2 μl lmg/ml glycogen, 60 μl 5 M sodium acetate, and 300 μl 100% ethanol. The probe is centrifuged for 20 min at 20,800xg, and the pellet is resuspended in 12 μl resuspension buffer, heated to 65C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below. In situ Hybridization

In situ hybridization is used to determine the expression of DNA helicase in sectioned tissue. With the digoxygenin protocol, fresh cryosections, 10 microns thick, are removed from the freezer, immediately immersed in 4% paraformaldehyde for 10 min, rinsed in PBS, and acetylated in 0.1 M TEA, pH 8.0, containing 0.25% (v/v) acetic anhydride. After the tissue equilibrated in 5 x SSC, it is prehybridized in hybridization buffer (50% formamide, 5 x SSC, 1 x Denhardt's solution, 10% dextran sulfate, and 1 mg/ml herring sperm DNA).

Digoxygenin-labeled DNA helicase-specific RNA probes, sense and antisense nucleotides selected from the cDNA of SEQ ID NO:l are produced using PCR. Approximately 500 ng/ml of probe is used in overnight hybridizations at 65C in hybridization buffer. Following hybridization, the sections are rinsed for 30 min in 2 x SSC at room temperature, 1 hr in 2 x SSC at 65C, and 1 hr in 0.1 x SSC at 65C. The sections are equilibrated in PBS, blocked for 30 min in 10% DIG kit blocker (Roche Molecular Biochemicals, Indianapolis IN) in PBS, then incubated overnight at 4C in 1:500 anti-DIG-AP. The following day, the sections are rinsed in PBS, equilibrated in detection buffer (0.1 M Tris, 0.1 M NaCl, 50 mM MgCl₂, pH 9.5), and then incubated in detection buffer containing 0.175 mg/ml NBT and 0.35 mg/ml BCIP. The reaction is terminated in TE, pH 8. Tissue sections are counterstained with 1 μg/ml DAPI and mounted in NECTASfflELD (Vector Laboratory, Burlingame CA).

With the [³⁵S]-UTP protocol, fresh cryosections, 7-10 microns thick, are fixed for 10-20 min in 4% paraformaldehyde, rinsed in PBS, incubated in 0.2 M HCl for 20 min, and washed in DEPC H₂O. Sections are then incubated in 2 x SSC for 30 min at 60C, washed in DEPC H₂O, and permeablized for 10 min with 10 μg/ml proteinase K in 25 mM Tris-HCl (pH 7.5) and 5 mM EDTA (pH 8.0). Following incubation in 0.2% glycine in PBS for 30 seconds, the sections are washed in PBS, fixed for 20 min at 4C in 4% paraformaldehyde in PBS, washed in PBS, washed in 0.1 M TEA (pH 8.0), and then acetylated in 0.25% acetic anhydride in TEA for 10 min. After another PBS wash, sections are dehydrated in an EtOH series (30%, 60%, 80%, 95%, 100% x 2) and air dried. Sections are incubated overnight at 45-60C in a humidified chamber with 50-100 μl of hybridization buffer plus probe (10⁵ cmp/μl) per slide. Hybridization buffer contains 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl (ρH7.5), 5 mM EDTA (pH 8.0), 1 x Denhardt's, 10% dextran sulfate, lmg/ml yeast tRNA, and 10 mM DTT.

The following day, the slides are washed in 5 x SSC with 10 mM DTT at 50C for 45 min, 2 x SSC with 10 mM DTT at 65C for 20-30 min, and then in 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0) at 37C three times for 10 min each. Sections are incubated in the above solution with 20 μg ml RNAse A at 37C for 30 min and rinsed in 2 x SSC with 20 mM DTT at 65C for 20-30 min. Slides are then dehydrated in an EtOH series (30% in 300 mM NH₄OAc, 60%, 80%, 95%, 100% x 2), air dried, and dipped in Kodak NT-B2 emulsion. After six days, slides are developed, stained with hematoxylin and eosin, dehydrated and mounted with Permount (ProSciTech, Queensland Australia). Probes are labeled with [³⁵S]-UTP by in vitro transcription with either T7 (antisense) or T3 (sense) RNA polymerase using 320 or 370 basepair fragment (nucleotide x to y, or x' to y') using the cDNA of SEQ ID NO: 2 as the template. The PCR-generated templates are each produced using two primers, one that contains the sequence for the T3 RNA polymerase promoter, and the other the sequence for the T7 RNA polymerase promoter. Membrane-based Hybridization

Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and lx high phosphate buffer (0.5 M NaCl, 0.1 M Na^ PO^, 5 mM EDTA, pH 7) at 55C for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55C for 16 hr. Following hybridization, the membrane is washed for 15 min at 25C in ImM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25C in ImM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to the membrane overnight at -70C, developed, and examined visually. Polymer Coated Slide-based Hybridization

The following method was used in the microarray analysis presented in Tables 1-3. Probe is heated to 65C for five min, centrifuged five min at 9400 rpm in a 5415C microcentrifuge (Eppendorf Scientific, Wes bury NY), and then 18 μl is aliquoted onto the array surface and covered with a coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5xSSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hr at 60C. The arrays are washed for 10 min at 45C in lxSSC, 0.1% SDS, and three times for 10 min each at 45C in O.lxSSC, and dried.

Hybridization reactions are performed in absolute or differential hybridization formats. In the absolute hybridization format, probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample. In the differential hybridization format, differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).

Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Melville NY). The slide containing the array is placed on a computer-controled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers. In the differential hybridization format, the two fluorophores are sequentially excited by the laser. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tubes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to- digital (A/D) conversion board (Analog Devices, Norwood MA) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore. A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS program (Incyte Genomics). QPCR Analysis

For QPCR analysis, cDNA was synthesized from 1 ug total RNA in a 25 ul reaction with 100 units M-MLN reverse transcriptase (Ambion, Austin TX), 0.5 mM dΝTPs (Epicentre, Madison WI), and 40 ng/ml random hexamers (Fisher Scientific, Chicago IL). Reactions were incubated at 25C for 10 minutes, 42C for 50 minutes, and 70C for 15 minutes, diluted to 500 ul, and stored at -30C. Alternatively, normal tissues were purchased from Clontech (Palo Alto CA) and Clinomics. PCR primers and probes (5' 6-FAM-labeled, 3' MGB/ΝFQ) were designed using PRIMER EXPRESS 1.5 software (ABI) and synthesized by ABI. QPCR reactions were performed using an PRISM 7900 detection system (ABI) in 25 ul total volume with 5 ul cDΝA template, lx TAQMAN UNJNERSAL PCR master mix (ABI), 100 nM each PCR primer, 200 nM probe, and lx NIC-labeled beta-2-microglobulin endogenous control (ABI). Reactions were incubated at 50C for 2 minutes, 95C for 10 minutes, followed by 40 cycles of incubation at 95C for 15 seconds and 60C for 1 minute. Emissions were measured once every cycle, and results were analyzed using SEQUENCE DETECTOR 1.7 software (ABI) and fold differences, relative concentration of mRNA as compared to standards, were calculated using the comparative C_τ method (ABI User Bulletin #2). VIII Complementary Molecules

Antisense molecules complementary to the cDNA, from about 5 bp to about 5000 bp in length, are used to detect or inhibit gene expression. Detection is described in Example NIL To inhibit transcription by preventing promoter binding, the complementary molecule is designed to bind to the most unique 5' sequence and includes nucleotides of the 5' UTR upstream of the initiation codon of the open reading frame. Complementary molecules include genomic sequences (such as enhancers or introns) and are used in triple helix base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. To inhibit translation, a complementary molecule is designed to prevent ribosomal binding to the mRΝA encoding the protein.

Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy. Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system.

Stable transformation of dividing cells with a vector encoding the complementary molecule produces a transgenic cell line, tissue, or organism (USPΝ 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough complementary molecules

IX Protein Expression, Cell Transfections and Fractionations

Expression and purification of the protein are achieved using either a cell expression system or an insect cell expression system. The pUB6/N5-His vector system (Invitrogen) is used to express protein in CHO cells. The vector contains the selectable bsd gene, multiple cloning sites, the promoter/enhancer sequence from the human ubiquitin C gene, a C-terminal N5 epitope for antibody detection with anti-V5 antibodies, and a C-terrninal polyhistidine (6xHis) sequence for rapid purification on PROBOΝD resin (Invitrogen). Transformed cells are selected on media containing blasticidin. Spodoptera frugiperda (Sf9) insect cells are infected with recombinant Autographica californica nuclear polyhedrosis virus (baculovirus). The polyhedrin gene is replaced with the cDΝA by homologous recombination and the polyhedrin promoter drives cDΝA transcription. The protein is synthesized as a fusion protein with 6xhis which enables purification as described above. Purified protein is used in following assays and to make antibodies. Cell Transfections RuvBLl Mutations Mutations were generated in RuvBLl using the QUICKCHARGE Mutagenesis kit

(Stratagene). HCTl 16 cells (ATCC) were transiently transfected with an expression vector containing the cDNA encoding a RuvBLl (WT) or one of four mutations in RuvBLl described in the Table at p. 16. The expression vector, pTRIEX-3 NEO (Novagen, Madison WI), contained the entire coding sequence and a C-terminal HlS-tag. Transfections were performed using the LIPOFECT AMINE 2000 transfection reagent (Invitrogen) according to the manufacturer's specifications. Forty-eight hours, or as indicated after transfection, cells were prepared as total cellular lysates for western analysis using an anti-HIS antibody (Novagen). RNAi Transfections

Embryonal kidney 293 cells were transfected as described above for HCTl 16 cells using one of four RNAi constructs from RuvBLl prepared as described in Paddison and Hannon (2002) Cancer Cell 17-23. Each of four short-helical RNA (shRNA) constructs were derived using a 19 nucleotide sequence of SEQ ID NO:2, and its complement, from 1) nucleotide T710 to nucleotide A729; 2) from nucleotide T898 to G916; 3) from nucleotide T923 to nucleotide A941; and 4) from nucleotide T1362 to nucleotide T1380 of SEQ ID NO:2. X Production of RUVBLl Specific Antibodies

The amino acid sequence of RuvBLl was analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity. An oligopeptide from amino acid residue R118 to E132 of RuvBLl was synthesized and conjugated to KLH (Sigma- Aldrich). A Rabbit was immunized with the oligopeptide-KLH complex in complete Freund's adjuvant, and the resulting antisera was tested for specific recognition of RuvBLl. Antisera that reacted positively with DNA helicase was affinity purified on a column containing beaded agarose resin to which the synthetic oligopeptide had been conjugated using the SULFOLINK kit (Pierce Chemical, Rockford EL). The column was equilibrated using 12 mL IMMUNOPURE Gentle Binding buffer (Pierce Chemical). Three mL of rabbit antisera was combined with one mL of binding buffer and added to the top of the column. The column was capped on the top and bottom, and antisera allowed to bind with gentle shaking at room temperature for 30 min. The column was allowed to settle for 30 min, drained by gravity flow, and washed with 16 mL binding buffer (4 x 4 mL additions of buffer). The antibody was eluted in one ml fractions with IMMUNOPURE Gentle Elution buffer (Pierce), and absorbance at 280 nm was determined. Peak fractions were pooled and dialyzed against 50 mM Tris, pH 7.4, 100 mM NaCl, and 10% glycerol. After dialysis, the concentration of the purified antibody was determined using the BCA assay (Pierce), aliquotted, and frozen. XI Immunopurification Using Antibodies

Naturally occurring or recombinantly produced protein is purified by immunoaffinity chromatography using antibodies which specifically bind the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the purified protein is collected. XII Western Analysis Electrophoresis and Blotting

Samples containing protein were mixed in 4 x loading buffer, heated to 95 C for 3-5 min, and loaded on 4-12% NUPAGE Bis-Tris precast gel (Invitrogen). Unless indicated, equal amounts of total protein were loaded into each well. The gel was electrophoresced in 1 x MES or MOPS running buffer (Invitrogen) at 200 V for approximately 45 min on an Xcell II apparatus (Invitrogen) until the RAINBOW marker (APB) had resolved, and dye front approached the bottom of the gel. The gel and its supports were removed from the apparatus and soaked in 1 x transfer buffer (Invitrogen) with 10% methanol for a few minutes; and the PVDF membrane was soaked in 100% methanol for a few seconds to activate it. The membrane, the gel, and supports were placed on the TRANSBLOT SD transfer apparatus (Biorad, Hercules CA) and a constant current of 350 mAmps was applied for 90 min. Conjugation with Antibody and Visualization

After the proteins were transferred to the membrane, it was blocked in 5% (w/v) non-fat dry milk in 1 x phosphate buffered saline (PBS) with 0.1% Tween 20 detergent (blocking buffer) on a rotary shaker for at least 1 hr at room temperature or at 4C overnight. After blocking, the buffer was removed, and 10 ml of primary antibody in blocking buffer was added and incubated on the rotary shaker for 1 hr at room temperature or overnight at 4C. The membrane was washed 3 x for 10 min each with PBS-Tween (PBST), and secondary antibody, conjugated to horseradish peroxidase, was added at a 1:3000 dilution in 10 ml blocldng buffer. The membrane and solution were shaken for 30 min at room temperature and then washed three times for 10 min each with PBST. The wash solution was carefully removed, and the membrane was moistened with ECL+ chemiluminescent detection system (APB) and incubated for approximately 5 min. The membrane, protein side down, was placed on BIOMAX M film (Eastman Kodak) and developed for approximately 30 seconds.

XIII Antibody Arrays Protei protein interactions

In an alternative to yeast two hybrid system analysis of proteins, an antibody array can be used to study protein-protein interactions and phosphorylation. A variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until protein: antibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest. In the alternative, a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex. The identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane. Proteomic Profiles

Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and gridded at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt, supra)

XIV Screening Molecules for Specific Binding with the cDNA or Protein

The cDNA, or fragments thereof, or the protein, or portions thereof, are labeled with ³²P- dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or with BIODIPY or FITC (Molecular Probes), respectively. Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled cDNA or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule. XV Two-Hybrid Screen

A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories), is used to screen for peptides that bind the protein of the invention. A cDNA encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. cojL cDNA, prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli to construct a cDNA library. The pLexA plasmid and pB42AD-cDNA library constructs are isolated fro E. coli and used in a 2:1 ratio to co-transform competent yeast EGY48[p8op-lacZ] cells using a polyethylene glycol/lithium acetate protocol. Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30C until the colonies have grown up and are counted. The colonies are pooled in a minimal volume of lx TE (pH 7.5), replated on SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies. Interaction between expressed protein and cDNA fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu). Interaction also activates expression of β- galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal.

Positive interactions between expressed protein and cDNA fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trp/-Ura liquid medium for 1 to 2 days at 30C. A sample of the culture is plated on SD/-Tφ/-Ura media and incubated at 30C until colonies appear. The sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SD containing histidine but not on media lacking histidine have lost the pLexA plasmid. Histidine-requiring colonies are grown on SD/Gal/Raf/X-Gal/-Trp/-Ura, and white colonies are isolated and propagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding a protein that physically interacts with the protein, is isolated from the yeast cells and characterized. XVI RuvBLl Activity Assay

DNA helicase activity or ATPase activity in RuvBLl is measured as described in Kanemaki et aL supra, p. 22438. All patents and publications mentioned in the specification are incorporated by reference herein. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

TABLE 1

Normal Tumor

Cy5/Cy3 Dn ID Tumor Description Dn ID

1.6 Pool Colon Tumor, AdenoCA Dn3649

1.3 Pool ColonTumor, Adenoma Dn3583

1.0 Pool Colon Tumor, Cancer Dn3647 -1.1 Pool Colon Tumor, Cancer Dn3647

-1.0 Dn3579 Colon Tumor, AdenoCA Dn3579

1. .5 Dn3580 Colon Tumor, AdenoCA Dn3580

1. .4 Dn3580 Colon Tumor, AdenoCA Dn3580

1. .4 Dn3580 Colon Tumor, AdenoCA Dn3580

Ol 1. .2 Dn3580 Colon Tumor, AdenoCA Dn3580

00

1. .2 Dn3580 Colon Tumor, AdenoCA Dn3580

1.1 Dn3582 Colon Tumor, AdenoCa Dn3582

1.6 Dn383 Colon Tumor, AdenoCA Dn3839

1.2 Dn4614 Colon Tumor, AdenoCA Dn4614

-1.2 Dn8309 Colon Tumor, AdenoCA Dn8309

2.1 Dn9573 Colon Tumor, AdenoCA Dn9573

3.0 Dn9574 Colon Tumor, Rectum, AdenoCA Dn9574

-2.3 Dn9575 Colon Tumor, Rectum, AdenoCA Dn9575

-1.1 Dn9576 Colon Tumor, Rectum, AdenoCA Dn9576

3.0 Dn9577 Colon Tumor, Rectum, AdenoCA Dn9577

3.5 Dn8403 Colon, . Tumor , Ulcerative AdenoCA Dn8403

1.3 Dn3581 Colon Tumor, Rectum, Cancer Dn3581

TABLE 2

Normal Tumor

Cy5/Cy3 Dn ID Tumor Description Dn ID

1.0 Dn7168 Lung Tumor, Squamous Cell CA Dn7168

1.9 Dn7189 Lung Tumor, Left Upper Lobe, AdenoCA Dn7189 2.5 Dn7189 Lung Tumor, Left Upper Lobe, AdenoCA Dn7189

-1.5 Dn7178 Lung Tumor, Left Upper Lobe, Scjuamous Cell CA Dn7178 -1.6 Dn71878 Lung Tumor, Left Upper Lobe, Squamous Cell CA Dn71878

2.0 Dn7197 Lung Tumor, Left, AdenoCA Dn7197

1.1 Dn7190 Lung Tumor, Left, Squamous Cell CA Dn7190 1.5 Dn71 0 Lung Tumor, Left, Squamous Cell CA Dn7190

2.1 Dn7191 Lung Tumor, Left, Squamous Cell CA Dn71 1 2.2 Dn7191 Lung Tumor, Left, Squamous Cell CA Dn7191

1.9 Dn7196 Lung Tumor, Left, Squamous Cell CA Dn7196 2.0 Dn7196 Lung Tumor, Left, Squamous Cell CA Dn7196

-1.5 On3969 Lung Tumor, AdenoCA Dn3969

-1.2 On5795 Lung Tumor, AdenoCA Dn5795

2.0 OΏ.5199 Lung Tumor, AdenoCA Dn5799

1.4 Dn3837 Lung Tumor Dn3837

1.3 Dn3838 Lung Tumor Dn3838

-1.2 Dn7164 Lung Tumor, Carcinoid Dn7164

4.0 Dn7162 Lung Tumor, Large Cell Endocrine Cancer Dn7162

1.3 Dn7962 Lung Tumor, Non-Small Cell Lung AdenoCA Dn7962

2.2 Dn7964 Lung Tumor, Non-Small Cell Lung AdenoCA Dn7964 2.6 Dn7964 Lung,. Tumor, Non-Small Cell Lung AdenoCA Dn7 64

-1.7 Dn7965 Lung Tumor, Non-Small Cell Lung AdenoCA Dn7965 -1.5 Dn7965 Lung, Tumor, Non-Small Cell Lung AdenoCA Dn7965

-1.3 Dn7966 Lung Tumor, Non-Small Cell Lung AdenoCA Dn7966 -1.2 Dn7966 Lung, Tumor, Non-Small Cell Lung AdenoCA Dn7966

1.7 Dn7967 Lung Tumor, Non-Small Cell Lung AdenoCA Dn7967 2.0 Dn7967 Lung, Tumor, Non-Small Cell Lung AdenoCA Dn7967

TABLE 2 (CON)

Normal Tumor

Cy5/Cy3 Dn ID Tumor Description Dn ID

2.7 Dn7969 Lung, umor Non-Small Cell Lung AdenoCA Dn7 69 2.9 Dn7969 Lung Tumor Non-Small Cell Lung AdenoCA Dn7969

1.1 Dn7 63 Lung Tumor , Non-Small Cell Lung CA Dn7963 1.2 Dn7963 Lung, umor , Non-Small Cell Lung CA Dn7963

1.5 Dn7971 Lung Tumor , Non-Small Cell Lung CA Dn7971 2.0 Dn7971 Lung, umor , Non-Small Cell Lung CA Dn7971

-1.1 Dn7973 Lung Tumor Non-Small Cell Lung CA Dn7973 1.5 Dn7973 Lung, Tumor , Non-Small Cell Lung CA Dn7973

3.8 Dn5792 Lung Tumor , Squamous Cell CA Dn5792

1.5 Dn5793 Lung Tumor _r Squamous Cell CA Dn5793

2.4 Dn5797 Lung Tumor Squamous Cell CA Dn5797

1.5 Dn5800 Lung Tumor Squamous Cell CA Dn5800 o -1.2 Dn7968 Lung Tumor Squamous Non-Small Cell Lung CA Dn7968 -1.1 Dn7968 Lung, Tumor , Squamous Non-Small Cell Lung CA Dn7968

-1.4 Dn7972 Lung Tumor Squamous Non-Small Cell Lung CA Dn7972 -1.2 Dn7972 Lung, Tumor Squamous Non-Small Cell Lung CA Dn7972

1.8 Dn7176 Lung Tumor Right Middle Lower Lobe, Adenosquamous Dn7176 2.6 Dn7176 Lung Tumor Right Middle Lower Lobe, Adenosqua ous Dn7176

1.6 Dn7175 Lung Tumor Right Upper Lobe, AdenoCA Dn7175 1.9 Dn7175 Lung Tumor Right Upper Lobe, AdenoCA Dn7175

2.2 Dn7179 Lung Tumor Right Upper Lobe, AdenoCA Dn7179 2.3 Dn7179 Lung Tumor Right Upper Lobe, AdenoCA Dn7179

1.5 Dn7188 Lung Tumor Right Upper Lobe, AdenoCA Dn7188 2.4 Dn7188 Lung Tumor Right Upper Lobe, AdenoCAδ Dn7188

2.3 Dn7194 Lung Tumor Right Upper Lobe, Squamous Cell CA Dn71 4

3.6 Dn7194 Lung Tumor Right Upper Lobe, Squamous Cell CA Dn7194

1.9 Dn7173 Lung Tumor Right, Squamous Cell CA Dn7173

TABLE 3

Normal Tumor

Cy5/Cy3 Dn ID Tumor Description Dn ID

2.20 3840 Ovary Tumor AdenoCA 3840

2.20 3840 Ovary Tumor AdenoCA 3840

2.20 3840 Ovary Tumor AdenoCA 3840

Claims

What is claimed is:

1 A purified protein comprising a polypeptide selected from: a) a variant of SEQ ID NO: 1 having N for D at amino acid residue 302; b) a variant of SEQ ID NO: 1 having R for K at amino acid residue 76; c) a variant of SEQ ID NO: 1 having A for K at amino acid residue 76, and d) a variant of SEQ ID NO: 1 having A for T at amino acid residue 77.

2. A composition comprising the protein of claim 1 and a labeling moiety.

3. A composition comprising the protein of claim 1 and a pharmaceutical carrier.

4. A substrate upon which the protein of claim 1 is immobilized.

5. An array element comprising the protein of claim 1.

6. A method for using a protein to diagnose a cancer comprising: a) performing an assay to quantify the expression of the protein of SEQ ID NO: 1 in a sample; and b) comparing the expression of the protein to standards, thereby diagnosing a cancer.

7. The method of claim 6 wherein the sample is selected from colon and lung.

8. The method of claim 6 wherein the protein is differentially expressed when compared with the standard and is diagnostic of a colon or lung cancer.

9. A method for using an antibody to diagnosis a cancer, the method comprising: a) combining an antibody specific for SEQ ID NO:l with a sample under conditions which allow the formation of antibody :protein complexes; b) detecting complex formation, wherein complex formation indicates expression of the protein in the sample; and c) comparing complex formation with standards, wherein the comparison is diagnostic of a cancer.

10. The method of claim 9 wherein the sample is from colon or lung.

11. The method of claim 9 wherein the cancer is a colon or lung cancer.

12. A method for delivering a therapeutic agent to a cell comprising: a) attaching the therapeutic agent to a multispecific molecule specific for SEQ ID NO: 1 ; and b) administering the multispecific molecule to a subject in need of therapeutic intervention, wherein the multispecific molecule specifically binds the protein having the amino acid sequence of SEQ ID NO:l thereby delivering the therapeutic agent to the cell.

13. The method of claim 12, wherein the cell is an epithelial cell of the colon.

14. An agonist that specifically binds the protein of SEQ ID NO:l.

15. A composition comprising an agonist of claim 14 and a pharmaceutical carrier.

16. An antagonist that specifically binds the protein of claim 14.

5 17. A composition comprising the antagonist of claim 16 and a pharmaceutical carrier.

18. A pharmaceutical agent that specifically binds the protein of claim 14.

19. A composition comprising the pharmaceutical agent of claim 18 and a pharmaceutical carrier.

20. A small drug molecule that specifically binds the protein of claim 14.

21. A composition comprising the small drug molecule of claim 20 and a pharmaceutical carrier. 10

22. A method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention the composition of claim 17.

23. A method for treating a colon or lung cancer comprising administering to a subject in need of therapeutic intervention the composition of claim 19.

24. A method for treating a colon or lung cancer comprising administering to a subject in need of 15 therapeutic intervention the composition of claim 21.