Genes expressed in treated human C3A liver cell cultures

[0001] The present invention relates to a composition comprising a plurality of cDNAs which are differentially expressed in treated human C3A liver cell cultures and which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of liver disorders such as hyperlipidemia.

[0002] Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. 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.

[0003] The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with hyperlipidemia may be compared with the levels and sequences expressed in normal tissue.

[0004] Toxicity testing is a mandatory and time-consuming part of drug development programs in the pharmaceutical industry. A more rapid screen to determine the effects upon metabolism and to detect toxicity of lead drug candidates may be the use of gene expression microarrays. For example, microarrays of various kinds may be produced using full length genes or gene fragments. These arrays can then be used to test samples treated with the drug candidates to elucidate the gene expression pattern associated with drug treatment. This gene pattern can be compared with gene expression patterns associated with compounds which produce known metabolic and toxicological responses.

[0005] The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells. C3A cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with -fetoprotein iii) convertion of ammonia to urea and glutamine; iv) metabolize aromatic amino acids; and v) are able to proliferate in glucose-free and insulin-free medium. The C3A cell line is now well established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol 272:G408-416).

[0006] Clofibrate is an hypolidemic drug which lowers elevated levels of serum triglycerides. In rodents, chronic treatment produces hepatomegaly and an increase in hepatic peroxisomes (peroxisome proliferation). Peroxisome proliferators (PPs) are a class of drugs which activate the PP-activated receptor in rodent liver, leading to enzyme induction, stimulation of S-phase, and a suppression of apoptosis (Hasmall and Roberts (1999) Pharmacol. Ther. 82:63-70). PPs include the fibrate class of hypolidemic drugs, phenobarbitone, thiazolidinediones, certain non-steroidal anti-inflarnmatory drugs, and naturally-occuring fatty acid-derived molecules (Gelman et al. (1999) Cell. Mol. Life Sci. 55:932-943). Clofibrate has been shown to increase levels of cytochrome P450 4A. It is also involved in transcription of -oxidation genes as well as induction of PP-activated receptors (Kawashima et al. (1997) Arch. Biochem. Biophys. 347:148-154). Peroxisome proliferation that is induced by both clofibrate and the chemically-related compound fenofibrate is mediated by a common inhibitory effect on mitochondrial membrane depolarization (Zhou and Wallace (1999) Toxicol. Sci. 48:82-89).

[0007] Captopril is an antihypertensive known as an angiotensin converting enzyme (ACE) inhibitor. ACE is a target for treatment of myocardial infarction and hypotension. ACE inhibitors can be classified into three broad groups based on chemical structure: i) sulfhydryl-containing ACE inhibitors, structurally related to captopril (e.g., fentiapril, pivalo-pril, zofenopril, alacepril); ii) dicarboxyl-containing ACE inhibitors, structurally related to enalapril (e.g., lisinopril, benazepril, quinapril, moexipril, ramipril, spirapril, perindopril, indolapril, pentopril, indala-pril, cilazapril); and iii) phosphorus-containing ACE inhibitors, structurally related to fosinopril. Many ACE inhibitors are ester-containing prodrugs that are 100 to 1000 times less potent as ACE inhibitors than their active metabolites, but have a much better oral bioavailability than the active molecules. Approximately 16 different ACE inhibitors are used world-wide. In general, ACE inhibitors differ with respect to potency; whether ACE inhibition is due to the drug itself or to activation of a prodrug; and pharmacokinetic properties. With the notable exceptions of fosinopril and spirapril (which display balanced elimination by the liver and kidneys), ACE inhibitors are cleared predominantly by the kidneys. Drugs that interfere with the renin-angiotensin system play a prominent role in the treatment cardiovascular disease and have been used as a therapy for a number of diseases including hypotension, left ventricular systolic dysfunction, myocardial infarction, progressive renal impairment, and scleroderma renal crisis.

[0008] Enalapril is a prodrug that is not highly active and, as such, it must be hydrolyzed by esterases in the liver to produce the active parent dicarboxylic acid, enalaprilat. Enalaprilat is a highly potent inhibitor of ACE with a Ki of 0.2 nM but differs from captopril in that it is an analog of a tripeptide rather than a dipeptide. Enalapril is rapidly absorbed when given orally and has an oral bioavailability of about 60% (not reduced by food). Although peak concentrations of plasma enalapril occur within an hour, enalaprilat concentrations do not peak until three to four hours. Enalapril has a half-life of only 1.3 hours. However, because it binds tightly to ACE, enalaprilat has a plasma half-life of about 11 hours. Nearly all the drug is eliminated by the kidneys either as intact enalapril or enalaprilat.

[0009] Dexamethasone and its derivatives, dexamethasone sodium phosphate and dexamethasone acetate, are synthetic glucocorticoids used as anti-inflammatory or immunosuppressive agents. Dexamethasone has little to no mineralocorticoid activity and is usually selected for management of cerebral edema because of its superior ability to penetrate the central nervous sytem. Glucocorticoids are naturally occurring hormones that prevent or suppress inflammation and immune responses when administered at pharmacological doses. Responses can include inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, and suppression of humoral immune responses. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A₂ inhibitory proteins, collectively called lipocortins. The numerous adverse effects related to corticosteroid use usually depend on the dose administered and the duration of therapy. Proposed mechanisms of action include decreased IgE synthesis, increased number of -adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction, such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances. Mast cell influx and activation, therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of adrenocorticoids. As with other corticosteroids, the effects upon liver metabolism and hormone clearance mechanisms are important to understand the pharmacodynamics of a drug.

[0010] Diethylstilbestrol (DES) is used for the palliative treatment of advanced, inoperable, metastatic carcinoma of the breast in post-menopausal women and in men. Estrogens are not used in the treatment of breast cancer in premenopausal women, because the drugs may stimulate tumor growth rather than inhibit it. In males, DES is used for the palliative treatment of advanced carcinoma of the prostate; however, the risk of adverse cardiovascular effects of estrogens are also considered. The specific role of estrogen therapy compared with other therapies (e.g., orchiectomy, treatment with analogs of gonadotropin releasing hormone) in the treatment of prostatic cancer has not been clearly determined. Hormonal manipulation with estrogens currently is considered a therapy of choice for patients with inoperable prostatic tumors, patients who refuse orchiectomy, and patients whose disease progresses despite orchiectomy in whom the benefits of estrogen use are considered to outweigh the risk of adverse effects. As with other steroid hormones, the effects upon liver metabolism and hormone clearance mechanisms are important to understand the pharmacodynamics of a drug.

[0011] The polycyclic aromatic hydrocarbon 3-methylcholanthrene (MCA) is a potent carcinogen that is often used in experimental cancer studies. MCA is also a strong inducer of the cytochrome P450 genes in humans. In animal models, MCA induces the upregulation of the cytochrome P450 CYP1A isoforms in the liver of treated rats.

[0012] Insulin resistance is central to the pathophysiology of type II diabetes and a number of other disease states. It has been known for some time that down-regulation and reduced tyrosine kinase activity of the insulin receptor play a role in insulin resistance. However, defects in the intracellular responses to insulin are also very important, in particular, tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) and IRS-1/phosphatidyinositol-3 (PI3) kinase interaction. Despite many advances in the field, understanding of how insulin stimulates glucose transport is fragmentary, in part because the major targets for insulin signaling to glucose transport is a complex membrane trafficking pathway that is likely to contain many unknown components. Understanding the fundamental physiological response of insulin will help to unravel the causes of insulin resistance in type II diabetes. LY294002 is a PI3 kinase-specific inhibitor that promotes cell cycle arrest of C3A cells and promotes differentiation. This inhibitor also appears to affect the metabolic activity of the cells, especially with respect to proteins such as cytochrome P450 molecules.

[0013] The present invention provides for a composition comprising a plurality of cDNAs for use in detecting changes in expression of genes encoding proteins that are associated with treated human C3A liver cell cultures. Such a composition can be employed for the diagnosis, prognosis or treatment of hyperlipidemia and other disorders, such as hypertension, type II diabetes, and tumors of the liver, correlated with differential gene expression. The present invention satisfies a need in the art in that it provides a set of differentially expressed genes which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of a subject with a disorder such as hyperlipidemia.

[0014] The present invention provides a composition comprising a plurality of cDNAs and their complements which are differentially expressed in brain tissues and which are selected from SEQ ID NOs:1-401 as presented in the Sequence Listing. In one embodiment, each cDNA is downregulated at least two-fold, SEQ ID NOs:3, 32, 94, 99, 100, 108, 137, 196, 274, 299, 380; in another embodiment, each cDNA is upregulated at least two-fold, SEQ ID NOs:9, 10, 70, 144, 145, 147, 164, 186, 190, 191, 203, 271, 305, 344. In one aspect, the composition is useful to diagnose a liver disorder selected from hyperlipidemia, hypertension, type II diabetes, and tumors of the liver. In another aspect, the composition is immobilized on a substrate.

[0015] The invention also provides a high throughput method to detect differential expression of one or more of the cDNAs of the composition. The method comprises hybridizing the substrate comprising the composition with the nucleic acids of a sample, thereby forming one or more hybridization complexes, detecting the hybridization complexes, and comparing the hybridization complexes with those of a standard, wherein differences in the size and signal intensity of each hybridization complex indicates differential expression of nucleic acids in the sample. In one aspect, the sample is from a subject with hyperlipidemia and differential expression determines an early, mid, and late stage of that disorder.

[0016] The invention further provides a high throughput method of screening a library or a plurality of molecules or compounds to identify a ligand. The method comprises combining the substrate comprising the composition with a library or a plurality of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand. The library or a plurality of molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acid molecules, mimetics, peptides, transcription factors, repressors, and other regulatory proteins.

[0017] The invention still further provides an isolated cDNA selected from SEQ ID NOs:23, 56, 59, 97, 136, 155, 157, 226, 255, 264, 303, 308, 310, 330, 353, 354, 364, 395 as presented in the Sequence Listing. The invention also provides a vector comprising the cDNA, a host cell comprising the vector, and a method for producing a protein comprising culturing the host cell under conditions for the expression of a protein and recovering the protein from the host cell culture. The invention additionally provides a method for purifying a ligand, the method comprising combining a cDNA of the invention with a sample under conditions which allow specific binding, recovering the bound cDNA, and separating the cDNA from the ligand, thereby obtaining purified ligand.

[0018] The present invention provides a purified protein encoded and produced by a cDNA of the invention. The invention also provides a high-throughput method for using a protein to screen a library or a plurality of molecules or compounds to identify a ligand. The method comprises combining the protein or a portion thereof with the library or a plurality of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. A library or a plurality of molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acid molecules, mimetics, peptides, proteins, agonists, antagonists, antibodies or their fragments, immunoglobulins, inhibitors, drug compounds, and pharmaceutical agents. The invention further provides for using a protein to purify a ligand. The method comprises combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and separating the protein from the ligand, thereby obtaining purified ligand. The invention still further provides a pharmaceutical composition comprising the protein. The invention yet still further provides a method for using the protein to produce an antibody. The method comprises immunizing an animal with the protein or an antigenically-effective epitope under conditions to elicit an antibody response, isolating animal antibodies, and screening the isolated antibodies with the protein to identify an antibody which specifically binds the protein. The invention yet still further provides a method for using the protein to purify antibodies which bind specifically to the protein.

[0019] The invention also provides a purified protein selected from SEQ ID NOs: 158, 311, 331.

[0020] A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0021] The Sequence Listing is a compilation of cDNAs obtained by sequencing and extension of clone inserts. Each sequence is identified by a sequence identification number (SEQ ID NO) and by the template number (TEMPLATE ID) from which it was obtained.

[0022] Table 1 lists the differential expression levels of cDNA as a function of the increase or decreased levels compared with levels in untreated human C3A liver cell cultures. Column 1 lists the clone present on the array (CLONE ID). Columns 2 through 10 list the compound used to treat the cell cultures, Clofibrate, Fenofibrate, Captopril, Enalapril, Dexamethasone, diethylstilbestrol (DES) 3-methylcholanthrene (MCA), LY294002, or insulin together with LY294002 (INS/LY294002), respectively.

[0023] Table 2 shows the nucleotide template sequence corresponding to the encoded protein template and to the clone present on the microarray. Columns 1, 2, 3, 4, and 5 show the clone number (CLONE ID), nucleotide SEQ ID NO, nucleotide TEMPLATE ID, protein SEQ ID NO, and protein TEMPLATE ID, respectively. Template IDs with the suffix c read on the complementary nucleotide strand.

[0024] Table 3 lists the functional annotation of the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the GenBank hit (GI Number), probability score (E-value), and functional annotation, respectively, as determined by BLAST analysis (version 1.4 using default parameters; Altschul (1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403410) of the cDNA against GenBank (release 116; National Center for Biotechnology Information (NCBI), Bethesda Md).

[0025] Table 4 shows Pfam annotations of the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the first residue (START), last residue (STOP), and reading frame (FRAME), respectively, for the segment of the cDNA identified by Pfam analysis. Columns 6, 7, and 8 show the Pfam ID, Pfam description, and E-values, respectively, corresponding to the polypeptide domain encoded by the cDNA segment.

[0026] Table 5 shows signal peptide and transmembrane regions predicted within the cDNAs of the present invention. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Columns 3, 4, and 5 show the first residue (START), last residue (STOP), and reading frame (FRAME), respectively, for a segment of the cDNA, and column 6 (HIT TYPE) identifies the polypeptide encoded by the segment as either a signal peptide (SP) or transmembrane (TM) domain.

[0027] Table 6 shows the region of each cDNA encompassed by the clone present on a microarray and identified as differentially expressed. Columns 1 and 2 show the SEQ ID NO and TEMPLATE ID, respectively. Column 3 shows the CLONE ID and columns 4 and 5 show the first residue (START) and last residue (STOP) encompassed by the clone on the template.

[0028] Table 7 lists the tissue distribution of the nucleotide templates. Columns 1 and 2 list the SEQ ID NO and TEMPLATE ID, respectively. Column 3 lists the predominant tissue distribution (TISSUE DISTRIBUTION) as a percentage of total tissues in the Incyte LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

[0029] Definitions

[0030] Array refers to an ordered arrangement of at least two cDNAs on a substrate. At least one of the cDNAs represents a control or standard sequence, and the other, a cDNA of diagnostic interest. The arrangement of from about two to about 40,000 cDNAs on the substrate assures that the size and signal intensity of each labeled hybridization complex formed between a cDNA and a sample nucleic acid is individually distinguishable.

[0031] The complement of a nucleic acid molecule of the Sequence Listing refers to a cDNA which is completely complementary over the full length of the sequence and which will hybridize to the nucleic acid molecule under conditions of high stringency.

[0032] A composition comprises at least two and up to 401 sequences of the Sequence Listing. cDNA refers to a chain of nucleotides, an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, be double-stranded or single-stranded, coding and/or noncoding, an exon with or without an intron from a genomic DNA molecule, and purified or combined with carbohydrate, lipids, protein or inorganic elements or substances. Preferably, the cDNA is from about 4000 to about 5000 nucleotides.

[0033] The phrase cDNA encoding a protein refers to a nucleic acid 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; Altschul (1993) J Mol Evol 36: 290-300; Altschul et al. (1990) J Mol Biol 215:403-410) which provides identity within the conserved region. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078) who analyzed BLAST for its ability to identify structural homologs by sequence identity found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40% is a reasonable threshold for alignments of at least 70 residues (Brenner et al., page 6076, column 2).

[0034] 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 morpholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer advantages such as longer lifespan or enhanced activity.

[0035] Differential expression refers to an increased, upregulated or present, or decreased, downregulated or absent, gene expression as detected by the absence, presence, or at least two-fold changes in the amount of transcribed messenger RNA or translated protein in a sample.

[0036] Disorder refers to conditions, diseases or syndromes of the liver, including hyperlipidemia, hypertension, type II diabetes, tumors of the liver, and disorders of the inflammatory and immune response.

[0037] Fragment refers to a chain of consecutive nucleotides from about 200 to about 700 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. Nucleic acids and their ligands identified in this manner are useful as therapeutics to regulate replication, transcription or translation.

[0038] 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. The degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.

[0039] Ligand refers to any agent, molecule, or compound which will bind specifically to a complementary site on a cDNA molecule or polynucleotide, or to an epitope or a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic or organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids.

[0040] 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. Substantially equivalent terms are amplimer, primer, and oligomer.

[0041] Portion refers to any part of a protein used for any purpose; but especially, to an epitope for the screening of ligands or for the production of antibodies.

[0042] Post-translational modification of a protein can involve lipi dation, 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.

[0043] Probe refers to a cDNA that hybridizes to at least one nucleic acid molecule 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.

[0044] Protein refers to a polypeptide or any portion thereof. A portion of a protein retains at least one biological or antigenic characteristic of a native protein. 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.

[0045] 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.

[0046] 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 fingerprint, buccal cells, skin, or hair; and the like.

[0047] Specific binding refers to a special and 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, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.

[0048] 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.

[0049] Substrate refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.

[0050] Variant refers to molecules that are recognized variations of a cDNA or a protein encoded by the cDNA. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. Single nucleotide polymorphism (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.

[0051] The Invention

[0052] The present invention provides for a composition comprising a plurality of cDNAs or their complements, SEQ ID NOs: 1-401 which may be used on a substrate to diagnose, to stage, to treat or to monitor the progression or treatment of a liver disorder. These cDNAs represent known and novel genes differentially expressed in C3A liver cells treated with clofibrate, fenofibrate, captopril, enalapril, dexamethasone, diethylstilbestrol (DES) 3-methylcholanthrene (MCA), LY294002, or insulin together with LY294002. The composition may be used in its entirety or in part, as subsets of cDNAs downregulated by fibrates, SEQ ID NOs:3, 32, 94, 137, 196, 274, and 380; of cDNAs upregulated by fibrates SEQ ID NOs:9, 10, 70, 147, 164, 186, 190, 191, 203, 271, and 344; of cDNAs downregulated by captopril, enalapril, and dexamethasone SEQ ID NOs:3, 99, 100, 108, and 299; and of cDNAs upregulated by captopril, enalapril, and dexamethasone SEQ ID NOs:9, 10, 70, 144, 145, 190, 203, 271, and 305. SEQ ID NOs:23, 56, 59, 97, 136, 155, 157, 226, 255, 264, 303, 308, 310, 330, 353, 354, 364, and 395 represent novel cDNAs associated with treatment of human C3A liver cells. Since the novel cDNAs were identified solely by their differential expression, it is not essential to know a priori the name, structure, or function of the gene or the protein encoded thereby. The usefulness of the novel cDNAs exists in their immediate value as diagnostics for disorders of liver metabolism and tumors of the liver.

[0053] The invention also provides isolated proteins SEQ ID NOs: 158, 311, 331 which are encoded by the cDNAs of SEQ ID NOs:157, 310, 330 as shown in Table 2.

[0054] Table 1 shows those genes on the array having differential expression (two-fold or greater increase or decrease) in treated human C3A liver cell cultures. Column 1 shows the clone ID and columns 2 through 10 show the measured expression levels of the cDNA in C3A cells treated with clofibrate, fenofibrate, captopril, enalapril, dexamethasone, diethylstilbestrol (DES) 3-methyl-cholanthrene (MCA), LY294002, and insulin together with LY294002, respectively. Table 2 shows the nucleotide template sequences and the respective encoded proteins sequences which correspond to the upregulated or downregulated clones present on the array. Table 3 shows the functional annotation of the template cDNAs as determined by BLAST analysis. Table 4 shows the functional annotation as determined by Pfam analysis. Table 5 shows the functional annotation as determined by Hidden Markov Model analysis for signal peptide or for transmembrane regions, column 6:SP or TM, respectively. Table 6 shows the positional information of the clone present on the array relative to the nucleotide template sequence. Table 7 shows the tissue distribution of the nucleotide template sequences.

[0055] The cDNAs of the invention define a differential expression pattern against which to compare the expression pattern of biopsied and/or in vitro treated human liver tissues. Experimentally, differential expression of the cDNAs can be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational discriminant analysis, clustering, transcript imaging and array technologies. These methods may be used alone or in combination.

[0056] The composition may be arranged on a substrate and hybridized with tissues from subjects with diagnosed liver disorders to identify those sequences which are differentially expressed in both hyperlipidemia and other liver disorders. This allows identification of those sequences of highest diagnostic and potential therapeutic value. In one embodiment, an additional set of cDNAs, such as cDNAs encoding signaling molecules, are arranged on the substrate with the composition. Such combinations may be useful in the elucidation of pathways which are affected in a particular liver disorder or to identify new, coexpressed, candidate, therapeutic molecules.

[0057] In another embodiment, the composition can be used for large scale genetic or gene expression analysis of a large number of novel, nucleic acid molecules. These samples are prepared by methods well known in the art and are from mammalian cells or tissues which are in a certain stage of development; have been treated with a known molecule or compound, such as a cytokine, growth factor, a drug, and the like; or have been extracted or biopsied from a mammal with a known or unknown condition, disorder, or disease before or after treatment. The sample nucleic acid molecules are hybridized to the composition for the purpose of defining a novel gene profile associated with that developmental stage, treatment, or disorder.

[0058] cDNAs and Their Uses

[0059] cDNAs can be prepared by a variety of synthetic or enzymatic methods well known in the art. cDNAs can be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp. Ser. (7)215-233). Alternatively, cDNAs can be produced enzymatically or recombinantly, by in vitro or in vivo transcription.

[0060] Nucleotide analogs can be incorporated into cDNAs by methods well known in the art. The only requirement is that the incorporated analog must base pair with native purines or pyrimidines. For example, 2,6-diaminopurine can substitute for adenine and form stronger bonds with thymidine than those between adenine and thymidine. A weaker pair is formed when hypoxanthine is substituted for guanine and base pairs with cytosine. Additionally, cDNAs can include nucleotides that have been derivatized chemically or enzymatically.

[0061] cDNAs can be synthesized on a substrate. Synthesis on the surface of a substrate may be accomplished using a chemical coupling procedure and a piezoelectric printing apparatus as described by Baldeschweiler et al. (PCT publication WO95/251116). Alternatively, the cDNAs can be synthesized on a substrate surface using a self-addressable electronic device that controls when reagents are added as described by Heller et al. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized directly on a substrate by sequentially dispensing reagents for their synthesis on the substrate surface or by dispensing preformed DNA fragments to the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions efficiently.

[0062] cDNAs can be immobilized on a substrate by covalent means such as by chemical bonding procedures or UV irradiation. In one method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another method, a cDNA is placed on a polylysine coated surface and UV cross-linked to it as described by Shalon et al. (WO95/35505). In yet another method, a cDNA is actively transported from a solution to a given position on a substrate by electrical means (Heller, supra). cDNAs do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure of the attached cDNA. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with a terminal group of the linker to bind the linker to the substrate. The other terminus of the linker is then bound to the cDNA. Alternatively, polynucleotides, plasmids or cells can be arranged on a filter. In the latter case, cells are lysed, proteins and cellular components degraded, and the DNA is coupled to the filter by UV cross-linking.

[0063] The cDNAs may be used for a variety of purposes. For example, the composition of the invention may be used on an array. The array, in turn, can be used in high-throughput methods for detecting a related polynucleotide in a sample, screening a plurality of molecules or compounds to identify a ligand, diagnosing a liver disorder, or inhibiting or inactivating a therapeutically relevant gene related to the cDNA.

[0064] When the cDNAs of the invention are employed on a microarray, the cDNAs are arranged in an ordered fashion so that each cDNA is present at a specified location. Because the cDNAs are at specified locations on the substrate, the hybridization patterns and intensities, which together create a unique expression profile, can be interpreted in terms of expression levels of particular genes and can be correlated with a particular metabolic process, condition, disorder, disease, stage of disease, or treatment.

[0065] Hybridization

[0066] The cDNAs or fragments or complements thereof may be used in various hybridization technologies. The cDNAs may be labeled using a variety of reporter molecules by either PCR , recombinant, or enzymatic techniques. For example, a commercially available vector containing the cDNA is transcribed in the presence of an appropriate polymerase, such as T7 or SP6 polymerase, and at least one labeled nucleotide. Commercial kits are available for labeling and cleanup of such cDNAs. Radioactive (Amersham Pharmacia Biotech (APB), Piscataway N.J.), fluorescent (Operon Technologies, Alameda Calif.), and chemiluminescent labeling (Promega, Madison Wis.) are well known in the art.

[0067] A cDNA may represent the complete coding region of an MRNA or be designed or derived from unique regions of the mRNA or genomic molecule, an intron, a 3 untranslated region, or from a conserved motif. The cDNA is at least 18 contiguous nucleotides in length and is usually single stranded. Such a cDNA may be used under hybridization conditions that allow binding only to an identical sequence, a naturally occurring molecule encoding the same protein, or an allelic variant. Discovery of related human and mammalian sequences may also be accomplished using a pool of degenerate cDNAs and appropriate hybridization conditions. Generally, a cDNA for use in Southern or northern hybridizations may be from about 400 to about 6000 nucleotides long. Such cDNAs have high binding specificity in solution-based or substrate-based hybridizations. An oligonucleotide, a fragment of the cDNA, may be used to detect a polynucleotide in a sample using PCR.

[0068] The stringency of hybridization is determined by GC content of the cDNA, salt concentration, and temperature. In particular, stringency is increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane based hybridizations, addition of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization may be performed with buffers, such as 5saline sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60 C., that permit the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed with buffers such as 0.2SSC with 0.1% SDS at either 45 C. (medium stringency) or 65-68 C. (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acid molecules are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide may be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals may be reduced by the use of detergents such as Sarkosyl or Triton X-100 (Sigma Aldrich, St. Louis Mo.) 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 et al. (1997, Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and 4-64.9).

[0069] Dot-blot, slot-blot, low density and high density arrays are prepared and analyzed using methods known in the art. cDNAs from about 18 consecutive nucleotides to about 5000 consecutive nucleotides in length are contemplated by the invention and used in array technologies. The preferred number of cDNAs on an array is at least about 100,000, a more preferred number is at least about 40,000, an even more preferred number is at least about 10,000, and a most preferred number is at least about 600 to about 800. The array may be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and SNPs. Such information may be used to determine gene function; to understand the genetic basis of a disorder; to diagnose a disorder; and to develop and monitor the activities of therapeutic agents being used to control or cure a disorder. (See, e.g., U.S. Pat. No. 5,474,796; WO95/11995; WO95/35505; U.S. Pat. No. 5,605,662; and U.S. Pat. No. 5,958,342.)

[0070] Screening and Purification Assays

[0071] A cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand which specifically binds the cDNA. Ligands may be DNA molecules, RNA molecules, peptide nucleic acid molecules, peptides, proteins such as transcription factors, promoters, enhancers, repressors, and other proteins that regulate replication, transcription, or translation of the polynucleotide in the biological system. The assay involves combining the cDNA or a fragment thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound cDNA to identify at least one ligand that specifically binds the cDNA.

[0072] In one embodiment, the cDNA may be incubated with a library of isolated and purified molecules or compounds and binding activity determined by methods such as a gel-retardation assay (U.S. Pat. No. 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. Protein binding may be confirmed by raising antibodies against the protein and adding the antibodies to the gel-retardation assay where specific binding will cause a supershift in the assay.

[0073] 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.

[0074] The cDNA may be used to purify a ligand from a sample. A method for using a cDNA to purify a ligand would involve combining the cDNA or a fragment thereof with a sample under conditions to allow specific binding, recovering the bound cDNA, and using an appropriate agent to separate the cDNA from the purified ligand.

[0075] Protein Production and Uses

[0076] The full length cDNAs or fragment thereof may be used to produce purified proteins using recombinant DNA technologies described herein and taught in Ausubel et al. (supra; Units 16.1-16.62). One of the advantages of producing proteins by these procedures is the ability to obtain highly-enriched sources of the proteins thereby simplifying purification procedures.

[0077] The proteins may contain amino acid substitutions, deletions or insertions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Such substitutions may be conservative in nature when the substituted residue has structural or chemical properties similar to the original residue (e.g., replacement of leucine with isoleucine or valine) or they may be nonconservative when the replacement residue is radically different (e.g., a glycine replaced by a tryptophan). Computer programs included in LASERGENE software (DNASTAR, Madison Wis.), MACVECTOR software (Genetics Computer Group, Madison Wis.) and RasMol software (www.umass.edu/microbio/rasmol) may be used to help determine which and how many amino acid residues in a particular portion of the protein may be substituted, inserted, or deleted without abolishing biological or immunological activity.

[0078] Expression of Encoded Proteins

[0079] Expression of a particular cDNA may be accomplished by cloning the cDNA into a vector and transforming this vector into a host cell. The cloning vector used for the construction of cDNA libraries in the LIFESEQ databases may also be used for expression. Such vectors usually contain a promoter and a polylinker useful for cloning, priming, and transcription. An exemplary vector may also contain the promoter for -galactosidase, an amino-terminal methionine and the subsequent seven amino acid residues of -galactosidase. The vector may be transformed into competent E. coli cells. Induction of the isolated bacterial strain with isopropylthiogalactoside (IPTG) using standard methods will produce a fusion protein that contains an N terminal methionine, the first seven residues of -galactosidase, about 15 residues of linker, and the protein encoded by the cDNA.

[0080] The cDNA may be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotides containing cloning sites and fragments of DNA sufficient to hybridize to stretches at both ends of the cDNA may be chemically synthesized by standard methods. These primers may then be used to amplify the desired fragments by PCR. The fragments may be digested with appropriate restriction enzymes under standard conditions and isolated using gel electrophoresis. Alternatively, similar fragments are produced by digestion of the cDNA with appropriate restriction enzymes and filled in with chemically synthesized oligonucleotides. Fragments of the coding sequence from more than one gene may be ligated together and expressed.

[0081] Signal sequences that dictate secretion of soluble proteins are particularly desirable as component parts of a recombinant sequence. For example, a chimeric protein may be expressed that includes one or more additional purification-facilitating domains. Such domains include, but are not limited to, metal-chelating domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex, Seattle Wash.). The inclusion of a cleavable-linker sequence such as ENTEROKINASEMAX (Invitrogen, San Diego Calif.) between the protein and the purification domain may also be used to recover the protein.

[0082] Suitable host cells may include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, plant cells such as Nicotiana tabacum, yeast cells such as Saccharomvces cerevisiae, and bacteria such as E. coli. For each of these cell systems, a useful vector may also include an origin of replication and one or two selectable markers to allow selection in bacteria as well as in a transformed eukaryotic host. Vectors for use in eukaryotic host cells may require the addition of 3 poly(A) tail if the cDNA lacks poly(A).

[0083] Additionally, the vector may contain promoters or enhancers that increase gene expression. Many promoters are known and used in the art. Most promoters are host specific and exemplary promoters includes SV40 promoters for CHO cells; T7 promoters for bacterial hosts; viral promoters and enhancers for plant cells; and PGH promoters for yeast. Adenoviral vectors with the rous sarcoma virus enhancer or retroviral vectors with long terminal repeat promoters may be used to drive protein expression in mammalian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of secreted soluble protein may be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art. An alternative method for the production of large amounts of secreted protein involves the transformation of mammalian embryos and the recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep, and the like.

[0084] In addition to recombinant production, proteins or portions thereof may be produced manually, using solid-phase techniques (Stewart et al. (1969) Solid-Phase Peptide Synthesis, W H Freeman, San Francisco Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machines such as the ABI431A peptide synthesizer (Applied Biosystems, Foster City Calif.). Proteins produced by any of the above methods may be used as pharmaceutical compositions to treat disorders associated with null or inadequate expression of the genomic sequence.

[0085] Screening and Purification Assays

[0086] A protein or a portion thereof encoded by the cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand with specific binding affinity or to purify a molecule or compound from a sample. The protein or portion thereof employed in such screening may be free in solution, affixed to an abiotic or biotic substrate, or located intracellularly. For example, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a protein on their cell surface can be used in screening assays. The cells are screened against a library or a plurality of ligands and the specificity of binding or formation of complexes between the expressed protein and the ligand may be measured. The ligands may be DNA, RNA, or PNA molecules, agonists, antagonists, antibodies, immunoglobulins, inhibitors, peptides, pharmaceutical agents, proteins, drugs, or any other test molecule or compound that specifically binds the protein. An exemplary assay involves combining the mammalian protein or a portion thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound protein to identify at least one ligand that specifically binds the protein.

[0087] 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 or oligopeptide or fragment thereof. One method for high throughput screening using very small assay volumes and very small amounts of test compound is described in U.S. Pat. No. 5,876,946. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.

[0088] The protein 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 or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and using an appropriate chaotropic agent to separate the protein from the purified ligand.

[0089] Production of Antibodies

[0090] A protein encoded by a cDNA of the invention may be used to produce specific antibodies. Antibodies may be produced using an oligopeptide or a portion of the protein with inherent immunological activity. Methods for producing antibodies include: 1) injecting an animal, usually goats, rabbits, or mice, with the protein, or an antigenically-effective portion or an oligopeptide thereof, to induce an immune response; 2) engineering hybridomas to produce monoclonal antibodies; 3) inducing in vivo production in the lymphocyte population; or 4) screening libraries of recombinant immunoglobulins. Recombinant immunoglobulins may be produced as taught in U.S. Pat. No. 4,816,567.

[0091] Antibodies produced using the proteins of the invention are useful for the diagnosis of prepathologic disorders as well as the diagnosis of chronic or acute diseases characterized by abnormalities in the expression, amount, or distribution of the protein. A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies specific for proteins are well known in the art. Immunoassays typically involve the formation of complexes between a protein and its specific binding molecule or compound and the measurement of complex formation. Immunoassays may employ a two-site, monoclonal-based assay that utilizes monoclonal antibodies reactive to two noninterfering epitopes on a specific protein or a competitive binding assay (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

[0092] Immunoassay procedures may be used to quantify expression of the protein in cell cultures, in subjects with a particular disorder or in model animal systems under various conditions. Increased or decreased production of proteins as monitored by immunoassay may contribute to knowledge of the cellular activities associated with developmental pathways, engineered conditions or diseases, or treatment efficacy. The quantity of a given protein in a given tissue may be determined by performing immunoassays on freeze-thawed detergent extracts of biological samples and comparing the slope of the binding curves to binding curves generated by purified protein.

[0093] Labeling of Molecules for Assay

[0094] A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various cDNA, polynucleotide, protein, peptide or antibody assays. Synthesis of labeled molecules may be achieved using commercial kits for incorporation of a labeled nucleotide such as ³²P-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as ³⁵S-methionine. Polynucleotides, cDNAs, proteins, or antibodies may be directly labeled with a reporter molecule by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).

[0095] The proteins and antibodies may be labeled for purposes of assay by joining them, either covalently or noncovalently, with a reporter molecule that provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported in the scientific and patent literature including, but not limited to U.S. Pat. No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0096] DIAGNOSTICS

[0097] The cDNAs, or fragments thereof, may be used to detect and quantify differential gene expression; absence, presence, or excess expression of mRNAs; or to monitor mRNA levels during therapeutic intervention. Disorders associated with altered expression include hyperlipidemia, hypertension, type II diabetes, and tumors of the liver. These cDNAs can also be utilized as markers of treatment efficacy against the disorders noted above and other liver disorders, conditions, and diseases over a period ranging from several days to months. 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 altered gene expression. Qualitative or quantitative methods for this comparison are well known in the art.

[0098] For example, the cDNA may be labeled by standard methods and added to a biological sample from a patient under conditions for hybridization complex formation. 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 the amount of label in the patient sample is significantly altered in comparison to the standard value, then the presence of the associated condition, disease or disorder is indicated.

[0099] In order to provide a basis for the diagnosis of a condition, disease or disorder associated with gene expression, a normal or standard expression profile is established. This may be accomplished by combining a biological sample taken from normal subjects, either animal or human, with a probe under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a substantially purified target sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular condition is used to diagnose that condition.

[0100] Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies and in clinical trial 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 months.

[0101] Gene Expression Profiles

[0102] A gene expression profile comprises a plurality of cDNAs and a plurality of detectable hybridization complexes, wherein each complex is formed by hybridization of one or more probes to one or more complementary sequences in a sample. The cDNA composition of the invention is used as elements on a microarray to analyze gene expression profiles. In one embodiment, the microarray is used to monitor the progression of disease. Researchers can assess and catalog the differences in gene expression between healthy and diseased tissues or cells. 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 microarray 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.

[0103] In another embodiment, animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disorder or disease; or treatment of the condition, disorder or disease. Novel treatment regimens may be tested in these animal models using microarrays to establish and then follow expression profiles over time. In addition, microarrays 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.

[0104] Assays Using Antibodies

[0105] Antibodies directed against epitopes on a protein encoded by a cDNA of the invention may be used in assays to quantify the amount of protein found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions. The antibodies may be used with or without modification, and labeled by joining them, either covalently or noncovalently, with a labeling moiety.

[0106] Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include ELISA, RIA, and fluorescent activated cell sorting (FACS). Such immunoassays typically involve the formation of complexes between the protein and its specific antibody and the measurement of such complexes. These and other assays are described in Pound (supra). The method may employ a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes, or a competitive binding assay. (See, e.g., Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience, New York N.Y.; Pound, supra)

[0107] Therapeutics

[0108] The cDNAs and fragments thereof 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 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, herpes 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 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(34):184Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics, Humana Press, Totowa N.J.; and August et al. (1997) Gene Therapy (Advances in Pharmacology. Vol. 40), Academic Press, San Diego Calif.).

[0109] In addition, expression of a particular protein can be regulated through the specific binding of a fragment of a cDNA to a genomic sequence or an mRNA which encodes the protein or directs its transcription or translation. The cDNA can be modified or derivatized to any RNA-like or DNA-like material including peptide nucleic acids, branched nucleic acids, and the like. These sequences can be produced biologically by transforming an appropriate host cell with a vector containing the sequence of interest.

[0110] Molecules which regulate the activity of the cDNA or encoded protein are useful as therapeutics for hyperlipidemia. Such molecules include agonists which increase the expression or activity of the polynucleotide or encoded protein, respectively; or antagonists which decrease expression or activity of the polynucleotide or encoded protein, respectively. In one aspect, an antibody which specifically binds the protein may be used directly as an antagonist or indirectly as a delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express the protein.

[0111] Additionally, any of the proteins, or their ligands, or complementary nucleic acid sequences may be administered as pharmaceutical compositions or in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to affect the treatment or prevention of the conditions and disorders associated with an immune response. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Further, the therapeutic agents may be combined with pharmaceutically-acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration used by doctors and pharmacists may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton Pa.).

[0112] Model Systems

[0113] 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, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of underexpression or overexpression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to overexpress a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.

[0114] Transgenic Animal Models

[0115] Transgenic rodents that overexpress or underexpress a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 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.

[0116] Embryonic Stem Cells

[0117] Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells such as the mouse 129/SvJ cell line are placed in a blastocyst from the C57BL/6 mouse strain, they resume normal development and contribute to tissues of the live-born animal. ES cells are preferred for use in the creation of experimental knockout and knockin animals. The method for this process is well known in the art and the steps are: the cDNA is introduced into a vector, the vector is transformed into ES cells, transformed cells are identified and microinjected into mouse cell blastocysts, blastocysts are surgically transferred to pseudopregnant dams. The resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.

[0118] Knockout Analysis

[0119] In gene knockout analysis, a region of a gene is enzymatically modified to include a non-natural intervening sequence 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.

[0120] Knockin Analysis

[0121] ES cells can be used to create knockin humanized animals or transgenic animal models 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. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on the progression and treatment of the analogous human condition.

[0122] As described herein, the uses of the cDNAs, provided in the Sequence Listing of this application, and their encoded proteins are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art. Furthermore, the cDNAs provided in this application may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known to the person of ordinary skill in the art, e.g., the triplet genetic code, specific base pair interactions, and the like. Likewise, reference to a method may include combining more than one method for obtaining or assembling full length cDNA sequences that will be known to those skilled in the art. It is also to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.

[0123] IConstruction of cDNA Libraries

[0124] RNA was purchased from Clontech Laboratories (Palo Alto Calif.) or isolated from various tissues. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL reagent (Life Technologies, Rockville Md.). The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated with either isopropanol or ethanol and sodium acetate, or by other routine methods.

[0125] Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In most 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, Valencia Calif.), or an OLIGOTEX mRNA purification kit (Qiagen). Alternatively, poly(A) RNA was isolated directly from tissue lysates using other kits, including the POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).

[0126] In some cases, Stratagene (La Jolla Calif.) was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies) using the recommended procedures or similar methods known in the art. (See Ausubel, supra, Units 5.1 through 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 the appropriate restriction enzyme or 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 the PBLUESCRIPT phagemid (Stratagene), PSPORT1 plasmid (Life Technologies), or PINCY plasmid (Incyte Genomics). Recombinant plasmids were transformed into XL1-BLUE, XL1-BLUEMRF, or SOLR competent E. coli cells (Stratagene) or DH5a, DH10B, or ELECTROMAX DH10B competent E. coli cells (Life Technologies).

[0127] In some cases, libraries were superinfected with a 5excess of the helper phage, M13K07, according to the method of Vieira et al. (1987, Methods Enzymol. 153:3-11) and normalized or subtracted using a methodology adapted from Soares (1994, Proc Natl Acad Sci 91:9228-9232), Swaroop et al. (1991, Nucl Acids Res 19:1954), and Bonaldo et al. (1996, Genome Research 6:791-806). The modified Soares normalization procedure was utilized to reduce the repetitive cloning of highly expressed high abundance cDNAs while maintaining the overall sequence complexity of the library. Modification included significantly longer hybridization times which allowed for increased gene discovery rates by biasing the normalized libraries toward those infrequently expressed low-abundance cDNAs which are poorly represented in a standard transcript image (Soares et al., supra).

[0128] II Isolation and Sequencing of cDNA Clones

[0129] Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using one of the following: the Magic or WIZARD MINIPREPS DNA purification system (Promega); the AGTC MINIPREP purification kit (Edge BioSystems, Gaithersburg Md.); the QIAWELL 8, QIAWELL 8 Plus, or QIAWELL 8 Ultra plasmid purification systems, or the REAL PREP 96 plasmid purification kit (QIAGEN). Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4 C.

[0130] 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 plasrnid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

[0131] cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 thermal cycler (Applied Biosystems) or the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.) in conjunction with the HYDRA microdispenser (Robbins Scientific, Sunnyvale Calif.) or the MICROLAB 2200 system (Hamilton, Reno Nev.). cDNA sequencing reactions were prepared using reagents provided by APB or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE cycle sequencing kit (Applied Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled cDNAs were carried out using the MEGABACE 1000 DNA sequencing system (APB); the ABI PRISM 373 or 377 sequencing systems (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, supra, Unit 7.7).

[0132] III Extension of cDNA Sequences

[0133] Nucleic acid sequences were extended using the cDNA clones 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 OLIGO 4.06 software (National Biosciences), or another appropriate program, 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 68 C. to about 72 C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.

[0134] Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. Preferred libraries are ones that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred because they will contain more sequences with the 5 and upstream regions of genes. A randomly primed library is particularly useful if an oligo d(T) library does not yield a full-length cDNA.

[0135] High fidelity amplification was obtained by PCR using methods well known in the art. 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 Mg² , (NH₄)₂SO₄, and -mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): Step 1: 94 C., 3 min; Step 2:94 C., 15 sec; Step 3:60 C., 1 min; Step 4:68 C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6:68 C., 5 min; Step 7: storage at 4 C. In the alternative, the parameters for primer pair T7 and SK(Stratagene) were as follows: Step 1:94 C., 3 min; Step 2:94 C., 15 sec; Step 3:57 C., 1 min; Step 4:68 C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times ; Step 6: 68 C., 5 min; Step 7: storage at 4 C.

[0136] The concentration of DNA in each well was determined by dispensing 100 l PICOGREEN reagent (0.25% reagent in 1x TE, v/v; Molecular Probes) and 0.5 l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton Mass.) and allowing the DNA to bind to the reagent. The plate was scanned in a FLUOROSKAN II (Labsystems Oy) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5,l to 10 l aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose mini-gel to determine which reactions were successful in extending the sequence.

[0137] The extended nucleic acids were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC18 vector (APB). For shotgun sequencing, the digested nucleic acids were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with AGARACE enzyme (Promega). Extended clones were religated using T4 DNA ligase (New England Biolabs, Beverly Mass.) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transformed into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 C. in 384-well plates in LB/2carbenicillin liquid media.

[0138] The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1:94 C., 3 min; Step 2:94 C., 15 sec; Step 60 C., 1 min; Step 4:72 C., 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step storage at 4 C. DNA was quantified using PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same 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 ABI PRISM BIGDYE terminator cycle sequencing kit (Applied Biosystems).

[0139] IV Assembly and Analysis of Sequences

[0140] Component nucleotide sequences from chromatograms were subjected to PHRED analysis (Phil Green, University of Washington, Seattle Wash.) and assigned a quality score. The sequences having at least a required quality score were subject to various pre-processing algorithms to eliminate low quality 3 ends, vector and linker sequences, polyA tails, Alu repeats, mitochondrial and ribosomal sequences, bacterial contamination sequences, and sequences smaller than 50 base pairs. Sequences were screened using the BLOCK 2 program (Incyte Genomics), a motif analysis program based on sequence information contained in the SWISS-PROT and PROSRIT databases (Bairoch et al. (1997) Nucleic Acids Res 25:217-221; Attwood et al. (1997) J Chem Inf Comput Sci 37:417-424).

[0141] Processed sequences were subjected to assembly procedures in which the sequences were assigned to bins, one sequence per bin. Sequences in each bin were assembled to produce consensus sequences, templates. Subsequent new sequences were added to existing bins using BLAST (Altschul (supra); Altschul et al. (supra); Karlin et al. (1988) Proc Natl Acad Sci 85-841-845), BLASTn (vers. 1.4, WashU), and CROSSMATCH software (Phil Green, supra). Candidate pairs were identified as all BLAST hits having a quality score greater than or equal to 150. Alignments of at least 82% local identity were accepted into the bin. The component sequences from each bin were assembled using PHRAP (Phil Green, supra). Bins with several overlapping component sequences were assembled using DEEP PHRAP (Phil Green, supra).

[0142] Bins were compared against each other, and those having local similarity of at least 82% were combined and reassembled. Reassembled bins having templates of insufficient overlap (less than 95% local identity) were re-split. Assembled templates were also subjected to analysis by STITCHER/EXON MAPPER algorithms which analyzed the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types, disease states, and the like. These resulting bins were subjected to several rounds of the above assembly procedures to generate the template sequences found in the LIFESEQ GOLD database (Incyte Genomics).

[0143] The assembled templates were annotated using the following procedure. Template sequences were analyzed using BLASTn (vers. 2.0, NCBI) versus GBpri (GenBank vers. 116). Hits were defined as an exact match having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs, or a homolog match having an E-value equal to or greater than 110⁸. (The E-value quantifies the statistical probability that a match between two sequences occurred by chance). The hits were subjected to frameshift FASTversus GENPEPT (GenBank version 109). In this analysis, a homolog match was defined as having an E-value of 110 ⁸. The assembly method used above was described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999, and the LIFESEQ GOLD user manual (Incyte Genomics).

[0144] Following assembly, template sequences were subjected to motif, BLAST, Hidden Markov Model (HMM; Pearson and Lipman (1988) Proc Natl Acad Sci 85:2444-2448; Smith and Waterman (1981) J Mol Biol 147:195-197), and functional analyses, and categorized in protein hierarchies using methods described in U.S. Ser. No. 08/812,290, filed Mar. 6, 1997; U.S. Ser. No. 08/947,845, filed Oct. 9, 1997; U.S. Pat. No. 5,953,727; and U.S. Ser. No. 09/034,807, filed Mar.4, 1998. Template sequences may be further queried against public databases such as the GenBank rodent, mammalian, vertebrate, eukaryote, prokaryote, and human EST databases.

[0145] V Selection of Sequences, Microarray Preparation and Use p Incyte clones represent template sequences derived from the LIFESEQ GOLD assembled human sequence database (Incyte Genomics). In cases where more than one clone was available for a particular template, the 5-most clone in the template was used on the microarray. The GENEALBUM GEM series 1-6 microarrays (Incyte Genomics) contain 52,616 array elements which represent 17,472 annotated clusters and 35,144 unannotated clusters. The HUMAN GENOME GEM series 1-3 microarrays (Incyte Genomics) contain 28,626 array elements which represent 10,068 annotated clusters and 18,558 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 microarray (Incyte Genomics) contains 7075 array elements which represent 4610 annotated genes and 2,184 unannotated clusters. Table 3 shows the GenBank annotations for SEQ ID NOs: 1-401 of this invention as produced by BLAST analysis.

[0146] To construct microarrays, cDNAs were amplified from bacterial cells using primers complementary to vector sequences flanking the cDNA insert. Thirty cycles of PCR increased the initial quantity of cDNAs from 1-2 ng to a final quantity greater than 5 g. Amplified cDNAs were then purified using SEPHACRYL-400 columns (APB). Purified cDNAs were immobilized on polymer-coated glass slides. Glass microscope slides (Corning, Corning N.Y.) were cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides were etched in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), washed thoroughly in distilled water, and coated with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol. Coated slides were cured in a 110 C. oven. cDNAs were applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522. One microliter of the cDNA at an average concentration of 100 ng/ul was loaded into the open capillary printing element by a high-speed robotic apparatus which then deposited about 5 nl of cDNA per slide.

[0147] Microarrays were UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene), and then washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites were blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (Tropix, Bedford Mass.) for 30 minutes at 60 C. followed by washes in 0.2% SDS and distilled water as before.

[0148] VI Preparation of Samples

[0149] Treatment of human C3A cell cultures

[0150] Early confluent C3A cells (ATTC, Manassas Va.) were treated with captopril (0.5, 2, 48, and 60 g/ml) for 1, 3, and 6 hours. Early confluent C3A cells were treated with enalapril (0.5, 2, 48, and 60 g/ml) for 1, 3, and 6 hours. Early confluent C3A cells were treated with LY294002 (0.5, 2, 15, and 25 g/ml) for 1, 3, and 6 hours. Early confluent C3A cells were starved of insulin for 3 days prior to treatment, then treated with LY294002 (10 M) for 2, 24, 36, and 72 hours in the presence of insulin. Early confluent C3A cells were treated with DES (1, 10, and 100 M) for 1, 3, and 6 hours. Early confluent C3A cells were treated with dexamethasone (1 and 20 M) for 0, 2, 6, and 24 hours. Early confluent C3A cells were treated with MCA (0.5 and 10 M) for 2, 6, and 24 hours. In all cases mRNA from untreated early confluent cells were prepared in parallel as described below.

[0151] Isolation and Labeling of Sample cDNAs

[0152] Cells were harvested and lysed in 1 ml of TRIZOL reagent (510⁶ cells/ml; Life Technologies). The lysates were vortexed thoroughly and incubated at room temperature for 2-3 minutes and extracted with 0.5 ml chloroform. The extract was mixed, incubated at room temperature for 5 minutes, and centrifuged at 15,000 rpm for 15 minutes at 4 C. The aqueous layer was collected and an equal volume of isopropanol was added. Samples were mixed, incubated at room temperature for 10 minutes, and centrifuged at 15,000 rpm for 20 minutes at 4 C. The supernatant was removed and the RNA pellet was washed with 1 ml of 70% ethanol, centrifuged at 15,000 rpm at 4 C., and resuspended in RNase-free water. The concentration of the RNA was determined by measuring the optical density at 260 nm.

[0153] Poly(A) RNA was prepared using an OLIGOTEX mRNA kit (QIAGEN) with the following modifications: OLIGOTEX beads were washed in tubes instead of on spin columns, resuspended in elution buffer, and then loaded onto spin columns to recover mRNA. To obtain maximum yield, the mRNA was eluted twice.

[0154] Each poly(A) RNA sample was reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/l oligo-d(T) primer (21mer), 1first strand buffer, 0.03 units/ul RNase inhibitor, 500 uM dATP, 500 uM dGTP, 500 uM dTTP, 40 uM dCTP, and 40 uM either dCTP-Cy3 or dCTP-Cy5 (APB). The reverse transcription reaction was performed in a 25 mnl volume containing 200 ng poly(A) RNA using the GEMBRIGHT kit (Incyte Genomics). Specific control poly(A) RNAs (YCFR06, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, control InRNAs (YCFR06, YCFR45, YCFR67, and YCFR85) at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse transcription reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100 (w/w) to sample mRNA, respectively. To sample differential expression patterns, control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were diluted into reverse transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA. Reactions were incubated at 37 C. for 2 hr, treated with 2.5 ml of 0.5M sodium hydroxide, and incubated for 20 minutes at 85 C. to the stop the reaction and degrade the RNA.

[0155] cDNAs were purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech). Cy3- and Cy5-labeled reaction samples were combined as described below and ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The cDNAs were then dried to completion using a SpeedVAC system (Savant Instruments, Holbrook N.Y.) and resuspended in 14 l 5SSC/0.2% SDS.

[0156] VII Hybridization and Detection

[0157] Hybridization reactions contained 9 l of sample mixture containing 0.2 g each of Cy3 and Cy5 labeled cDNA synthesis products in 5SSC, 0.2% SDS hybridization buffer. The mixture was heated to 65 C. for 5 minutes and was aliquoted onto the microarray surface and covered with an 1.8 cm² coverslip. The microarrays were transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber was kept at 100% humidity internally by the addition of 140 l of 5SSC in a corner of the chamber. The chamber containing the microarrays was incubated for about 6.5 hours at 60 C. The microarrays were washed for 10 min at 45 C. in low stringency wash buffer (1SSC, 0.1% SDS), three times for 10 minutes each at 45 C. in high stringency wash buffer (0.1SSC), and dried.

[0158] Reporter-labeled hybridization complexes were detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light was focused on the microarray using a 20microscope objective (Nikon, Melville N.Y.). The slide containing the microarray was placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm1.8 cm microarray used in the present example was scanned with a resolution of 20 micrometers.

[0159] In two separate scans, the mixed gas multiline laser excited the two fluorophores sequentially. Emitted light was split, based on wavelength, into two photomultiplier tube detectors (PMT R1477; Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Appropriate filters positioned between the microarray and the photomultiplier tubes were used to filter the signals. The emission maxima of the fluorophores used were 565 nm for Cy3 and 650 nm for Cy5. Each microarray was typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus was capable of recording the spectra from both fluorophores simultaneously.

[0160] The sensitivity of the scans was calibrated using the signal intensity generated by a cDNA control species. Samples of the calibrating cDNA were separately labeled with the two fluorophores and identical amounts of each were added to the hybridization mixture. A specific location on the microarray contained 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.

[0161] The output of the photomultiplier tube was 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 were displayed as an image where the signal intensity was mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data was also analyzed quantitatively. Where two different fluorophores were excited and measured simultaneously, the data were first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.

[0162] A grid was superimposed over the fluorescence signal image such that the signal from each spot was centered in each element of the grid. The fluorescence signal within each element was then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis was the GEMTOOLS gene expression analysis program (Incyte Genornics). Significance was defined as signal to background ratio exceeding 2and area hybridization exceeding 40%.

[0163] VIII Data Analysis and Results

[0164] Array elements that exhibited at least 2-fold change in expression at one or more time points, a signal intensity over 250 units, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed using the GEMTOOLS program (Incyte Genomics). The cDNAs that are differentially expressed are shown in Table 1. Table I identifies upregulated and downregulated cDNAs. The cDNAs are further identified by their SEQ ID NO and TEMPLATE ID, and by the description associated with at least a fragment of a polynucleotide found in GenBank as shown in Tables 2 and 3. The descriptions were obtained using the sequences of the Sequence Listing and BLAST analysis.

[0165] IX Other Hybridization Technologies and Analyses

[0166] Other hybridization technologies utilize a variety of substrates such as nylon membranes, capillary tubes, etc. Arranging cDNAs on polymer coated slides is described in Example V; sample cDNA preparation and hybridization and analysis using polymer coated slides is described in examples VI and VII, respectively.

[0167] The cDNAs are applied to a membrane 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 37 C. 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 2SSC for 10 min each. The membrane is then LTV irradiated in a STRATALINKER UV-crosslinker (Stratagene).

[0168] In the second method, cDNAs 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 g. 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 LTV irradiation as described above.

[0169] Hybridization probes derived from 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 l TE buffer, denaturing by heating to 100 C. 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 microliters of ³²PdCTP is added to the tube, and the contents are incubated at 37 C. for 10 min. The labeling reaction is stopped by adding 5 l of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100 C. for five min and then snap cooled for two min on ice. Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and 1high phosphate buffer (0.5 M NaCl, 0.1 M Na₂HP O₄, 5 mM EDTA, pH 7) at 55 C. for two hr. The probe, dilut fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55 C. for 16 hr. Following hybridization, the membrane is washed for 15 min at 25 C. in 1 mM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25 C. in 1 mM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed to the membrane overnight at 70 C., developed, and examined.

[0170] X Further Characterization of Differentially Expressed cDNAs and Proteins

[0171] Clones were blasted against the LIFESEQ Gold 5.1 database (Incyte Genomics) and an Incyte template and its sequence variants were chosen for each clone. The template and variant sequences were blasted against GenBank database to acquire annotation. The nucleotide sequences were translated into amino acid sequence which was blasted against the GenPept and other protein databases to acquire annotation and characterization, i.e., structural motifs.

[0172] Percent sequence identity can be determined electronically for two or more amino acid or nucleic acid sequences using the MEGALIGN program (DNASTAR). The percent identity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage identity.

[0173] Sequences with conserved protein motifs may be searched using the BLOCKS search program. This program analyses sequence information contained in the Swiss-Prot and PROSITE databases and is useful for determining the classification of uncharacterized proteins translated from genomic or cDNA sequences (Bairoch et al.(supra); Attwood et al. (supra). PROSITE database is a useful source for identifying functional or structural domains that are not detected using motifs due to extreme sequence divergence. Using weight matrices, these domains are calibrated against the SWISS-PROT database to obtain a measure of the chance distribution of the matches.

[0174] The PRINTS database can be searched using the BLIMPS search program to obtain protein family fingerprints. The PRINTS database complements the PROSITE database by exploiting groups of aconserved motifs within sequence alignments to build characteristic signatures of different protein families. For both BLOCKS and PRINTS analyses, the cutoff scores for local similarity were: >1300strong, 1000-1300suggestive; for global similarity were: p<exp-3; and for strength (degree of correlation) were: >1300strong, 1000-1300weak.

[0175] X Expression of the Encoded Protein

[0176] Expression and purification of a protein encoded by a cDNA of the invention is achieved using bacterial or virus-based expression systems. For expression in bacteria, cDNA is subcloned into a vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into bacterial hosts, such as BL21(DE3). Antibiotic resistant bacteria express the protein upon induction with IPTG. Expression in eukaryotic cells is achieved by infecting Spodontera frugiperda (Sf9) insect cells with recombinant baculovirus, Autographica californica nuclear polyhedrosis virus. The polyhedrin gene of baculovirus is replaced with the cDNA by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of transcription.

[0177] For ease of purification, the protein is synthesized as a fusion protein with glutathione-S-transferase (GST; APB) or a similar alternative such as FLAG. The fusion protein is purified on immobilized glutathione under conditions that maintain protein activity and antigenicity. After purification, the GST moiety is proteolytically cleaved from the protein with thrombin. A fusion protein with FLAG, an 8-amino amino acid peptide, is purified using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.).

[0178] XI Production of Specific Antibodies

[0179] A denatured protein from a reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits following standard protocols. About 100 g is used to immunize a mouse, while up to 1 mg is used to immunize a rabbit. The denatured protein is radioiodinated and incubated with murine B-cell hybridomas to screen for monoclonal antibodies. About 20 mg of protein is sufficient for labeling and screening several thousand clones.

[0180] In another approach, the amino acid sequence translated from a cDNA of the invention is analyzed using PROTEAN software (DNASTAR) to determine regions of high antigenicity, essentially antigenically-effective epitopes of the protein. The optimal sequences for immunization are usually at the C-terminus, the N-terminus, and those intervening, hydrophilic regions of the protein that are likely to be exposed to the external environment when the protein is in its natural conformation. Typically, oligopeptides about 15 residues in length are synthesized using an ABI 431 Peptide synthesizer (Applied Biosystems) using Fmoc-chemistry and then coupled to keyhole limpet hemocyanin (KLH; Sigma Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester. If necessary, a cysteine may be introduced at the N-termninus of the peptide to permit coupling to KLH. Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG.

[0181] Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with radioiodinated protein to identify those fusions producing a monoclonal antibody specific for the protein. In a typical protocol, wells of 96 well plates (FAST, Becton-Dickinson, Palo Alto Calif.) are coated with affinity-purified, specific rabbit-anti-mouse (or suitable anti-species Ig) antibodies at 10 mg/ml. The coated wells are blocked with 1% BSA and washed and exposed to supernatants from hybridomas. After incubation, the wells are exposed to radiolabeled protein at 1 mg/ml. Clones producing antibodies bind a quantity of labeled protein that is detectable above background.

[0182] Such clones are expanded and subjected to 2 cycles of cloning at 1 cell/3 wells. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on protein A (APB). Monoclonal antibodies with affinities of at least 10⁸ M¹, preferably 10⁹ to 10¹⁰ M¹ or stronger, are made by procedures well known in the art.

[0183] XII Purification of Naturally Occurring Protein Using Specific Antibodies

[0184] Naturally occurring or recombinant protein is substantially purified by immunoaffinity chromatography using antibodies specific for 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 protein is collected.

[0185] XIII Screening Molecules for Specific Binding with the cDNA or Protein

[0186] The cDNA or fragments thereof and the protein or portions thereof are labeled with ³²P-dCTP, Cy3-dCTP, Cy5-dCTP (APB), or BIODIPY or FITC (Molecular Probes), respectively. Candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled nucleic or amino acid. After incubation under conditions for either a cDNA or a protein, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed. The binding molecule is identified by its arrayed position on the substrate. 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. High throughput screening using very small assay volumes and very small amounts of test compound is fully described in Burbaum et al. U.S. Pat. No. 5,876,946.

[0187] All patents and publications mentioned in the specification are incorporated herein by reference. 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
Clone ID	Clofibrate	Fenofibrate	Captopril	Enalapril	Dexamethasone	DES	MCA	LY294002	Ins/LY294002
26474					4.2
60123						2.6
63038	3.95	2.9	3.16	2.97	2.36
72713							3
85606							2.96
86390	2.83
118501	4.87		4.36	6.06	2.79		4.54		2.4
121785							3.3
136073							3.1
160822			2.5	2.39
167081					2.14
172023					5.8	2.85		3.38
211389			2.52
237027					2.85
293477						2.83
271299									2.91
279249									2.15
279898				4.08			5.48	6.7
280932	2.8	2.67
293477						2
311346							4.59
318486							4.11
341884	2.32								3.33
348143	2.11
388964									2.77
389362									2.88
407032	3.05
408886							4.93
419492									2.47
437481		2.44				2.24
442723									2.51
443991		2.47				2.62
450856		2.49					2.89
452321		3					4.3
454839							3.4
459372								4.65	2.63
460779	2.26
462069							2.41
480791		2.27
481402							3.22
510056						2.73	5.65
511448							3.05
560115							2.61
604019							3.28
630625	2.53	3.95	2.81	2.51	2.52		2.25
669498							2.97
701484							2.51
758192	3.11
773154									2.39
818192	2.87
872017							2.85
891322						3.81
963536									2.93
970905		4.06				2.25
990375					2.5
1213932									6.31
1259841									3.15
1272483						2.51
1306814									2.83
1308112	2.19			2.14			2.28
1315663	3.18	4.02	2.84					3.27
1316801							3.38
1326255						2.7
1368834			2.47	2.43
1379063	2.38		4	2.44	3.42		3.63
1381654			1.57		2.57		2.64
1395143									3.09
1435374									3.67
1441245					2.3
1448718	2.95		3.93	3.91	3.1
1454436									2.62
1457424							2.93
1457718									2.92
1464613	2.39
1468660					3.3			2.28
1482116									3.44
1495382									2.77
1500245									2.92
1511658	3.56
1519431			2.36
1519683	2.12
1522880							2.74
1530595									3.52
1559665	4.058
1559756			1.56		2.47
1560906							3.13
1577614									3.3
1616783					2.55		2.63
1619292	3.07	5	4.3		4.4		2.8
1619980									2.97
1623214					2.25		3.12
1630990					2.86		3.34
1696224	11.91		3.28	3.97	2.83		4.4
1705208	4.2	2.83
1711151	2.4		3.05	2.48
1732221									2.85
1756875							2.99
1786554					2.66		2.67
1822716									2.45
1833362					3.7		2.33
1834236					2.3
1838114	2.65
1845046	2.5
1846209			1.81		2.2
1846463	3.62	6.58
1861456				2.51
1867614									5.81
1869130									2.72
1871340									2.43
1874037									3.63
1874307									2.57
1890576					2.5		3.07
1890791	2.7
1920215	2.59
1922468	3.03
1926883	2.94
1930235	2.73		3.25	3			2.93
1956982						2.13
1958226	2.52
1963081									4.16
1966517	2.8
1969563	3.78	6.8
1975268		2.42		2.75	3.35		2.51	3.45
1998269	2.7	2.31	2.99	2.27	2.44		2.46	2.46
2042056	2.5	5.46							3.56
2046717					2.8
2048551					2.91				5.55
2055569	2.76	3.12	3.33	3.1
2055867		3.4	2.61			2.97	2.67
2120743					3.25		2.97
2121863									2.76
2123516	8.29	4.6	3.89	3.71	2.66		3.83
2132285									2.94
2132774									2.7
2160794					2.51
2195427					2.3		2.32
2201708									2.61
2208780									2.41
2232658							2.25
2234853		2.7
2241825						2.39
2242817									3.4
2252107							2.44
2273944	2.48
2278688	2.37
2293496							2.88
2311213						2.34
2343348					2.18		2.89
2352645	3.77
2360580		4.5	2.71
2365335	2.67
2382192	2.54
2382195		2.87
2383269									2.23
2394990									2.83
2399162		2.5
2446289			2.15
2448149		2.35
2453558									2.4
2470485	2.45				2.8
2495131									2.22
2511277							3.07
2513883		4.47		3.82	3.93		3.67		17.42
2513883									3.35
2514988									4.03
2516448									2.88
2517254									2.65
2520894							3.21
2517386									2.51
2545486						2.49
2550767						2.1
2579218							2.42
2607921		3.2
2538878									2.31
2636043									4.58
2660756					2.21		2.25
2675232							3.09
2695371							2.68
2708055							2.45
2740665	2.54
2756333							2.66
2757583	2.8	4.75	4.03	4.13	4.93		4.31		33.5
2765271							2.25
2769888	2.79	3.17	2.01					3.37
2813255									2.72
2820337			2.29
2822027					3		2.96
2825358							2.44
2830828									3.15
2831490							2.48
2860918							3.2
2879068									2.67
2884613	2.54		2.46	2.51			2.2
2890336	2.38
2891601							2.56
2899419									2.43
2912637			2.38				4.29
2912830									3.8
2921194									3.4
2921991	3.1		5.21	4.67	2.95		3.43
2925373					2.63
2929484		2.89
2933775	4		5.14	4.73	4.32				6.47
2953987									3.13
2955163				2.33			3.24
2956444						2.1	3.04
2957205	2.94	2.44							2.35
2991027									2.9
2992044									3.07
2999855	2.4
3026540									2.65
3028719					2.6
3038508	2.57
3070625									2.67
3074113							3.03
3084204					2.47
3108506							3.02
3109384					2.6
3120209									2.53
3121380									3.32
3121871									7.45
3123731									3.07
3128810									4.34
3129338									2.86
3136857	3.24
3158828							2.67
3170010					3.67
3208425									2.54
3222802	2.71	2.55				2.4	3.23
3225977			2.89				2.59
3240708			2.02
3272165		2.57
3284411					2.4
3345528									4.11
3380034	3.15
3381870			2.62				2.3
3407653							2.7
3427373								4.2
3472927							2.77
3493381							2.62
3493710									2.46
3494714	2.53
3606046							3.24
3679667			2.09
3715059					3.17			4.3
3792988							3.23
3815422			3.78
4019706						2.99	3.55
4066764	2.3
4070979							2.44
4087621	3.55
4091186					2.45		2.64
4092112							3.55
4107126			2.88
4110976	2.18
4203937							7.13
4246966	8.12						3.03
4254855							2.3
4284384							2.73
4287327	3.47	3.14				2.66	2.54		2.14
4403805							2.4
4508879					2.86		2.68
4549259							2.53
4556538		2.53		3.02				2.45
4715924						2.21
4721130	4.25						3.57
4795635	2.38
5047895	2.44
5077219	2.63
5093071							2.36
5102731							2.33
5266015							2.47
5266376							2.54
5293028						2.3
5398014	2.67
5398701	3.55
5399371					2.63	2.63
5512044							2.26
5541949	2.38

[0188]

TABLE 2
	NUCLEOIDE	NUCLEOTIDE	PROTEIN	PROTEIN
CLONE	SEQ ID	TEMPLATE	SEQ ID	TEMPLATE
ID	NO:	ID	NO:	ID
26474	1	220060.4
60123	2	016238.1
63038	3	1266683.1
72713	4	129384.1c
85606	5	3201389CB1	6	3201389CD1
86390	7	086390CB1	8	086390CD1
118501	9	1102322.16
118501	10	1545176CB1	11	1545176CD1
121785	12	978222.4
121785	13	978222.5
136073	14	1720920CB1	15	1720920CD1
160822	16	1857017CB1	17	1857017CD1
3493710	16	1857017CB1	17	1857017CD1
167081	18	2114865CB1	19	2114865CD1
172023	20	2700132CB1	21	2700132CD1
2470485	20	2700132CB1	21	2700132CD1
211389	22	238349.2
211389	23	238349.4c
237027	24	402917.3c
259054	25	406330.1
271299	26	2516070CB1	27	2516070CD1
2517386	26	2516070CB1	27	2516070CD1
279249	28	167507CB1	29	167507CD1
279898	30	3860413CB1	31	3860413CD1
3121871	30	3860413CB1	31	3860413CD1
280932	32	3393861CB1	33	3393861CD1
293477	34	2517374CB1	35	2517374CD1
311346	36	030850.7
318486	37	237416.12c
318486	38	237416.14
341884	39	1269631CB1	40	1269631CD1
348143	41	961189CB1	42	961189CD1
388964	43	246946.1
389362	44	017958.1
407032	45	985556.1
408886	46	476301CB1	47	476301CD1
419492	48	996427.2
437481	49	2989375CB1	50	2989375CD1
442723	51	236359.2
443991	52	011112.1c
450856	53	198268.1
452321	54	978740.3
454839	55	400197.1
459372	56	235687.5c
460779	57	2797839CB1	58	2797839CD1
462069	59	978690.6
480791	60	348072.5
481402	61	085596CB1	62	085596CD1
510056	63	103917CB1	64	103917CD1
511448	65	3603037CB1	66	3603037CD1
560115	67	088564CB1	68	088564CD1
604019	69	040429.1
630625	70	407096.2
669498	71	209265.54
701484	72	701484CB1	73	701484CD1
758192	74	251859.2
773154	75	3766715CB1	76	3766715CD1
818192	77	2049950CB1	78	2049950CD1
818192	79	231588.6c
872017	80	152298.2
891322	81	199507.1
963536	82	1434821CB1	83	1434821CD1
970905	84	289671.27
990375	85	1282225CB1	86	1282225CD1
1213932	87	263336.57
1259841	88	464689.40
1272483	89	155943.1
1306814	90	243794.19c
1306814	91	243794.23
1308112	92	159309CB1	93	159309CD1
1315663	94	1273641CB1	95	1273641CD1
1316801	96	403717.1
1326255	97	047593.1
1368834	98	347055.4
1379063	99	898899.11
1379063	100	898899.32
1381654	101	2047630CB1	102	2047630CD1
1395143	103	1039889.8
1435374	104	1272969CB1	105	1272969CD1
1441245	106	282397.85c
1441245	107	282397.94
1448718	108	1448817CB1	109	1448817CD1
1454436	110	1100769.2
1457424	111	332521.1
1457718	112	225080.16
1464613	113	334851.5
1468660	114	995529.7
1468660	115	995529.8
1482116	116	201851.1
1495382	117	059509CB1	118	059509CD1
1500245	119	481231.14
1511658	120	280276CB1	121	280276CD1
1519431	122	4675668CB1	123	4675668CD1
1519683	124	153825.1
1522880	125	403484.2c
1522880	126	1459432CB1	127	1459432CD1
1530595	128	1096583.1
1559665	129	516300CB1	130	516300CD1
1559756	131	627856CB1	132	627856CD1
1560906	133	1823159CB1	134	1823159CD1
1577614	135	232567.4
1616783	136	218419.1
1619292	137	1630551CB1	138	1630551CD1
1619980	139	360961.19
1623214	140	809809CB1	141	809809CD1
1630990	142	2558815CB1	143	2558815CD1
1696224	144	242010.16
1696224	145	1678695CB1	146	1678695CD1
1705208	147	988653.1
1711151	148	1250434CB1	149	1250434CD1
1732221	150	236196.3
1756875	151	442308.1
1786554	152	060957.1
1822716	153	014284CB1	154	014284CD1
1833362	155	1095192.1
1834236	156	233003.20
1834236	157	1911808CB1	158	1911808CD1
1838114	159	978276.8
1845046	160	405844.21
1845046	161	405844.22
1846209	162	2705515CB1	163	2705515CD1
1846463	164	2023119CB1	165	2023119CD1
1861456	166	1000084.27
3679667	166	1000084.27
1867614	167	220134.1
1869130	168	216331.1
1871340	169	206044.1
1874037	170	382906.16
1874307	171	331306.1
1890576	172	1094829.20
1890576	173	1094829.38
1890791	174	1135580.4
1920215	175	196623.3
1922468	176	048488.32
1926883	177	2767012CB1	178	2767012CD1
1930235	179	1651724CB1	180	1651724CD1
1956982	181	206397.1
1958226	182	461707.40
1963081	183	2706645CB1	184	2706645CD1
1966517	185	474372.7
1969563	186	3592543CB1	187	3592543CD1
1975268	188	048612.12c
1975268	189	048612.13
1998269	190	245259.16
2042056	191	522433CB1	192	522433CD1
2046717	193	1040667.43
2048551	194	2048551CB1	195	2048551CD1
2055569	196	1969731CB1	197	1969731CD1
2055867	198	1326983.14
2120743	199	2120743CB1	200	2120743CD1
2121863	201	3551330CB1	202	3551330CD1
2123516	203	1440032CB1	204	1440032CD1
2132285	205	1000133.1
2132774	206	4020439CB1	207	4020439CD1
2160794	208	2507087CB1	209	2507087CD1
2195427	210	239996.1
2201708	211	1097380.1
2208780	212	021524.2c
2208780	213	021524.9
2232658	214	253987.16
2234853	215	344553.1
2241825	216	410785.1
2242817	217	237623.6
2252107	218	076047.1
2273944	219	1099500.15
2273944	220	1099500.18
2278688	221	2278688CB1	222	2278688CD1
2293496	223	380283.1
2311213	224	1720847CB1	225	1720847CD1
2343348	226	333776.1c
2352645	227	3478236CB1	228	3478236CD1
2360580	229	147541.17
2365335	230	331120.16c
2382192	231	575983CB1	232	575983CD1
2382195	233	413268.6
2383269	234	1989186CB1	235	1989186CD1
2394990	236	337448.1c
2399162	237	228304.19
2446289	238	420527.25
2448149	239	998034.3
2453558	240	474165.26
2495131	241	697785CB1	242	697785CD1
2511277	243	346209.3
2513883	244	167772CB1	245	167772CD1
2514988	246	2514988CB1	247	2514988CD1
2516070	248	481231.16
2516070	249	481231.17
2516104	249	481231.17
2516261	249	481231.17
2516448	249	481231.17
2517254	250	1045853.2
5398014	250	1045853.2
2520894	251	336615.1
2527879	252	1328423.2
2545486	253	085282.1
2550767	254	1081605.3
2579218	255	1053517.1
2607921	256	480169.76
2636043	257	2636043CB1	258	2636043CD1
2641522	259	2993696CB1	260	2993696CD1
2660756	261	240518.21
2660756	262	240518.34
2663164	263	001322.4c
2675232	264	350502.3
2675232	265	350502.4c
2695371	266	253783.3
2708055	267	085119.1
2740665	268	902559.1
2756333	269	4113161CB1	270	4113161CD1
2757583	271	2757583CB1	272	2757583CD1
2765271	273	198317.1
2769888	274	1508254CB1	275	1508254CD1
2813255	276	474691.3
2820337	277	2457215CB1	278	2457215CD1
2822027	279	201395.4c
2825358	280	233189.21
2830828	281	196606.6c
2830828	282	196606.8c
2831490	283	1040190.3
2860918	284	1427459CB1	285	1427459CD1
2879068	286	480453.16c
2884613	287	1095604.1
2890336	288	241291.28
2891601	289	230611.1
2899419	290	3993708CB1	291	3993708CD1
2899419	292	1000133.12
2912637	293	400253.17c
2912637	294	400253.5
2912830	295	030882CB1	296	030882CD1
2921194	297	898779CB1	298	898779CD1
2921991	299	3727408CB1	300	3727408CD1
2925373	301	984236.1c
2925373	302	984236.2c
2929484	303	348082.5
2929484	304	348082.7
2933775	305	1097910.1
2953987	306	246841.1
2955163	307	351241.1
2956444	308	2790762CB1	309	2790762CD1
2957205	310	2253717CB1	311	2253717CD1
2991027	312	2655184CB1	313	2655184CD1
2991027	314	363000.9c
2992044	315	232818.15
2999855	316	347781.10
2999855	317	2477616CB1	318	2477616CD1
3026540	319	360532.1
3026540	320	360532.9
3028719	321	110245.1
3038508	322	478620.53
3038508	323	1813444CB1	324	1813444CD1
3070625	325	474588.21
3074113	326	407838.1
3084204	327	994387.19
3108506	328	347796.7
3109384	329	406498.4c
3120209	330	3346307CB1	331	3346307CD1
3121380	332	4005778CB1	333	4005778CD1
3123731	334	995575.17
3128810	335	863406CB1	336	863406CD1
3129338	337	413864.17
3136857	338	350106.16
3158828	339	399785.1
3170010	340	010498.19
3208425	341	255824.39
3208425	342	2706606CB1	343	2706606CD1
3222802	344	118006.1
3225977	345	1039889.26
3240708	346	481480.7
3272165	347	662575CB1	348	662575CD1
3284411	349	027619.3
3345528	350	235447.5
3380034	351	331104.2
3381870	352	348390.2
3407653	353	127004.1
3427373	354	026190.1
3472927	355	250330.1
3493381	356	480375.28
3494714	357	364726.10
3494714	358	364726.12
3606046	359	1505038CB1	360	1505038CD1
3715059	361	903508.12
3792988	362	346716.17c
3792988	363	346716.21c
3815422	364	330776.1
4019706	365	407999.1c
4066764	366	1719478CB1	367	1719478CD1
4070979	368	351157.2
4087621	369	088957CB1	370	088957CD1
5398701	369	088957CB1	370	088957CD1
4091186	371	980446.1
4092112	372	198827.1
4107126	373	1102297.22
4110976	374	215112.1
4203937	375	171495.1
4246966	376	242010.43
4254855	377	5834958CB1	378	5834958CD1
4284384	379	335648.1c
4287327	380	333840.1
4403805	381	480885.2
4508879	382	998106.8c
4549259	383	400701.4
4556538	384	1100320.4
4715924	385	246727.11
4715924	386	246727.17
4721130	387	1102322.12c
4721130	388	1102322.18
4795635	389	2070610CB1	390	2070610CD1
5047895	391	336733.3
5077219	392	1326902.13
5077219	393	1326902.6
5093071	394	013521.16
5102731	395	985369.1
5266015	396	002455.1
5266376	397	372647.1
5293028	398	208075.1
5399371	399	209279.1
5512044	400	381058.1
5541949	401	046977.1

[0189]

TABLE 3
SEQ ID	TEMPLATE	GI
NO:	ID	Number	E-value	Annotation
1	220060.4	g847737	0	Human transthyretin precursor mRNA, complete cds.
2	016238.1	g187326	0	Human macrophage mannose receptor (MRC1) gene, exon 24.
3	1266683.1	g1103903	0	Human spermidine/spermine N1-acetyltransferase (SSAT) gene,
				complete cds.
4	129384.1c	g508989	0	Human (oct-6) mRNA, complete cds.
5	3201389CB1	g927210	0	Human mRNA for adrenergic receptor alpha 1C
				isoform 3, complete cds.
6	3201389CD1	g927210	0	Human mRNA for adrenergic receptor alpha 1C
				isoform 3, complete cds.
7	086390CB1	g337749	0	Human serum amyloid A protein mRNA, complete cds.
8	086390CD1	g337749	0	Human serum amyloid A protein mRNA, complete cds.
9	1102322.16	g313283	0	African green monkey hsp70 mRNA.
10	1545176CB1	g313283	0	African green monkey hsp70 mRNA.
11	1545176CD1	g313283	0	African green monkey hsp70 mRNA.
12	978222.4	g6006501	0	Human mRNA for basic-helix-loop-helix protein, bHLH (Hey2 gene).
13	978222.5	g600650l	0	Human mRNA for basic-helix-loop-helix protein, bHLH (Hey2 gene).
14	1720920CB1	g1617313	0	Human mRNA for melanoma-associated chondroitin sulfate proteoglycan
				(MCSP).
15	1720920CD1	g1617313	0	Human mRNA for melanoma-associated chondroitin sulfate proteoglycan
				(MCSP).
16	1857017CB1	g184243	0	Human 3-hydroxy-3-methylglutaryl CoA reductase mRNA, complete cds.
17	1857017CD1	g184243	0	Human 3-hydroxy-3-methylglutaryl CoA reductase mRNA, complete cds.
18	2114865CB1	g177808	0	Human alpha-1-antichymotrypsin (AACT) mRNA, complete cds.
19	2114865CD1	g177808	0	Human alpha-1-antichymotrypsin (AACT) mRNA, complete cds.
20	2700132CB1	g415818	0	Human mki67a mRNA (long type) for antigen of monoclonal
				antibody Ki-67.
21	2700132CD1	g415818	0	Human mki67a mRNA (long type) for antigen of monoclonal
				antibody Ki-67.
22	238349.2	g4324682	E104	late gestation lung protein 1 Rattus norvegicus
23	238349.4c	g7644416	0.4	Mus platythrix TSPY gene, intron 1.
24	402917.3c			Incyte Unique
25	406330.1	g182103	0	Human enkephalin B (enkB) gene, exon 4 and 3 flank and
				complete cds.
26	2516070CB1	g28771	0	Human mRNA for apolipoprotein AI (apo AI).
27	2516070CD1	g28771	0	Human mRNA for apolipoprotein AI (apo AI).
28	167507CB1	g177889	0	Human alpha-2-thiol protease inhibitor mRNA, complete coding
				sequence.
29	167507CD1	g177889	0	Human alpha-2-thiol protease inhibitor mRNA, complete coding
				sequence.
30	3860413CB1	g187530	0	Human metallothionein-II pseudogene (mt-IIps).
31	3860413CD1	g187530	0	Human metallothionein-II pseudogene (mt-IIps).
32	3393861CB1	g182429	0	Human fibrinogen beta-chain mRNA, partial cds.
33	3393861CD1	g182429	0	Human fibrinogen beta-chain mRNA, partial cds.
34	2517374CB1	g24444	0	Human mRNA for alpha1-acid glycoprotein (orosomucoid).
35	2517374CD1	g24444	0	Human mRNA for alpha1-acid glycoprotein (orosomucoid).
36	030850.7			Incyte Unique
37	237416.12c	g396704	5.00E92	Human integrin associated protein mRNA, complete cds,.
38	237416.14	g396704	0	Human integrin associated protein mRNA, complete cds,.
39	1269631CB1	g5030423	0	Human gp250 precursor, mRNA, complete cds.
40	1269631CD1	g5030423	0	Human gp250 precursor, mRNA, complete cds.
41	961189CB1	g286008	0	Human mRNA for KIAA0020 gene, complete cds.
42	961189CD1	g286008	0	Human mRNA for KIAA0020 gene, complete cds.
43	246946.1	g4107230	3.00E34	Human mRNA for lipophilin B.
44	017958.1			Incyte Unique
45	985556.1			Incyte Unique
46	476301CB1	g1232174	0	Human mRNA for transketolase-like protein (2418 bp).
47	476301CD1	g1232174	0	Human mRNA for transketolase-like protein (2418 bp).
48	996427.2	g179892	0	Human cAMP phosphodiesterase PDE7 (PDE7A1) mRNA, complete cds.
49	2989375CB1			Incyte Unique
50	2989375CD1			Incyte Unique
51	236359.2			Incyte Unique
52	011112.1c	g3183903	0	Human partial mRNA; ID YG40-1B.
53	198268.1	g4337095	0	Human MSH55 gene, partial cds; and CLIC1, DDAH, G6b, G6c, G5b,
				G6d, G6e, G6f, BAT5, G5b, CSK:
54	978740.3			Incyte Unique
55	400197.1	g1457944	5.00E10	Human desmoglein 3 gene, promoter region.
56	235687.5c			Incyte Unique
57	2797839CB1	g189421	0	Human proliferating-cell nucleolar protein P120 mRNA, complete cds.
58	2797839CD1	g189421	0	Human proliferating-cell nucleolar protein P120 mRNA, complete cds.
59	978690.6	g3287264	e145	Rattus norvegicus mRNA for STOP protein.
60	348072.5	g288562	0	Human mRNA for inter-alpha-trypsin inhibitor heavy chain H3.
61	085596CB1	g184391	0	Human histidine-rich glycoprotein mRNA, complete cds.
62	085596CD1	g184391	0	Human histidine-rich glycoprotein mRNA, complete cds.
63	103917CB1	g183030	0	Human grancalcin mRNA, complete cds.
64	103917CD1	g183030	0	Human grancalcin mRNA, complete cds.
65	3603037CB1	g181986	0	Human early growth response 2 protein (EGR2) mRNA, complete cds.
66	3603037CD1	g181986	0	Human early growth response 2 protein (EGR2) mRNA, complete cds.
67	088564CB1	g1778716	0	Human chemokine exodus-1 mRNA, complete cds.
68	088564CD1	g1778716	0	Human chemokine exodus-1 mRNA, complete cds.
69	040429.1			Incyte Unique
70	407096.2	g1237037	0	Human mRNA for thioredoxin reductase.
71	209265.54	g2160718	0	Human amphiphysin II mRNA, complete cds.
72	701484CB1	g5926690	0	Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 2/20.
73	701484CD1	g5926690	0	Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 2/20.
74	251859.2	g1145815	0	Human 54 kDa progesterone receptor-associated immunophilin FKBP54
				mRNA, partial cds.
75	3766715CB1	g4914599	0	Human mRNA; cDNA DKFZp564A126 (from clone DKFZp564A126); partial cds.
76	3766715CD1	g4914599	0	Human mRNA; cDNA DKFZp564A126 (from clone DKFZp564A126); partial cds.
77	2049950CB1	g183038	0	Human gamma-glutamylcysteine synthetase (GCS) mRNA, complete cds.
78	2049950CD1	g183038	0	Human gamma-glutamylcysteine synthetase (GCS) mRNA, complete cds.
79	231588.6c	g183038	0	Human gamma-glutamylcysteine synthetase (GCS) mRNA, complete cds.
80	152298.2			Incyte Unique
81	199507.1			Incyte Unique
82	1434821CB1	g35706	0	Human pS2 mRNA induced by estrogen from Human breast cancer cell
				line MCF-7.
83	1434821CD1	g35706	0	Human pS2 mRNA induced by estrogen from Human breast cancer cell
				line MCF-7.
84	289671.27	g31896	0	Human GPx-3 mRNA for plasma glutathione peroxidase.
85	1282225CB1	g182355	0	Human liver fatty acid binding protein (FABP) mRNA, complete cds.
86	1282225CD1	g182355	0	Human liver fatty acid binding protein (FABP) mRNA, complete cds.
87	263336.57	g187538	0	Human metallothionein-Ie gene (hMT-Ie).
88	464689.40	g30257	0	Human CST3 gene for cystatin C.
89	155943.1	g6453599	0	Human mRNA; cDNA DKFZp434K098 (from clone DKFZp434K098); partial cds.
90	243794.19c	g550026	0	Human ribosomal protein S29 mRNA, complete cds.
91	243794.23	g550026	0	Human ribosomal protein S29 mRNA, complete cds.
92	159309CB1	g510689	0	Human Ki nuclear autoantigen mRNA, complete cds.
93	159309CD1	g510689	0	Human Ki nuclear autoantigen mRNA, complete cds.
94	1273641CB1			Incyte Unique
95	1273641CD1			Incyte Unique
96	403717.1			Incyte Unique
97	047593.1			Incyte Unique
98	347055.4	g410027	0	Human 3-hydroxy-3-methylglutaryl CoA synthase mRNA, complete cds.
99	898899.11	g183976	0	Human hepatocyte growth factor-like protein mRNA, complete cds.
100	898899.32	g1311660	0	Human hepatocyte growth factor-like protein gene, complete cds.
101	2047630CB1	g179099	0	Human asparagine synthetase mRNA, complete cds.
102	2047630CD1	g179099	0	Human asparagine synthetase mRNA, complete cds.
103	1039889.8	g178042	0	Human cytoskeletal gamma-actin gene, complete cds.
104	1272969CB1	g286028	0	Human mRNA for XPAC protein.
105	1272969CD1	g286028	0	Human mRNA for XPAC protein.
106	282397.85c	g3660662	0	Human D15F37 pseudogene, S3 allele, mRNA sequence.
107	282397.94	g6683696	0	Human mRNA for KIAA0393 protein, partial cds.
108	1448817CB1	g183117	0	Human insulin-like growth factor binding protein mRNA, complete cds.
109	1448817CD1	g183117	0	Human insulin-like growth factor binding protein mRNA, complete cds.
110	1100769.2	g296451	0	Human mRNA for ribosomal protein S26.
111	332521.1			Incyte Unique
112	225080.16	g951313	0	Human 2,3-oxidosqualene-lanosterol cyclase mRNA, complete cds.
113	334851.5	g5912050	0	Human mRNA; cDNA DKFZp434P1550 (from clone DKFZp434P1550); partial cds.
114	995529.7	g3126638	0	Human mRNA for CDC2 delta T, complete cds.
115	995529.8	g29838	0	Human CDC2 gene involved in cell cycle control.
116	201851.1	g2104768	0	Human echinoderm microtubule-associated protein homolog HuEMAP mRNA,
				complete cds.
117	059509CB1	g1149557	0	Human TNF-related apoptosis inducing ligand TRAIL mRNA, complete cds.
118	059509CD1	g1149557	0	Human TNF-related apoptosis inducing ligand TRAIL mRNA, complete cds.
119	481231.14	g28771	0	Human mRNA for apolipoprotein AI (apo AI).
120	280276CB1	g182406	0	Human fibrinogen alpha subunit and fibrinogen alpha subunit precursor,
				genes, complete cds.
121	280276CD1	g182406	0	Human fibrinogen alpha subunit and fibrinogen alpha subunit precursor,
				genes, complete cds.
122	4675668CB1	g1160618	0	Human bystin mRNA, complete cds.
123	4675668CD1	g1160618	0	Human bystin mRNA, complete cds.
124	153825.1	g456256	0	Human stromelysin-3 mRNA.
125	403484.2c	g1737178	0	Human somatostatin receptor-like protein (GPR24) gene, complete cds.
126	1459432CB1	g1737178	0	Human somatostatin receptor-like protein (GPR24) gene, complete cds.
127	1459432CD1	g1737178	0	Human somatostatin receptor-like protein (GPR24) gene, complete cds.
128	1096583.1	g30340	9.00E96	Human gene for cytochrome P(1)-450.
129	516300CB1	g1098616	0	Human CD94 protein mRNA, complete cds.
130	516300CD1	g1098616	0	Human CD94 protein mRNA, complete cds.
131	627856CB1	g2665518	0	Human tyrosyl-tRNA synthetase mRNA, complete cds.
132	627856CD1	g2665518	0	Human tyrosyl-tRNA synthetase mRNA, complete cds.
133	1823159CB1	g1378039	0	Human myotubularin (MTM1) mRNA, complete cds.
134	1823159CD1	g1378039	0	Human myotubularin (MTM1) mRNA, complete cds.
135	232567.4	g182718	0	Human follistatin gene, exons 1-5.
136	218419.1			Incyte Unique
137	1630551CB1	g30302	0	Human mRNA for cytochrome c1.
138	1630551CD1	g30302	0	Human mRNA for cytochrome c1.
139	360961.19	g896282	0	Human methionine adenosyltransferase alpha subunit gene fragment.
140	809809CB1	g3252871	0	Human BRCA1-associated protein 2 (BRAP2) mRNA, complete cds.
141	809809CD1	g3252871	0	Human BRCA1-associated protein 2 (BRAP2) mRNA, complete cds.
142	2558815CB1	g1049218	0	Human gamma-aminobutyraldehyde dehydrogenase mRNA, complete cds.
143	2558815CD1	g1049218	0	Human gamma-aminobutyraldehyde dehydrogenase mRNA, complete cds.
144	242010.16	g188487	0	Human MHC class III HSP70-1 gene (HLA), complete cds.
145	1678695CB1	g5926690	0	Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 2/20.
146	1678695CD1	g5926690	0	Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 2/20.
147	988653.1	g31129	0	Human mRNA for early growth response protein 1 (hEGR1).
148	1250434CB1	g1144012	0	Human MOP1 mRNA, complete cds.
149	1250434CD1	g1144012	0	Human MOP1 mRNA, complete cds.
150	236196.3	g4003383	0	Human genomic DNA of 8p21.3-p22 anti-oncogene of hepatocellular
				colorectal and non-small cell lung c
151	442308.1	g3955193	0	Human homeodomain protein (Nkx2.2) gene, exon 2 and complete cds.
152	060957.1			Incyte Unique
153	014284CB1	g1006656	0	Human mRNA for cathepsin C.
154	014284CD1	g1006656	0	Human mRNA for cathepsin C.
155	1095192.1	g57216	2.00E75	Rat brain mRNA for sodium channel protein I.
156	233003.20			Incyte Unique
157	1911808CB1			Incyte Unique
158	1911808CD1			Incyte Unique
159	978276.8			Incyte Unique
160	405844.21	g179320	0	Human B61 mRNA, complete cds.
161	405844.22	g179320	0	Human B61 mRNA, complete cds.
162	2705515CB1	g30820	0	Human mRNA for IFN-inducible gamma2 protein.
163	2705515CD1	g30820	0	Human mRNA for IFN-inducible gamma2 protein.
164	2023119CB1	g306769	0	Human leukemia virus receptor 1 (GLVR1) mRNA, complete cds.
165	2023119CD1	g306769	0	Human leukemia virus receptor 1 (GLVR1) mRNA, complete cds.
166	1000084.27	g3719220	0	Human vascular endothelial growth factor mRNA, complete cds.
167	220134.1	g1546096	0	Human hbc647 mRNA sequence.
168	216331.1			Incyte Unique
169	206044.1	g4529920	3.50E17	serine protease inhibitor
170	382906.16	g180142	0	Human CD53 glycoprotein mRNA, complete cds.
171	331306.1	g219862	0	Human mRNA for HM145.
172	1094829.20	g181040	0	Human cAMP response element regulatory protein (CREB2) mRNA,
				complete cds.
173	1094829.38	g220087	0	Human mRNA for DNA binding protein TAXREB67.
174	1135580.4	g2887408	0	Human KIAA0417 mRNA, complete cds.
175	196623.3	g4929830	0	Human peroxisomal D3,D2-enoyl-CoA isomerase (PEC1) mRNA, complete cds.
176	048488.32	g1503985	0	Human mRNA for KIAA0201 gene, complete cds.
177	2767012CB1	g306713	0	Human heat shock protein, E. coli DnaJ homologue mRNA, complete cds.
178	2767012CD1	g306713	0	Human heat shock protein, E. coli DnaJ homologue mRNA, complete cds.
179	1651724CB1	g35135	0	Human odc1 mRNA for ornithine decarboxylase.
180	1651724CD1	g35135	0	Human odc1 mRNA for ornithine decarboxylase.
181	206397.1	g3719360	2.00E34	Human CC chemokine gene cluster, complete sequence.
182	461707.40	g6841321	0	Human HSPC336 mRNA, partial cds.
183	2706645CB1	g337728	0	Human S100 protein beta-subunit gene, exon 3.
184	2706645CD1	g337728	0	Human S100 protein beta-subunit gene, exon 3.
185	474372.7	g1066790	0	Human protein kinase-related oncogene (PIMI) mRNA, complete cds.
186	3592543CB1	g186624	0	Human c-jun proto oncogene (JUN), complete cds, clone hCJ-1.
187	3592543CD1	g186624	0	Human c-jun proto oncogene (JUN), complete cds, clone hCJ-1.
188	048612.12c	g307332	0	Human phosphoenolpyruvate carboxykinase (PCK1) gene, complete cds
				with repeats.
189	048612.13	g189944	0	Human (clone lamda-hPEC-3) phosphoenolpyruvate carboxykinase (PCK1)
				mRNA, complete cds.
190	245259.16	g36031	0	Human rhoB gene mRNA.
191	522433CB1	g1813326	0	Human mRNA for TGF-beta superfamily protein, complete cds.
192	522433CD1	g1813326	0	Human mRNA for TGF-beta superfamily protein, complete cds.
193	1040667.43	g28338	5.00E94	Human mRNA for cytoskeletal gamma-actin.
194	2048551CB1	g188709	0	Human metallothionein I-B gene, exon 3.
195	2048551CD1	g188709	0	Human metallothionein I-B gene, exon 3.
196	1969731CB1	g2344811	0	Human mRNA for Drg1 protein.
197	1969731CD1	g2344811	0	Human mRNA for Drg1 protein.
198	1326983.14			Incyte Unique
199	2120743CB1			Incyte Unique
200	2120743CD1			Incyte Unique
201	3551330CB1	g719268	0	Human cysteine-rich heart protein (hCRHP) mRNA, complete cds.
202	3551330CD1	g719268	0	Human cysteine-rich heart protein (hCRHP) mRNA, complete cds.
203	1440032CB1	g35221	0	Human heat-shock protein HSP70B gene.
204	1440032CD1	g35221	0	Human heat-shock protein HSP70B gene.
205	1000133.1	g339660	0	Human thymosin beta 10 mRNA, complete cds.
206	4020439CB1	g296451	0	Human mRNA for ribosomal protein S26.
207	4020439CD1	g296451	0	Human mRNA for ribosomal protein S26.
208	2507087CB1	g6807670	0	Human mRNA; cDNA DKFZp434F205 (from clone DKFZp434F205); complete cds.
209	2507087CD1	g6807670	0	Human mRNA; cDNA DKFZp434F205 (from clone DKFZp434F205); complete cds.
210	239996.1			Incyte Unique
211	1097380.1	g6594626	0	Human pRGR1 mRNA, partial cds.
212	021524.2C	g598639	7.00E77	Human HepG2 3 region Mbol cDNA, clone hmd2d06m3.
213	021524.9	g598640	2.00E09	Human HepG2 partial cDNA, clone hmd2d06m5.
214	253987.16	g313212	0	Human Id3 gene for HLH type transcription factor.
215	344553.1	g469095	0	Human RNA for MTP.
216	410785.1	g187133	0	Human liver glucose transporter-like protein (GLUT2), complete cds.
217	237623.6	g402482	0	Human secretory protein (P1.B) mRNA, complete cds.
218	076047.1	g1688257	0	Human collagenase and stromelysin genes, complete cds, and
				metalloelastasegene, partial cds.
219	1099500.15	g3287488	0	Human Hsp89-alpha-delta-N mRNA, complete cds.
220	1099500.18	g32487	0	Human mRNA for 90-kDa heat-shock protein.
221	2278688CB1	g4210725	0	Human mRNA for puromycin sensitive aminopeptidase, partial.
222	2278688CD1	g4210725	0	Human mRNA for puromycin sensitive aminopeptidase, partial.
223	380283.1	g2564321	0	Human mRNA for KIAA0287 gene, partial cds.
224	1720847CB1	g4218425	6.00E11	Human pex3 gene (joined cds, promoter and exon 1).
225	1720847CD1	g4218425	6.00E11	Human pex3 gene (joined cds, promoter and exon 1).
226	333776.1c			Incyte Unique
227	3478236CB1	g179039	0	Human amphiregulin (AR) mRNA, complete cds, clones lambda-AR1 and
				lambda-AR2.
228	3478236CD1	g179039	0	Human amphiregulin (AR) mRNA, complete cds, clones lambda-AR1 and
				lambda-AR2.
229	147541.17	g6899845	0	Human mRNA for cisplatin resistance-associated overexpressed protein,
				complete cds.
230	331120.16c	g663009	0	Human PHKLA mRNA.
231	575983CB1	g2546963	0	Human mRNA for diubiquitin.
232	575983CD1	g2546963	0	Human mRNA for diubiquitin.
233	413268.6	g541677	0	Human HBZ17 mRNA.
234	1989186CB1	g2708328	0	Human atrophin-1 interacting protein 4 (AIP4) mRNA, partial cds.
235	1989186CD1	g2708328	0	Human atrophin-1 interacting protein 4 (AIP4) mRNA, partial cds.
236	337448.1c	g5912019	0	Human mRNA; cDNA DKFZp434H0735 (from clone DKFZp434H0735); partial cds.
237	228304.19			Incyte Unique
238	420527.25	g186757	0	Human protein kinase mRNA.
239	998034.3	g927597	0	Human transcription factor TFIIIB 90 kDa subunit (hTFIIIB90) mRNA,
				complete cds.
240	474165.26	g3005586	0	Human Ser/Arg-related nuclear matrix protein (SRM160) mRNA,
				complete cds.
241	697785CB1	g187109	0	Human 14 kd lectin mRNA, complete cds.
242	697785CD1	g187109	0	Human 14 kd lectin mRNA, complete cds.
243	346209.3	g4240220	3.00E14	Human mRNA for KIAA0866 protein, complete cds.
244	167772CB1	g3954884	0	Human mRNA for Ig kappa light chain, anti-RhD, therad 7.
245	167772CD1	g3954884	0	Human mRNA for Ig kappa light chain, anti-RhD, therad 7.
246	2514988CB1	g178848	0	Human apolipoprotein E mRNA, complete cds.
247	2514988CD1	g178848	0	Human apolipoprotein E mRNA, complete cds.
248	481231.16	g28771	0	Human mRNA for apolipoprotein AI (apo AI).
249	481231.17	g28771	0	Human mRNA for apolipoprotein AI (apo AI).
250	1045853.2	g763428	0	Human mRNA clone with similarity to L-glycerol-3-phosphate:NAD
				oxidoreductase and albumin gene se
251	336615.1	g2072161	0	Human tubby related protein 1 (TULP1) mRNA, complete cds.
252	1328423.2	g682747	0	Human mRNA for Apol_Human (MER5(Aopl-Mouse)-like protein), complete cds.
253	085282.1			Incyte Unique
254	1081605.3	g6466185	0	Human zinc finger protein ZNF228 (ZNF228) mRNA, complete cds.
255	1053517.1	g7339817	0.15	Mus musculus DNA methyltransferase (Dnmt1) gene, exon 28.
256	480169.76	g2921872	0	Human spleen mitotic checkpoint BUB3 (BUB3) mRNA, complete cds.
257	2636043CB1			Incyte Unique
258	2636043CD1			Incyte Unique
259	2993696CB1	g1143491	0	Human mRNA for BiP protein.
260	2993696CD1	g1143491	0	Human mRNA for BiP protein.
261	240518.21	g6841489	1.00E80	Human HSPC134 mRNA, complete cds.
262	240518.34	g6841489	0	Human HSPC134 mRNA, complete cds.
263	001322.4c			Incyte Unique
264	350502.3	g3978170	4.00E36	Mus musculus lysyl oxidase-related protein 2 (Lor2) mRNA,
265	350502.4c	g2661055	6.00E25	Human clone 23863 mRNA, partial cds.
266	253783.3	g2664429	6.40E43	hypothetical protein
267	085119.1	g1000863	0	Human DNA-binding protein (Fli-1) gene, 5 end of cds.
268	902559.1	g183990	0	Human epidermal growth factor receptor (HER3) mRNA, complete cds.
269	4113161CB1	g3360429	0	Human clone 23929 mRNA sequence.
270	4113161CD1	g3360429	0	Human clone 23929 mRNA sequence.
271	2757583CB1	g187542	0	Human metallothionein (MT)I-F gene, complete cds.
272	2757583CD1	g187542	0	Human metallothionein (MT)I-F gene, complete cds.
273	198317.1	g183398	3.00E36	Human guanine nucleotide-binding protein alpha-subunit gene
				(G-s-alpha), exon 2.
274	1508254CB1	g587201	0	Human HK2 mRNA for hexokinase II.
275	1508254CD1	g587201	0	Human HK2 mRNA for hexokinase II.
276	474691.3			Incyte Unique
277	2457215CB1	g38457	0	Human mRNA for PTB-associated splicing factor.
278	2457215CD1	g38457	0	Human mRNA for PTB-associated splicing factor.
279	201395.4c	g2224626	0	Human mRNA for KIAA0343 gene, complete cds.
280	233189.21	g189869	0	Human phosphoglycerate mutase 2 (muscle specific isozyme) (PGAM2)
				gene, 5 end.
281	196606.6C	g2924334	0	Human mRNA for exportin (tRNA).
282	196606.8c	g2924334	0	Human mRNA for exportin (tRNA).
283	1040190.3	g187518	0	Human MEM-102 glycoprotein mRNA, complete cds.
284	1427459CB1	g2437832	0	Human mRNA for RNF3A (DONG1) ring finger protein.
285	1427459CD1	g2437832	0	Human mRNA for RNF3A (DONG1) ring finger protein.
286	480453.16c	g4512253	0	Human gene for JKTBP2, JKTBP1, complete cds.
287	1095604.1	g1143491	0	Human mRNA for BiP protein.
288	241291.28	g3327107	0	Human mRNA for KIAA0647 protein, partial cds.
289	230611.1	g1321847	4.00E28	Human mRNA for U61 small nuclear RNA.
290	3993708CB1	g339660	0	Human thymosin beta 10 mRNA, complete cds.
291	3993708CD1	g339660	0	Human thymosin beta 10 mRNA, complete cds.
292	1000133.12	g264772	0	thymosin beta-10 Human, metastatic melanoma cell line, mRNA,
				453 nt.
293	400253.17c	g6016843	5.00E23	Human genomic DNA, chromosome 22q11.2, clone KB1561E1.
294	400253.5	g2826476	1.00E13	IL-17 receptor Homo sapiens
295	030882CB1	g1617087	0	Human mRNA for hBD-1 protein.
296	030882CD1	g1617087	0	Human mRNA for hBD-1 protein.
297	898779CB1	g179530	0	Human IgE-binding protein (epsilon-BP) mRNA, complete cds.
298	898779CD1	g179530	0	Human IgE-binding protein (epsilon-BP) mRNA, complete cds.
299	3727408CB1	g189944	0	Human (clone lamda-hPEC-3) phosphoenolpyruvate carboxykinase (PCK1)
				mRNA, complete cds.
300	3727408CD1	g189944	0	Human (clone lamda-hPEC-3) phosphoenolpyruvate carboxykinase (PCK1)
				mRNA, complete cds.
301	984236.1c			Incyte Unique
302	984236.2c			Incyte Unique
303	348082.5	g3868777		Rattus norvegicus mRNA for atypical PKC specific binding
304	348082.7	g3868778	0	atypical PKC specific binding protein Rattus norvegicus
305	1097910.1	g181275	0	Human cytochrome P1-450 (TCDD-inducible) mRNA, complete cds.
306	246841.1	g6453594	0	Human mRNA; cDNA DKFZp566M0947 (from clone DKFZp566M0947).
307	351241.1	g2935483	4.00E56	Human minisatellite ceb 1 repeat region.
308	2790762CB1			Incyte Unique
309	2790762CD1			Incyte Unique
310	2253717CB1	g7542489		Homo sapiens FK506 binding protein precursor (FKBP22)
311	2253717CD1			Incyte Unique
312	2655184CB1	g5531903	0	Human pre-mRNA splicing factor (SFRS3) mRNA, complete cds.
313	2655184CD1	g5531903	0	Human pre-mRNA splicing factor (SFRS3) mRNA, complete cds.
314	363000.9c	g5531903	0	Human pre-mRNA splicing factor (SFRS3) mRNA, complete cds.
315	232818.15	g3329377	0	Human vacuolar H()-ATPase subunit mRNA, complete cds.
316	347781.10	g1051169	0	Human GAP SH3 binding protein mRNA, complete cds.
317	2477616CB1	g1051169	0	Human GAP SH3 binding protein mRNA, complete cds.
318	2477616CD1	g1051169	0	Human GAP SH3 binding protein mRNA, complete cds.
319	360532.1	g37207	0	Human mRNA for slow skeletal troponin C (TnC).
320	360532.9	g37207	0	Human mRNA for slow skeletal troponin C (TnC).
321	110245.1	g3213194	0	Human serine-threonine kinase (BTAK) gene, partial cds.
322	478620.53	g386156	0	TLS translocated in liposarcoma Human, mRNA, 1824 nt.
323	1813444CB1	g386158	0	TLS/CHOP hybrid gene translocation breakpoint Human,
				myxoid liposarcomas cells, mRNA Mutant,
324	1813444CD1	g386158	0	TLS/CHOP hybrid gene translocation breakpoint Human,
				myxoid liposarcomas cells, mRNA Mutant,
325	474588.21	g339700	0	Human polyadenylate binding protein (TIA-1) mRNA, complete cds.
326	407838.1			Incyte Unique
327	994387.19	g6808610	2.00E14	Human 88-kDa Golgi protein (GM88) mRNA, complete cds.
328	347796.7	g710405	2.8	35 kDa protein Bartonella henselae
329	406498.4c			Incyte Unique
330	3346307CB1			Incyte Unique
331	3346307CD1			Incyte Unique
332	4005778CB1	g182513	0	Human ferritin L chain mRNA, complete cds.
333	4005778CD1	g182513	0	Human ferritin L chain mRNA, complete cds.
334	995575.17	g189066	0	Human NAP (nucleosome assembly protein) mRNA, complete cds.
335	863406CB1	g3327203	0	Human mRNA for KIAA0695 protein, complete cds.
336	863406CD1	g3327203	0	Human mRNA for KIAA0695 protein, complete cds.
337	413864.17			Incyte Unique
338	350106.16	g183059	0	Human glutamate dehydrogenase (GDH) mRNA, complete cds.
339	399785.1			Incyte Unique
340	010498.19	g4240316	0	Human mRNA for KIAA0914 protein, complete cds.
341	255824.39	g28596	0	Human fibroblast mRNA for aldolase A.
342	2706606CB1	g178350	0	Human aldolase A mRNA, complete cds.
343	2706606CD1	g178350	0	Human aldolase A mRNA, complete cds.
344	118006.1	g2290764	5.00E86	Human gonadotropin releasing hormone receptor (GNRHR) gene, exon 1.
345	1039889.26	g28338	0	Human mRNA for cytoskeletal gamma-actin.
346	481480.7	g561665	0	Human cysteine protease CPP32 isoform alpha mRNA, complete cds.
347	662575CB1			Incyte Unique
348	662575CD1			Incyte Unique
349	027619.3			Incyte Unique
350	235447.5	g37432	0	Human mRNA for transferrin receptor.
351	331104.2	g451209	0	Human mRNA for histidase, complete cds.
352	348390.2	g36502	0	Human mRNA for enteric smooth muscle gamma-actin.
353	127004.1			Incyte Unique
354	026190.1			Incyte Unique
355	250330.1	g456587	4.00E80	Human granulocyte-macrophage colony stimulating factor (GM-CSF)
				receptor alpha subunit gene, exon 1
356	480375.28	g5360203	5.00E27	Human A-kinase anchor protein (AKAP100) mRNA, complete cds.
357	364726.10	g498012	0	Human X104 mRNA, complete cds.
358	364726.12	g498012	0	Human X104 mRNA, complete cds.
359	1505038CB1	g536897	0	Human follistatin-related protein precursor mRNA, complete cds.
360	1505038CD1	g536897	0	Human follistatin-related protein precursor mRNA, complete cds.
361	903508.12	g5262490	0	Human mRNA; cDNA DKFZp564D0462 (from clone DKFZp564D0462).
362	346716.17c	g1147782	0	Human myosin-IXb mRNA, complete cds.
363	346716.21c			Incyte Unique
364	330776.1			Incyte Unique
365	407999.1c			Incyte Unique
366	1719478CB1	g758109	0	Human mRNA for voltage-activated sodium channel.
367	1719478CD1	g758109	0	Human mRNA for voltage-activated sodium channel.
368	351157.2	g31139	6.00E64	Human EMX1 mRNA.
369	088957CB1	g763428	0	Human mRNA clone with similarity to L-glycerol-3-phosphate:NAD
				oxidorcductase and albumin gene sc
370	088957CD1	g763428	0	Human mRNA clone with similarity to L-glycerol-3-phosphate:NAD
				oxidoreductase and albumin gene sc
371	980446.1	g2769702	0.088	chondroitin-6-sulfotransferase Homo sapiens
372	198827.1	g1O17792	0	Human substance P beta-PPT-A mRNA, complete cds.
373	1102297.22	g28335	0	Human ACTB mRNA for mutant beta-actin (beta-actin).
374	215112.1	g4240476	0	Human short chain L-3-hydroxyacyl-CoA dehydrogenase precursor
				(HADHSC) gene, nuclear gene encod
375	171495.1	g5102577	0	Human mRNA full length insert cDNA clone EUROIMAGE 345330.
376	242010.43	g5926690	0	Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 2/20.
377	5834958CB1	g881474	0	Human pephBGT-1 betaine-GABA transporter mRNA, complete cds.
378	5834958CD1	g881474	0	Human pephBGT-1 betaine-GABA transporter mRNA, complete cds.
379	335648.1c	g36712	0	Human mRNA for tyrosine aminotransferase (TAT) (EC 2.6.1.5).
380	333840.1	g452443	0	Human glucose-6-phosphatase mRNA, complete cds.
381	480885.2	g2394309	0	Human homeobox protein MEIS2 (MEIS2) mRNA, partial cds.
382	998106.8c	g174918	5.00E13	Human Ala-tRNA.
383	400701.4			Incyte Unique
384	1100320.4	g1209060	0	Human cytoplasmic dynein light chain 1 (hdlc1) mRNA, complete cds.
385	246727.11	g337456	0	Human ribonucleoprotein (La) mRNA, 3 end.
386	246727.17	g178686	0	Human La protein mRNA, complete cds.
387	1102322.12c	g32466	0	Human hsc70 gene for 71 kd heat shock cognate protein.
388	1102322.18	g313283	0	African green monkey hsp70 mRNA.
389	2070610CB1	g338696	0	Human thyroxine-binding globulin mRNA, complete cds.
390	2070610CD1	g338696	0	Human thyroxine-binding globulin mRNA, complete cds.
391	336733.3	g6049603	0	Human dickkopf-1 (DKK-1) mRNA, complete cds.
392	1326902.13	g219909	0	Human mRNA for lipocortin II, complete cds.
393	1326902.6	g219909	0	Human mRNA for lipocortin II, complete cds.
394	013521.16	g37611	0	Human urf-ret mRNA.
395	985369.1	g310099	2.00E36	Rattus norvegicus developmentally regulated protein mRNA,
396	002455.1			Incyte Unique
397	372647.1			Incyte Unique
398	208075.1	g23915	3.00E49	Human 7SK RNA gene and flanking regions.
399	209279.1	g2342725	3.6	hypothetical protein Arabidopsis thaliana
400	381058.1	g1021027	0	Human CpG island DNA genomic Mse1 fragment, clone 181h1, reverse
				read cpg181h1.rt1c.
401	046977.1	g4028582	1.00E12	Human connective tissue growth factor related protein WISP-2 (WISP2)
				mRNA, complete cds.

[0190]

TABLE 4
SEQ ID
NO:	TEMPLATE II	START	STOP	FRAME	Pfam ID	Pfam Description	E-value
1	220060.4	1	441	forward 1	Transthyretin	Transthyretin precursor (formerly prealbumin)	8.20E103
6	3201389CD1	43	326		7tm_1	7 transmembrane receptor (rhodopsin family)	4.20E103
8	086390CD1	21	130		SAA_proteins	Serum amyloid A protein	3.00E85
9	1102322.16	3	803	forward 3	HSP70	Hsp70 protein	2.40E12
11	1545176CD1	6	612		HSP70	Hsp70 protein	0.00E00
12	978222.4	1	159	forward 1	HLH	Helix-loop-helix DNA-binding domain	1.10E10
15	1720920CD1	55	181		laminin_G	Laminin G domain	2.00E25
17	1857017CD1	475	871		HMG-CoA_re	Hydroxymethylglutaryl-coenzyme A reductase	1.10E298
19	2114865CD1	46	420		serpin	Serpins (serine protease inhibitors)	1.60E216
21	2700132CD1	27	91		FHA	FHA domain	4.30E21
22	238349.2	379	837	forward 1	SCP	SCP-like extracellular protein	1.40E34
27	2516070CD1	2	265		Apolipoprotein	Apolipoprotein A1/A4/E family	2.00E137
29	167507CD1	266	370		cystatin	Cystatin domain	3.40E39
31	3860413CD1	1	61		metalthio	Metallothionein	2.10E25
33	3393861CD1	234	484		fibrinogen_C	Fibrinogen beta and gamma chains, C-terminal	3.00E179
						globular domain
35	2517374CD1	38	183		lipocalin	Lipocalin/cytosolic fatty-acid binding	2.10E33
						protein family
36	030850.7	1	396	forward 1	arf	ADP-ribosylation factor family	1.30E05
40	1269631CD1	1651	1735		fn3	Fibronectin type III domain	9.40E10
40	1269631CD1	1197	1237		ldl_recept_a	Low-density lipoprotein receptor domain class A	2.50E17
40	1269631CD1	888	931		ldl_recept_b	Low-density lipoprotein receptor repeat class B	2.00E06
47	476301CD1	28	586		transketolase	Transketolase	7.20E124
54	978740.3	1182	1487	forward 3	PH	PH domain	5.10E06
54	978740.3	516	1049	forward 3	RhoGEF	RhoGEF domain	1.10E23
58	2797839CD1	300	585		Nol1_Nop2_S	NOl1/NOP2/sun family	2.80E157
60	348072.5	860	1411	forward 2	vwa	von Willebrand factor type A domain	8.80E13
62	085596CD1	17	126		cystatin	Cystatin domain	3.10E25
66	3603037CD1	340	364		zf-C2H2	Zinc finger, C2H2 type	5.50E07
68	088564CD1	24	89		IL8	Small cytokines (intecrine/chemokine),	2.50E10
						interleukin-8 like
70	407096.2	1111	1953	forward 1	pyr_redox	Pyridine nucleotide-disulphide oxidoreductase	7.60E05
						class-I
70	407096.2	593	1489	forward 2	pyr_redox	Pyridine nucleotide-disulphide oxidoreductase	3.20E06
						class-I
70	407096.2	786	1730	forward 3	pyr_redox	Pyridine nucleotide-disulphide oxidoreductase	1.70E09
						class-I
71	209265.54	2041	2250	forward 1	SH3	SH3 domain	1.30E05
73	701484CD1	8	614		HSP70	Hsp70 protein	0.00E00
74	251859.2	348	632	forward 3	FKBP	FKBP-type peptidyl-prolyl cis-trans isomerases	9.40E49
76	3766715CD1	170	198		TPR	TPR Domain	7.70E04
83	1434821CD1	30	71		trefoil	Trefoil (P-type) domain	1.00E24
84	289671.27	1273	1614	forward 1	GSHPx	Glutathione peroxidases	4.40E68
86	1282225CD1	2	127		lipocalin	Lipocalin/cytosolic fatty-acid binding protein	6.90E25
						family
87	263336.57	55	171	forward 1	metalthio	Metallothionein	8.20E06
88	464689.40	443	631	forward 2	cystatin	Cystatin domain	6.90E21
91	243794.23	270	434	forward 3	Ribosomal_S1	Ribosomal protein S14p/S29e	6.80E19
98	347055.4	279	1649	forward 3	HMG_CoA_sy	Hydroxymethylglutaryl-coenzyme A synthase	0.00E00
99	898899.11	661	1266	forward 1	trypsin	Trypsin	2.10E39
99	898899.11	281	517	forward 2	kringle	Kringle domain	1.50E50
100	898899.32	1222	1383	forward 1	kringle	Kringle domain	5.60E09
100	898899.32	379	609	forward 1	PAN	PAN domain	1.50E06
100	898899.32	1367	1543	forward 2	kringle	Kringle domain	1.10E07
100	898899.32	2141	2785	forward 2	trypsin	Trypsin	4.50E46
100	898899.32	723	965	forward 3	kringle	Kringle domain	1.40E21
102	2047630CD1	206	557		Asn_synthase	Asparagine synthase	9.00E261
102	2047630CD1	2	148		GATase_2	Glutamine amidotransferases class-II	9.90E65
103	1039889.8	265	1002	forward 1	actin	Actin	2.40E83
103	1039889.8	968	1204	forward 2	actin	Actin	5.30E43
103	1039889.8	1281	1694	forward 3	actin	Actin	4.40E67
105	1272969CD1	79	391		filament	Intermediate filament proteins	4.30E157
109	1448817CD1	30	89		IGFBP	insulin-like growth factor binding proteins	2.20E23
109	1448817CD1	176	251		thyroglobulin	Thyroglobulin type-1 repeat	5.50E40
110	1100769.2	262	603	forward 1	Ribosomal_S2	Ribosomal protein S26e	9.20E75
110	1100769.2	663	884	forward 3	Ribosomal_S2	Ribosomal protein S26e	9.80E30
112	225080.16	510	641	forward 3	prenyltrans	Prenyltransferase and squalene oxidase repeat	6.50E13
113	334851.5	138	452	forward 3	CH	Calponin homology (CH) domain	2.00E25
114	995529.7	46	726	forward 1	pkinase	Eukaryotic protein kinase domain	6.00E46
115	995529.8	53	766	forward 2	pkinase	Eukaryotic protein kinase domain	1.00E87
115	995529.8	795	872	forward 3	pkinase	Eukaryotic protein kinase domain	2.70E07
116	201851.1	1634	1750	forward 2	WD40	WD domain, G-beta repeat	1.20E08
118	059509CD1	153	280		TNF	TNF (Tumor Necrosis Factor) family	4.00E15
119	481231.14	112	573	forward 1	Apolipoprotein	Apolipoprotein A1/A4/E family	5.80E34
124	153825.1	820	951	forward 1	hemopexin	Hemopexin	3.80E14
124	153825.1	42	497	forward 3	Peptidase_M 1	Matrixin	5.70E13
127	1459432CD1	57	311		7tm_1	7 transmembrane receptor (rhodopsin family)	2.30E64
130	516300CD1	87	174		lectin_c	Lectin C-type domain	7.50E05
132	627856CD1	143	239		tRNA_bind	Putative tRNA binding domain	5.80E46
135	232567.4	902	1042	forward 2	kazal	Kazal-type serine protease inhibitor domain	2.30E17
139	360961.19	1186	1266	forward 1	S-AdoMet_syn	S-adenosylmethionine synthetase	1.80E22
139	360961.19	149	604	forward 2	S-AdoMet_syn	S-adenosylmethionine synthetase	4.00E87
141	809809CD1	264	303		zf-C3HC4	Zinc finger, C3HC4 type (RING finger)	8.10E06
143	2558815CD1	45	511		aldedh	Aldehyde dehydrogenase family	3.50E216
144	242010.16	217	2037	forward 1	HSP70	Hsp70 protein	0.00E00
146	1678695CD1	6	612		HSP70	Hsp70 protein	0.00E00
147	988653.1	1295	1369	forward 2	zf-C2H2	Zinc finger, C2H2 type	1.00E06
149	1250434CD1	303	346		PAC	PAC motif	1.60E10
151	442308.1	294	464	forward 3	homeobox	Homeobox domain	2.70E27
154	014284CD1	231	458		Peptidase_C1	Papain family cysteine protease	8.30E106
160	405844.21	133	498	forward 1	Ephrin	Ephrin	7.60E80
161	405844.22	157	573	forward 1	Ephrin	Ephrin	1.30E96
163	2705515CD1	158	381		tRNA-synt_1b	tRNA synthetases class I (W and Y)	1.10E37
163	2705515CD1	12	68		WHEP-TRS	WHEP-TRS domain containing proteins	2.90E30
165	2023119CD1	39	665		PHO4	Phosphate transporter family	0.00E00
166	1000084.27	152	1423	forward 2	tubulin	Tubulin/FtsZ family	2.40E279
169	206044.1	248	532	forward 2	serpin	Serpins (serine protease inhibitors)	7.10E25
170	382906.16	182	226	forward 2	transmembrane	Transmembrane 4 family	4.40E04
171	331306.1	967	1029	forward 1	7tm_1	7 transmembrane receptor (rhodopsin family)	8.90E08
171	331306.1	312	1001	forward 3	7tm_1	7 transmembrane receptor (rhodopsin family)	2.50E88
172	1094829.20	1365	1559	forward 3	bZIP	bZIP transcription factor	2.90E19
173	1094829.38	1692	1886	forward 3	bZIP	bZIP transcription factor	2.90E19
175	196623.3	511	984	forward 1	ECH	Enoyl-CoA hydratase/isomerase family	3.80E06
175	196623.3	159	413	forward 3	ACBP	Acyl CoA binding protein	6.10E40
176	048488.32	348	2465	forward 3	HSP70	Hsp70 protein	2.70E220
178	2767012CD1	6	68		DnaJ	DnaJ domain	1.40E34
178	2767012CD1	220	346		DnaJ_C	DnaJ C terminal region	2.50E07
178	2767012CD1	121	207		DnaJ_CXXCX	DnaJ central domain (4 repeats)	5.80E43
180	1651724CD1	40	400		Orn_DAP_Arg	Pyridoxal-dependent decarboxylase	2.30E202
184	2706645CD1	53	81		efhand	EF hand	5.90E06
184	2706645CD1	4	47		S_100	S-100/ICaBP type calcium binding domain	3.60E23
185	474372.7	406	1164	forward 1	pkinase	Eukaryotic protein kinase domain	3.80E88
187	3592543CD1	250	314		bZIP	bZIP transcription factor	4.90E22
189	048612.13	203	1984	forward 2	PEPCK	Phosphoenolpyruvate carboxykinase	0.00E00
190	245259.16	398	955	forward 2	ras	Ras family	6.90E91
192	522433CD1	211	308		TGF-beta	Transforming growth factor beta like domain	6.80E19
195	2048551CD1	1	61		metalthio	Metallothionein	1.80E24
198	1326983.14	518	1768	forward 2	Aa_trans	Transmembrane amino acid transporter protein	9.50E15
198	1326983.14	518	1768	forward 2	Aa_trans	Transmembrane amino acid transporter protein	9.50E15
202	3551330CD1	4	61		LIM	LIM domain containing proteins	2.10E19
204	1440032CD1	8	614		HSP70	Hsp70 protein	0.00E00
205	1000133.1	199	321	forward 1	Thymosin	Thymosin beta-4 family	1.90E21
207	4020439CD1	1	114		Ribosomal_S2	Ribosomal protein S26e	4.70E67
213	021524.9	318	434	forward 3	WD40	WD domain, G-beta repeat	1.80E06
215	344553.1	159	1820	forward 3	Vitellogenin	Lipoprotein amino terminal region	1.80E160
216	410785.1	72	1535	forward 3	sugar_tr	Sugar (and other) transporter	2.40E200
217	237623.6	155	280	forward 2	trefoil	Trefoil (P-type) domain	2.00E25
219	1099500.15	466	1116	forward 1	HSP90	Hsp90 protein	1.20E128
219	1099500.15	263	472	forward 2	HSP90	Hsp90 protein	5.50E41
219	1099500.15	102	287	forward 3	HSP90	Hsp90 protein	1.00E41
220	1099500.18	1187	1630	forward 2	HSP90	Hsp90 protein	1.70E113
220	1099500.18	480	2609	forward 3	HSP90	Hsp90 protein	0.00E00
222	2278688CD1	54	441		Peptidase_M1	Peptidase family M1	2.90E234
223	380283.1	111	392	forward 3	SCAN	SCAN domain	2.80E11
223	380283.1	2889	2957	forward 3	zf-C2H2	Zinc finger, C2H2 type	3.20E06
232	575983CD1	8	79		ubiquitin	Ubiquitin family	7.90E09
235	1989186CD1	447	752		HECT	HECT-domain (ubiquitin-transferase).	5.70E130
235	1989186CD1	289	318		WW	WW domain	3.00E16
238	420527.25	924	1871	forward 3	pkinase	Eukaryotic protein kinase domain	5.90E79
239	998034.3	931	1152	forward 1	transcript_fac2	Transcription factor TFIIB repeat	5.80E19
240	474165.26	244	465	forward 1	PWI	PWI domain	2.60E41
242	697785CD1	22	126		Gal-bind_lecti	Vertebrate galactoside-binding lectins	2.90E65
243	346209.3	285	2861	forward 3	Myosin_tail	Myosin tail	2.00E181
245	167772CD1	1	61		metalthio	Metallothionein	2.20E23
247	2514988CD1	2	284		Apolipoprotein	Apolipoprotein A1/A4/E family	9.20E144
248	481231.16	77	823	forward 2	Apolipoprotein	Apolipoprotein A1/A4/E family	2.90E123
249	481231.17	829	1599	forward 1	Apolipoprotein	Apolipoprotein A1/A4/E family	2.20E130
249	481231.17	216	986	forward 3	Apolipoprotein	Apolipoprotein A1/A4/E family	1.60E103
250	1045853.2	955	1713	forward 1	NAD_Gly3P	NAD-dependent glycerol-3-phosphate dehydrogenase	2.00E11
250	1045853.2	1889	2413	forward 2	transport_prot	Serum albumin family	1.20E89
251	336615.1	86	874	forward 2	Tub	Tub family	3.00E195
254	1081605.3	177	365	forward 3	KRAB	KRAB box	4.10E29
254	1081605.3	2649	2717	forward 3	zf-C2H2	Zinc finger, C2H2 type	3.90E08
256	480169.76	337	453	forward 1	WD40	WD domain, G-beta repeat	2.80E07
260	2993696CD1	30	636		HSP70	Hsp70 protein	0.00E00
266	253783.3	976	1185	forward 1	rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	6.40E21
						domain)
266	253783.3	976	1185	forward 1	rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	6.40E21
						domain)
268	902559.1	733	1191	forward 1	Furin-like	Furin-like cysteine rich region	1.70E97
268	902559.1	358	732	forward 1	Recep_L_dom	Receptor L domain	6.50E60
268	902559.1	2238	2996	forward 3	pkinase	Eukaryotic protein kinase domain	3.20E61
272	2757583CD1	1	61		metalthio	Metallothionein	1.20E24
275	1508254CD1	16	463		hexokinase	Hexokinase	0.00E00
278	2457215CD1	299	364		rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	8.40E16
						domain)
285	1427459CD1	22	60		zf-C3HC4	Zinc finger, C3HC4 type (RING finger)	5.60E14
288	241291.28	3067	3267	forward 1	FYVE	FYVE zinc finger	9.30E21
291	3993708CD1	2	42		Thymosin	Thymosin beta-4 family	1.80E24
292	1000133.12	76	198	forward 1	Thymosin	Thymosin beta-4 family	1.80E24
298	898779CD1	136	239		Gal-bind_lecti	Vertebrate galactoside-binding lectins	3.80E50
304	348082.7	686	946	forward 2	PDZ	PDZ domain (Also known as DHR or GLGF).	1.20E19
305	1097910.1	53	835	forward 2	p450	Cytochrome P450	4.60E107
311	2253717CD1	48	141		FKBP	FKBP-type peptidyl-prolyl cis-trans isomerases	1.40E27
318	2477616CD1	11	133		NTF2	Nuclear transport factor 2 (NTF2) domain	5.30E67
318	2477616CD1	342	402		rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	1.70E12
						domain)
319	360532.1	574	660	forward 1	efhand	EF hand	3.00E08
320	360532.9	473	559	forward 2	efhand	EF hand	3.00E08
322	478620.53	924	1163	forward 3	rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	1.10E17
						domain)
322	478620.53	1329	1424	forward 3	zf-RanBP	Zn-finger in Ran binding protein and others.	1.50E11
325	474588.21	2263	2460	forward 1	rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	9.20E21
						domain)
325	474588.21	1863	2075	forward 3	rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	6.90E09
						domain)
333	4005778CD1	13	169		ferritin	Ferritins	6.90E99
334	995575.17	439	1260	forward 1	NAP_family	Nucleosome assembly protein (NAP)	8.80E191
336	863406CD1	15	717		Cullin	Cullin family	2.50E234
337	413864.17	910	1008	forward 1	ank	Ank repeat	2.30E07
338	350106.16	574	1905	forward 1	GLFV_dehydr	Glutamate/Leucine/Phenylalanine/Valine dehydrogenase	1.70E200
341	255824.39	568	1317	forward 1	glycolytic_enz	Fructose-bisphosphate aldolase class-I	9.60E192
341	255824.39	276	581	forward 3	glycolytic_enz	Fructose-bisphosphate aldolase class-I	2.70E65
343	2706606CD1	15	364		glycolytic_enz	Fructose-bisphosphate aldolase class-I	7.60E270
345	1039889.26	515	1279	forward 2	actin	Actin	3.90E190
345	1039889.26	117	491	forward 3	actin	Actin	2.40E89
346	481480.7	112	348	forward 1	ICE_p10	ICE-like protease (caspase) p10 domain	6.40E41
351	331104.2	481	2220	forward 1	PAL	Phenylalanine and histidine ammonia-lyases	0.00E00
352	348390.2	108	413	forward 3	actin	Actin	9.40E59
357	364726.10	333	647	forward 3	Guanylate_kin	Guanylate kinase	7.10E17
358	364726.12	2317	2631	forward 1	Guanylate_kin	Guanylate kinase	5.60E10
358	364726.12	223	483	forward 1	PDZ	PDZ domain (Also known as DHR or GLGF).	9.20E20
360	1505038CD1	54	98		kazal	Kazal-type serine protease inhibitor domain	7.20E12
367	1719478CD1	1177	1445		ion_trans	Ion transport protein	2.00E100
368	351157.2	3	134	forward 3	homeobox	Homeobox domain	6.60E13
370	088957CD1	28	202		transport_prot	Serum albumin family	1.20E89
373	1102297.22	1354	2481	forward 1	actin	Actin	1.70E286
375	171495.1	67	237	forward 1	homeobox	Homeobox domain	4.10E34
376	242010.43	2	1267	forward 2	HSP70	Hsp70 protein	5.00E129
378	5834958CD1	36	575		SNF	Sodium:neurotransmitter symporter family	0.00E00
384	1100320.4	237	503	forward 3	Dynein_light	Dynein light chain type I	1.10E62
386	246727.17	469	678	forward 1	rrm	RNA recognition motif, (a.k.a. RRM, RBD, or RNP	1.00E13
						domain)
388	1102322.18	5	751	forward 2	HSP70	Hsp70 protein	3.80E05
390	2070610CD1	39	412		serpin	Serpins (serine protease inhibitors)	2.70E194
392	1326902.13	270	473	forward 3	annexin	Annexin	5.20E18
393	1326902.6	919	1122	forward 1	annexin	Annexin	5.20E18
393	1326902.6	459	662	forward 3	annexin	Annexin	1.10E24
394	013521.16	486	734	forward 3	PH	PH domain	1.50E09