Genes associated with vascular disease

Abstract

The present invention relates to a combination comprising a plurality of cDNAs which are differentially expressed in vascular endothelium and which may be used in their entirety or in part to diagnose, to stage, to treat, or to monitor the treatment of a subject with a vascular disorder.

Description

FIELD OF THE INVENTION

[0002] The present invention relates to a combination comprising a plurality of cDNAs which are differentially expressed in vascular endothelium and which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of disorders of the vascular system including atherosclerosis, diabetic retinopathy, endometriosis, obesity, and cancer.

BACKGROUND OF THE INVENTION

[0003] The blood vessel wall is a complex tissue that plays a critical role in numerous physiological processes such as maintaining blood pressure, controlling selective permeability, directing vessel remodeling and angiogenesis, and modulating inflammatory and immune responses. Vessel walls are composed of three morphologically distinct layers, the intima, media, and adventitia, arranged concentrically from the lumenal surface outward. The intima is comprised of an endothelial cell (EC) layer in direct contact with the blood, surrounded by a layer of subendothelial connective tissue, within which lies the internal elastic lamina. The media is comprised of vascular smooth muscle cells (VSMC) and elastic fibrous components. The adventitia is comprised of the external elastic lamina surrounded on the outside by a layer of extracellular matrix components interspersed with fibroblasts and smooth muscle cells.

[0004] During embryonic development, blood vessels are formed de novo by vasculogenesis, the assembly of endothelial precursors. Later, the primitive vascular tree is expanded by angiogenesis, the development of new vessels from preexisting vessels. New vessels are formed both by sprouting of branches (sprouting angiogenesis) and by the partitioning of preexisting vessels via insertion of interstitial tissue columns (intussuseption). In adults, angiogenesis occurs normally during wound healing and during the menstrual cycle when the corpus luteum is formed. A key stimulus for angiogenesis is the production of a signal from hypoxic or otherwise stressed tissues that is detected by receptors on nearby microvascular endothelial cells. The first such signal to be identified was the angiogenesis inducing molecule vascular endothelial growth factor (VEGF).

[0005] Blood vessel structure and function vary considerably in different parts of the vascular tree. For example, vessel diameter and wall thickness decrease, along with changes in wall composition with increasing distance from the heart. Arterial walls are relatively thick compared with their venous counterparts, enabling them to withstand the higher blood pressures and shear forces to which they are exposed. The walls of the largest “elastic arteries” close to the heart have a thick medial layer, rich in elastic fibers which allow it to expand and contract with the heart. These elastic components are lacking in the small arteriole walls which play a major role in systemic blood pressure regulation. At the level of the microvascular capillary beds, where vessels have a diameter of about 7-8 μm, vessels are composed of a simple endothelial cell layer with a basement membrane composed primarily of type IV collagen.

[0006] Different types of capillary walls can be distinguished based on the degree of continuity and basement membrane composition.

[0007] Differences in vessel wall structure and function influence the diseases with which particular vessels are associated. For example, tumor angiogenesis and diabetic retinopathy are pathological conditions associated with abnormal growth of microvascular endothelium. By contrast, atherosclerosis affects the large elastic and muscular arteries such as the aorta and carotid, iliac, coronary, and popliteal arteries, but does not affect small muscular arteries or arterioles, large or small veins, or microvascular tissues.

[0008] Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common causes of death in industrialized nations. Atherosclerosis is a progressive disease characterized by a chronic local inflammatory response within the vessel wall of large arteries (Lusis (2000) Nature 407:233-241). Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and identifies all potential therapeutic targets for this complex disease has not been achieved. Pathological angiogenesis occurs in numerous conditions such as diabetic retinopathy, endometriosis, obesity, and cancer (Carmeliet et al. (2000) Nature 407:249-257). The demonstration that tumor induced angiogenesis is a critical stage in tumor progression, potentially amenable to clinical intervention, has led to a great deal of interest in factors which mediate tumor angiogenesis. Molecules involved in angiogenesis act to stimulate endothelial proliferation, migration, and morphogenesis, and remodel extracellular matrix.

[0009] A number of primary endothelial cell lines are available to study vascular endothelial cell function. Primary cells derived from the endothelia of human aorta (HAEC), coronary artery (HCAEC), iliac artery (HIAEC), brain microvasculature, (HMVECB), neonatal dermal microvasculature (HMVECD), pulmonary artery (HPAEC), umbilical artery (HUAEC), umbilical vein (HUVEC), and uterine myometrium microvasculature (UtMVEC) have all been used as experimental models for investigating in vitro the role of the endothelium in human vascular biology.

[0010] Array technology can provide a simple way to explore the expression of a single polymorphic gene or to produce an expression profile of a large number of related or unrelated genes. Arrays provide a platform for examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder. The 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 vascular endothelium from subjects with atherosclerosis may be compared with the levels and sequences expressed in normal vascular tissue.

[0011] The present invention provides for a combination comprising a plurality of cDNAs for use in detecting changes in expression of genes encoding proteins that are associated with a vascular disorder. The present invention satisfies a need in the art by identifying 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 vascular disorder such as atherosclerosis, diabetic retinopathy, endometriosis, obesity, and cancer.

SUMMARY

[0012] The present invention provides a combination comprising a plurality of cDNAs and their complements which are differentially expressed in vascular endothelial tissues and which are selected from SEQ ID NOs:1-292 as presented in the Sequence Listing. In one embodiment, each cDNA is expressed at higher levels in microvascular endothelium, SEQ ID NOs:1-209; in another embodiment, each cDNA is expressed at higher levels in large artery endothelium, SEQ ID NOs:210-292. In one aspect, the combination is useful to diagnose a vascular disorder selected from atherosclerosis, hemangioma, hemangioendothelioma, diabetic retinopathy, warts, pyogenic granulomas, Kaposi's sarcoma, scar keloids, allergic oedema, neoplasms, psoriasis, ulcers, follicular cysts, endometriosis, peritoneal sclerosis, and obesity. In another aspect, the combination is immobilized on a substrate.

[0013] The invention also provides a high throughput method to detect differential expression of one or more of the cDNAs of the combination. The method comprises hybridizing the substrate comprising the combination 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 a vascular disorder and differential expression determines an early, mid, and late stage of the disorder.

[0014] 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 combination 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 plurality of molecules or compounds are selected from DNA molecules, enhancers, mimetics, peptide nucleic acids, proteins, repressors, regulatory proteins, RNA molecules, and transcription factors. 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.

[0015] The invention provides an isolated cDNA selected from SEQ ID NOs:36, 51, 61, 83, 87, 110, 111, 136, 159, 198, and 244 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 provides a method for using an isolated cDNA to detect differential gene expression in a sample, the method comprising hybridizing a cDNA having a nucleotide sequence selected from SEQ ID NOs:36, 51, 61, 83, 87, 110, 111, 136, 159, 198, and 244 with a sample containing nucleic acids, thereby forming a hybridization complex, and comparing hybridization complex formation with complex formation in a normal standard, wherein differences in complex formation indicate differential expression of the nucleic acid in the sample. In one aspect, the sample is from vascular tissue or a patient with a vascular disorder. In another aspect, the nucleic acids of the sample are amplified prior to hybridization.

[0016] 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 plurality of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. The library or plurality of molecules or compounds is selected from agonists, antagonists, antibodies, DNA molecules, small molecule drugs, immunoglobulins, inhibitors, mimetics, peptide nucleic acids, peptides, pharmaceutical agents, proteins, RNA molecules, and ribozymes.

[0017] The invention provides a method for using the protein to produce an antibody which specifically binds the protein. The method for preparing a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies. The method for preparing a monoclonal antibodies comprises immunizing a animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating monoclonal antibodies from culture.

[0018] The invention provides purified antibodies which bind specifically to a protein. The invention also provides a method for using an antibody to detect expression of a protein in a sample, the method comprising combining the antibody with a sample under conditions for formation of antibody:protein complexes, and detecting complex formation, wherein complex formation indicates expression of the protein in the sample. In one aspect, the amount of complex formation when compared to standards is diagnostic of vascular disorders.

[0019] The invention provides a method for immunopurification of a protein comprising attaching an antibody to a substrate, exposing the antibody to a sample containing protein under conditions to allow antibody:protein complexes to form, dissociating the protein from the complex, and collecting purified protein. The invention also provides an array upon which a cDNA encoding a protein, the protein, or an antibody which specifically binds the protein are immobilized. The invention also provides a composition comprising a cDNA, a protein, an antibody, or a ligand which has agonistic or antagonistic activity.

DESCRIPTION OF THE COMPACT DISC-RECORDABLE (CD-R)

[0020] CD-R 1 is labeled: “PA-0049 US, Copy 1,” was created on Apr. 25, 2002 and contains: the Sequence Listing formatted in plain ASCII text. The file for the Sequence Listing is entitled pa0049sl.txt, created on Apr. 25, 2002 and is 716 KB in size.

[0021] CD-R 2 is an exact copy of CD-R 1. CD-R 2 is labeled: “PA-0049 US, Copy 2,” and was created on Apr. 25, 2002.

[0022] The CD-R labeled as: “PA-0049 US, CRF,” contains the Sequence Listing formatted in plain ASCII text. The file for the Sequence Listing is entitled pa0049sl.txt, was created on Apr. 25, 2002 and is 716 KB in size.

[0023] The content of the Sequence Listing named above and as described below, submitted in duplicate on two (2) CD-Rs (labeled “PA-0049 US, Copy 1” and “PA-0049 US, Copy 2”), and the CRF (labeled “PA-0049 US, CRF”) containing the Sequence Listing, are incorporated by reference herein, in their entirety.]

DESCRIPTION OF THE SEQUENCE LISTING AND TABLES

[0024] 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.

[0025] The Sequence Listing is a compilation of cDNAs obtained by sequencing and extending clone inserts. Each sequence is identified by a sequence identification number (SEQ ID NO) and by the Incyte template number (Incyte ID) from which it was obtained.

[0026] Tables 1, 2, and 3 show the differential expression of clones representing the cDNAs of the present invention. Each clone in Tables 1 and 2 represents a gene expressed at higher levels in microvascular endothelium relative to non-microvascular endothelium. Each clone in Table 3 represents a gene expressed at higher levels in large artery endothelium relative to microvascular and umbilical vein endothelium. Ratios are reported as log base 2; negative values indicate greater expression in the specific endothelial sample relative to the control. In Tables 1 and 3, column 1 shows the Clone ID for the clone present on the microarray and columns 2-10 show the differential expression value for the clone in specific regions of vascular endothelium relative to a vascular endothelium pooled control. In Table 2, column 1 shows the Clone ID for the clone present on the microarray, column 2 shows the average expression of the cDNA in microvascular endothelium and umbilical vein relative to the pooled control, column 3 shows the average expression in large artery endothelium relative to the pooled control, and column 4 shows the difference between microvascular/umbilical vein and large artery expression.

[0027] Table 4 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 5 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 nearest GenBank homolog (hit; GI Number), probability score (E-value), and functional annotation, respectively, as determined by BLAST analysis (version 2.0 using default parameters; Altschul et al. (1997) Nucleic Acids Res 25:3389-3402; Altschul (1993) J Mol Evol 36:290-300; and Altschul et al. (1990) J Mol Biol 215:403-410) of the cDNA against GenBank (release 122; National Center for Biotechnology Information (NCBI), Bethesda Md.).

DESCRIPTION OF THE INVENTION

[0029] Definitions

[0030] “Antibody” refers to intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an F(ab)2 fragment, an Fv fragment; and an antibody-peptide fusion protein.

[0031] “Antigenic determinant” refers to an antigenic or immunogenic epitope, structural feature, or region of an oligopeptide, peptide, or protein which is capable of inducing formation of an antibody which specifically binds the protein. Biological activity is not a prerequisite for immunogenicity.

[0032] “Array” refers to an ordered arrangement of at least two cDNAs, proteins, or antibodies on a substrate. At least one of the cDNAs, proteins, or antibodies represents a control or standard, and the other cDNA, protein, or antibody of diagnostic or therapeutic interest. The arrangement of at least two and up to about 40,000 cDNAs, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each cDNA and at least one nucleic acid, each protein and at least one ligand or antibody, or each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.

[0033] A “combination” comprises at least two and up to about 584 sequences selected from the group consisting of SEQ ID NOs:1-292 and their complements as presented in the Sequence Listing.

[0034] “cDNA” refers to an isolated polynucleotide, nucleic acid, or a fragment thereof, that contains from about 400 to about 12,000 nucleotides. It may have originated recombinantly or synthetically, may be double-stranded or single-stranded, represents coding and noncoding 3′ or 5′ sequence, generally lacks introns and may be purified or combined with carbohydrate, lipids, protein or inorganic elements or substances.

[0035] The phrase “cDNA encoding a protein” refers to a nucleic acid whose sequence closely aligns with sequences that encode conserved regions, motifs or domains identified by employing analyses well known in the art. These analyses include BLAST (Basic Local Alignment Search Tool; Altschul, supra; Altschul (1990) supra) 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, page 6076, column 2).

[0036] “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 longer lifespan or enhanced activity.

[0037] “Differential expression” refers to an increased or upregulated or a decreased or downregulated expression as detected by absence, presence, or at least two-fold change in the amount of transcribed messenger RNA or translated protein in a sample.

[0038] “Disorder” refers to conditions, diseases or syndromes of the vascular endothelium including disorders of increased vascularization such as atherosclerosis, hemangioma, hemangioendothelioma, diabetic retinopathy, warts, pyogenic granulomas, Kaposi's sarcoma, scar keloids, allergic oedema, neoplasms, follicular cysts, endometriosis, peritoneal sclerosis, and obesity; disorders of insufficient vascularization such as ulcers; and disorders of abnormal remodeling such as psoriasis.

[0039] An “expression profile” is a representation of gene expression in a sample. A nucleic acid expression profile is produced using sequencing, hybridization, or amplification technologies and mRNAs or cDNAs from a sample. A protein expression profile, although time delayed, mirrors the nucleic acid expression profile and uses PAGE, ELISA, FACS, or arrays and labeling moieties or antibodies to detect expression in a sample. The nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art.

[0040] “Fragment” refers to a chain of consecutive nucleotides from about 60 to about 5000 base pairs in length. Fragments may be used in PCR, hybridization or array technologies to identify related nucleic acids and in binding assays to screen for a ligand. Such ligands are useful as therapeutics to regulate replication, transcription or translation.

[0041] 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.

[0042] “Identity” 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), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul (1997) supra). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. “Similarity” as applied to proteins uses the same algorithms but takes into account conservative substitutions of nucleotides or residues.

[0043] “Isolated” or “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.

[0044] “Labeling moiety” refers to any reporter molecule whether a visible or radioactive label, stain or dye that can be attached to or incorporated into a cDNA or protein. Visible labels and dyes include but are not limited to anthocyanins, β glucuronidase, BIODIPY, Coomassie blue, Cy3 and Cy5, digoxigenin, FITC, green fluorescent protein, luciferase, spyro red, silver, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.

[0045] “Large artery endothelium” includes vascular endothelium obtained from aorta, coronary artery, pulmonary artery, and iliac artery.

[0046] “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.

[0047] “Microvascular endothelium” includes vascular endothelium obtained from neonatal dermis and uterine myometrium.

[0048] “Neoplasms” may include adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, esophagus, gall bladder, ganglia, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, rectum, salivary glands, skin, small intestine, spleen, stomach, testis, thymus, thyroid, and uterus.

[0049] “Non-microvascular endothelium” includes large artery endothelium and vascular endothelium obtained from umbilical artery and vein.

[0050] “Oligonucleotide” refers a single stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms include amplimer, primer, and oligomer.

[0051] “Portion” refers to any part of a protein used for any purpose which retains at least one biological or antigenic characteristic of a native protein, but especially, to an epitope for the screening of ligands or for production of antibodies.

[0052] “Post-translational modification” of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.

[0053] “Probe” refers to a cDNA that hybridizes to at least one nucleic acid in a sample. Where targets are single stranded, probes are complementary single strands. Probes can be labeled for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.

[0054] “Protein” refers to a polypeptide or any portion thereof. 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.

[0055] “Sample” is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like. A sample may comprise a bodily fluid such as ascites, blood, lymph, saliva, semen, spinal, sputum, tears, and urine; 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 or tissue biopsy; a tissue print; buccal cells, skin, a hair or its follicle; and the like.

[0056] “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.

[0057] “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.

[0058] A “transcript image” (TI) is a profile of gene transcription activity in a particular tissue at a particular time. TI provides assessment of the relative abundance of expressed polynucleotides in the cDNA libraries of an EST database as described in U.S. Pat. No. 5,840,484, incorporated herein by reference.

[0059] “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.

[0060] The Invention

[0061] The present invention provides for a combination comprising a plurality of cDNAs and their complements, SEQ ID NOs:1-292, which may be used to diagnose, to stage, to treat, or to monitor the progression or treatment of a vascular disorder. These cDNAs represent known and novel genes differentially expressed in vascular endothelium from microvascular and non-microvascular tissues. The combination may be used in its entirety or in part, as subsets of cDNAs expressed at higher levels in microvascular endothelium, SEQ ID NOs:1-209, or of cDNAs expressed at higher levels in large artery endothelium, SEQ ID NOs:210-292. SEQ ID NOs:36, 51, 61, 83, 87, 110, 111, 136, 159, 198, and 244 represent novel cDNAs differentially expressed in vascular endothelium. Since the novel cDNAs were identified solely by their differential expression, it is not essential to know a priori the name, structure, or 40 function of the gene or its encoded protein. The usefulness of the novel cDNAs exists in their immediate value as diagnostics for vascular disorders.

[0062] Tables 1, 2, and 3 show the differential expression of clones representing the cDNAs of the present invention on microarrays. Each clone in Tables 1 and 2 represents a gene expressed at higher levels in microvascular endothelium relative to non-microvascular endothelium. Each clone in Table 3 represents a gene expressed at higher levels in large artery endothelium relative to microvascular endothelium and umbilical vein. Ratios are reported as log base 2; negative values indicate greater expression in the specific endothelial sample relative to the control.

[0063] In Tables 1 and 3, column 1 shows the clone ID and columns 2-10 show the differential expression value for the clone in specific regions of vascular endothelium relative to a vascular endothelium pooled control. The vascular endothelium represented in columns 2-10 are aorta (HAEC), coronary artery (HCAEC), pulmonary artery (HPAEC), iliac artery (HIAEC), uterine myometrium microvasculature (UtMVEC), neonatal dermal microvasculature (HMVECD), umbilical artery (HUAEC), and umbilical vein (HUVEC; three separate samples), respectively. In Table 2, column 1 shows the Clone ID for the clone present on the microarray, column 2 shows the average expression of the cDNA in microvascular and umbilical vein endothelium relative to the pooled control, column 3 shows the average expression in large artery endothelium relative to the pooled control, and column 4 shows the difference between microvascular/umbilical vein and large artery expression.

[0064] Table 4 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.

[0065] Table 5 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 homolog (hit), probability score (E-value), and functional annotation, respectively, as determined by BLAST analysis (version 2.0 using default parameters; Altschul (1997) supra) of the cDNA against GenBank (release 122; NCBI).

[0066] The cDNAs of the invention define a differential expression pattern against which to compare the expression pattern of biopsied and/or in vitro treated vascular 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.

[0067] The combination may be arranged on a substrate and hybridized with tissues from subjects with diagnosed vascular disorders to identify those sequences which are differentially expressed in, e.g., both atherosclerosis and other vascular 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 combination. Such combinations may be useful in the elucidation of pathways which are affected in a particular disorder or to identify new, coexpressed, candidate, therapeutic molecules.

[0068] In another embodiment, the combination can be used for large scale genetic or gene expression analysis of a large number of novel, nucleic acids. 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 acids are hybridized to the combination for the purpose of defining a novel gene profile associated with that developmental stage, treatment, or disorder.

[0069] cDNAs and Their Uses

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The cDNAs may be used for a variety of purposes. For example, the combination 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 atherosclerosis, or inhibiting or inactivating a therapeutically relevant gene related to the cDNA.

[0075] 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.

[0076] Hybridization

[0077] 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 Bisociences (APB), Piscataway N.J.), fluorescent (Qiagen-Operon, Alameda Calif.), and chemiluminescent labeling (Promega, Madison Wis.) are well known in the art.

[0078] 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.

[0079] The stringency of hybridization is determined by G+C 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 5×saline 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 0.2×SSC 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 acids 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 4-6-4.9).

[0080] 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.)

[0081] Screening and Purification Assays

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Protein Production and Uses

[0087] The full length cDNAs or fragment thereof may be used to produce purified proteins using recombinant DNA technologies described herein and taught in Ausubel (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.

[0088] 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.) and algorithms included in RasMol software (University of Massachusetts, Amherst Mass.) 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.

[0089] Expression of Encoded Proteins

[0090] 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 (Incyte Genomics, Palo Alto Calif.) 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 β-galactosidase, an amino-terminal methionine and the subsequent seven amino acid residues of β-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 β-galactosidase, about 15 residues of linker, and the protein encoded by the cDNA.

[0091] 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.

[0092] 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.

[0093] 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 Saccharomyces 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).

[0094] 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.

[0095] 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 431A peptide synthesizer (Applied Biosystems (ABI), 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.

[0096] Screening and Purification Assays

[0097] 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 agonists, antagonists, antibodies, DNA molecules, small molecule drugs, immunoglobulins, inhibitors, mimetics, peptide nucleic acids, peptides, pharmaceutical agents, proteins, RNA molecules, ribozymes 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.

[0098] 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.

[0099] 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.

[0100] Production of Antibodies

[0101] 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 amtigenic determinant 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.

[0102] 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.).

[0103] 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.

[0104] Antibody Arrays

[0105] In an alternative to yeast two hybrid system analysis of proteins, an antibody array can be used to study protein-protein interactions and phosphorylation. A variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until protein:antibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest. In the alternative, a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex. The identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane.

[0106] Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and gridded at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt et al. (2000) Nat Biotechnol 18:989-94).

[0107] Labeling of Molecules for Assay

[0108] 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.).

[0109] 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. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0110] Diagnostics

[0111] 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 disorders of increased vascularization such as atherosclerosis, hemangioma, hemangioendothelioma, warts, pyogenic granulomas, Kaposi's sarcoma, scar keloids, allergic oedema, neoplasms, follicular cysts, endometriosis, peritoneal sclerosis, diabetic retinopathy, and obesity; disorders of insufficient vascularization such as ulcers; and disorders of abnormal remodeling such as psoriasis. These cDNAs can also be utilized as markers of treatment efficacy against the disorders noted above and other 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.

[0112] 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.

[0113] 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 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.

[0114] 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.

[0115] Gene Expression Profiles

[0116] 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 nucleic acids in a sample. The cDNAs of the invention are used as elements on a array to analyze gene expression profiles. In one embodiment, the array is used to monitor the progression of disease. Researchers and clinicians can 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 array is employed to improve the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.

[0117] Experimentally, expression profiles can also be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational discriminant analysis, transcript imaging, and by protein or antibody arrays. Expression profiles produced by these methods may be used alone or in combination. The correspondence between mRNA and protein expression has been discussed by Zweiger (2001, Transducing the Genome. McGraw-Hill, San Francisco, Calif.) and Glavas et al. (2001; T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342) among others.

[0118] 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 arrays to establish and then follow expression profiles over time. In addition, arrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug.

[0119] Assays Using Antibodies

[0120] 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.

[0121] The antibodies may be used with or without modification, and labeled by joining them, either covalently or noncovalently, with a labeling moiety.

[0122] Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include ELISA, RIA, fluorescent activated cell sorting (FACS) and arrays. 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).

[0123] Therapeutics

[0124] 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(3-4):184-192; Weiss 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 Pharmacolo, Vol. 40), Academic Press, San Diego Calif.).

[0125] 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 5 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.

[0126] Molecules which regulate the activity of the cDNA or encoded protein are useful as therapeutics for vascular disorders such as atherosclerosis, diabetic retinopathy, neoplasms, and obesity. 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. 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 (Mack Publishing, Easton Pa.).

[0127] Model Systems

[0128] 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.

[0129] Transgenic Animal Models

[0130] 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.

[0131] Embryonic Stem Cells

[0132] 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.

[0133] Knockout Analysis

[0134] 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.

[0135] Knockin Analysis

[0136] 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.

[0137] 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.

[0138] 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.

EXAMPLES

[0139] I Construction of cDNA Libraries

[0140] 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 (Invitrogen). 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.

[0141] 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.).

[0142] 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 (Invitrogen) 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 (Invitrogen), 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 (Invitrogen).

[0143] In some cases, libraries were superinfected with a 5×excess 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, Nucleic Acids Res 19:1954), and Bonaldo et al. (1996, Genome Res 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, supra).

[0144] II Isolation and Sequencing of cDNA Clones

[0145] 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.

[0146] Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao (1994) Anal Biochem 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

[0147] cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the CATALYST 800 thermal cycler (ABI) 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 sequencing kits such as the PRISM BIGDYE cycle sequencing kit (ABI). Electrophoretic separation of cDNA sequencing reactions and detection of labeled cDNAs were carried out using the MEGABACE 1000 DNA sequencing system (APB); the PRISM 373 or 377 sequencing systems (ABI) in conjunction with standard 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).

[0148] III Extension of cDNA Sequences

[0149] 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 primer analysis software (Molecular Biology Insights, Cascade Colo.), 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.

[0150] 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.

[0151] 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 mmol of each primer, reaction buffer containing Mg2+, (NH4)2SO4, and β-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): 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.

[0152] The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN reagent (0.25% reagent in 1×TE, v/v; Molecular Probes) and 0.5 μl 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 μl to 10 μl 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.

[0153] 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/2×carbenicillin liquid media.

[0154] 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 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: 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 PRISM BIGDYE terminator cycle sequencing kit (ABI).

[0155] IV Assembly and Analysis of Sequences

[0156] The nucleic acid sequences of the cDNAs presented in the Sequence Listing may contain occasional sequencing errors and unidentified nucleotides (N) that reflect state-of-the-art technology at the time the cDNA was sequenced. Occasional sequencing errors and Ns may be resolved and SNPs verified either by resequencing the cDNA or using algorithms to compare multiple sequences; these techniques are well known to those skilled in the art who wish to practice the invention. The sequences may be analyzed using a variety of algorithms described in Ausubel (supra, unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).

[0157] 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 PROSITE databases (Bairoch et al. (1997) Nucleic Acids Res 25:217-221; Attwood et al. (1997) J Chem Inf Comput Sci 37:417-424).

[0158] 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 (1990, supra); Altschul (1993, supra); Karlin et al. (1988) Proc Natl Acad Sci 85:841-845), BLASTn (vers.1.4, WashU), and CROSSMATCH software (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 (Green, supra). Bins with several overlapping component sequences were assembled using DEEP PHRAP (Green, supra).

[0159] 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).

[0160] 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 1×10−8. (The “E-value” quantifies the statistical probability that a match between two sequences occurred by chance). The hits were subjected to frameshift FASTx versus GENPEPT (GenBank version 109). In this analysis, a homolog match was defined as having an E-value of 1×10−8. 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).

[0161] 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, supra), 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.

[0162] V Selection of Sequences, Microarray Preparation and Use

[0163] 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 HUMAN GENOME GEM series 1-5 microarrays (Incyte Genomics) contain 45,320 array elements which represent 22,632 annotated clusters and 22,688 unannotated clusters. For the UNIGEM series microarrays (Incyte Genomics), Incyte clones were mapped to non-redundant Unigene clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med 75:694-698), and the 5′ clone with the strongest BLAST alignment (at least 90% identity and 100 bp overlap) was chosen, verified, and used in the construction of the microarray. The UNIGEM V 2.0 microarray (Incyte Genomics) contains 8,502 array elements which represent 8,372 annotated genes and 130 unannotated clusters. Table 5 shows the GenBank annotations (where available) for SEQ ID NOs:1-292 of this invention as produced by BLAST analysis.

[0164] 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 μg. 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.

[0165] 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.

[0166] VI Preparation of Samples

[0167] Human coronary artery endothelial cells (HCAEC), human iliac artery endothelial cells (HIAEC), human pulmonary artery endothelial cells (HPAEC), human aortic endothelial cells (HAEC), human umbilical artery endothelial cells (HUAEC), human uterine myometium microvasculature (UtMVEC), human dermal microvasculature (HMVECD), and human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics Corporation (San Diego, Calif.). Cells were grown in EGM-2 medium at 37° C., 5% CO2 using procedures supplied by Clonetics. All cells were grown to approximately 85% confluency and harvested without treatment.

[0168] Isolation and Labeling of Sample cDNAs

[0169] Cells were harvested and lysed in 1 ml of TRIZOL reagent (5×106 cells/ml; Invitrogen). 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 16,000×g 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 16,000×g for 20 minutes at 4° C. The supernatant was removed, and the RNA pellet was washed with 1 ml of 70% ethanol, centrifuged at 16,000×g at 4° C., and resuspended in RNAse-free water. The concentration of the RNA was determined by measuring the optical density at 260 nm. 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. The pooled control was prepared using an equal amount of mRNA from HCAEC, HUAEC, UtMVEC, HIAEC, HMVECD, and HUVEC.

[0170] Each poly(A) RNA sample was reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oligo-d(T) primer (21mer), 1×first 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 ml volume containing 200 ng poly(A) RNA using the GEMBRIGHT kit (Incyte Genomics). Specific control poly(A) RNAs (YCFRO6, 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 mRNAs (YCFRO6, 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 rmRNAs (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. cDNAs were purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech). Cy3- and CyS-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 μl 5×SSC, 0.2% SDS.

[0171] VII Hybridization and Detection

[0172] Hybridization reactions contained 9 μl of sample mixture containing 0.2 μg each of Cy3-labeled pooled control and Cy5 labeled source-specific vascular endothelium cDNA synthesis products in 5×SSC, 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 μl of 5×SSC 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 (1×SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in high stringency wash buffer (0.1×SSC), and dried.

[0173] 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 CyS. The excitation laser light was focused on the microarray using a 20×microscope 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 cm×1.8 cm microarray used in the present example was scanned with a resolution of 20 micrometers.

[0174] 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.

[0175] 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.

[0176] The output of the photomultiplier tube was digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood, Mass.) 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.

[0177] 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 Genomics). Significance was defined as signal to background ratio exceeding 2× and area hybridization exceeding 40%.

[0178] VIII Data Analysis and Results

[0179] Array elements that exhibited at least 1.5-fold change in expression in one or more vascular endothelial sample, 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). Differential expression values were converted to log base 2 scale; negative values indicate higher expression in the specific endothelial sample relative to the pooled control. Expression in large arteries (HCAEC, HIAEC, HPAEC, HAEC) was compared with expression in microvasculature (UtMVEC, HMVECD). The clones representing cDNAs that are expressed at higher levels in microvasculature are shown in Tables 1 and 2. The clones representing cDNAs that are expressed at higher levels in large artery endothelium are shown in Table 3.

[0180] IX Further Characterization of Differentially Expressed cDNAs and Proteins

[0181] 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. Table 4 provides a map between the clones present on microarrays and identified in Tables 1, 2, and 3, and the cDNAs identified in the Sequence Listing. 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. Table 5 shows the annotations provided by BLAST analysis for each cDNA of the present invention.

[0182] Percent sequence identity can be determined electronically for two or more amino acid or nucleic acid sequences using the MEGALIGN program, a component of LASERGENE software (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.

[0183] 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 (supra); Attwood (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.

[0184] 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 conserved 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: >1300=strong, 1000-1300=suggestive; for global similarity were: p<exp-3; and for strength (degree of correlation) were: >1300=strong, 1000-1300=weak.

[0185] X Other Hybridization Technologies and Analyses

[0186] 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.

[0187] 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 2×SSC for 10 min each. The membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene).

[0188] 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 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above.

[0189] 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 μl 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 [32P]dCTP is added to the tube, and the contents are incubated at 37° C. for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100° C. for five min and then snap cooled for two min on ice.

[0190] Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and 1×high phosphate buffer (0.5 M NaCl, 0.1 M Na2HPO4, 5 mM EDTA, pH 7) at 55° C. for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 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.

[0191] XI Expression of the Encoded Protein

[0192] 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.

[0193] 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 Spodoptera 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.

[0194] Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of transcription. 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 acid peptide, is purified using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.).

[0195] XII Production of Specific Antibodies

[0196] 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 μg 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.

[0197] In another approach, the amino acid sequence translated from a cDNA of the invention is analyzed using PROTEAN software (DNASTAR) to determine antigenic determinants 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 431 peptide synthesizer (ABI) 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-terminus 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.

[0198] 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.

[0199] 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 M−1, preferably 109 to 1010 M−1 or stronger, are made by procedures well known in the art.

[0200] XIII Purification of Naturally Occurring Protein Using Specific Antibodies

[0201] Naturally occurring or recombinant protein is 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.

[0202] XIV Screening Molecules for Specific Binding With the cDNA or Protein

[0203] 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 U.S. Pat. No. 5,876,946.

[0204] 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
								1368	1497	1569
CloneID	HAEC	HCAEC	HPAEC	HIAEC	UtMVEC	HMVECD	HUAEC	HUVEC	HUVEC	HUVEC
311197	ND	0.28	ND	0.12	0.15	−1.43	0.18	−0.11	0.04	0.16
2586533	ND	0.03	ND	ND	−0.17	−0.66	−0.05	ND	ND	ND
2539263	−0.35	−0.25	−0.49	−0.10	−0.48	−0.65	−0.54	ND	−0.48	−0.14
2228990	ND	0.16	ND	−0.04	−0.50	−0.61	−0.12	−0.25	0.09	0.09
3126622	ND	−0.50	ND	0.23	0.26	−0.64	−0.17	−0.16	0.19	0.14
1612384	ND	−0.38	ND	−0.36	ND	−0.60	ND	ND	ND	−0.38
924936	ND	0.03	ND	−0.28	0.17	−0.62	0.36	−0.26	ND	−0.06
1655416	ND	ND	ND	ND	ND	−1.25	ND	ND	ND	ND
1746640	ND	−0.13	ND	−0.18	−0.18	−0.79	−0.27	−0.12	−0.14	−0.19
2325661	−0.07	−0.24	−0.08	0.05	−0.16	−0.59	−0.08	ND	0.15	−0.06
2373263	ND	−0.33	ND	−0.26	ND	−0.59	ND	ND	ND	−0.17
2510835	ND	−0.28	ND	−0.26	−0.35	−0.78	−0.19	−0.12	−0.18	−0.05
3206154	ND	−0.49	ND	0.10	−0.19	−0.59	−0.33	−0.31	−0.32	0.10
3249851	ND	0.02	ND	ND	ND	−0.63	ND	ND	ND	ND
3600367	ND	ND	ND	ND	ND	−0.61	ND	ND	ND	ND
3891867	ND	−0.01	ND	−0.23	0.03	−0.59	−0.06	ND	−0.22	−0.26
3972234	−0.21	−0.22	−0.24	−0.08	−0.14	−0.61	−0.33	ND	−0.35	−0.25
3973887	ND	−0.09	ND	−0.01	−0.02	−0.61	−0.06	−0.08	−0.17	0.03
4514944	ND	0.38	ND	0.40	−0.42	−0.89	0.02	0.07	0.06	−0.02
797406	ND	−0.23	ND	−0.10	−0.08	−0.59	−0.40	0.00	0.02	−0.13
1655734	ND	ND	ND	ND	ND	−0.82	ND	ND	ND	ND
1817646	ND	−0.16	ND	0.14	0.19	−0.71	0.09	0.25	0.34	0.19
2278772	ND	−0.29	ND	−0.46	−0.13	−0.70	−0.30	ND	−0.41	−0.12
2739076	ND	−0.44	ND	−0.30	−0.61	−0.71	−0.52	−0.35	ND	−0.46
2828159	ND	0.01	ND	−0.02	−0.04	−0.65	−0.07	0.02	−0.16	0.05
3068978	ND	−0.15	ND	−0.49	−0.16	−0.69	−0.17	ND	−0.03	0.20
3172265	ND	0.57	ND	−0.12	0.29	−0.72	0.77	ND	0.29	0.53
3639751	−0.02	−0.17	0.21	0.22	0.29	−0.60	0.31	ND	0.45	0.31
3734094	ND	−0.38	ND	−0.24	−0.49	−0.60	−0.46	−0.07	ND	−0.47
3804843	ND	−0.36	ND	−0.19	−0.11	−0.58	−0.34	−0.28	−0.24	−0.09
4092750	ND	0.04	ND	ND	0.09	−0.65	0.08	−0.05	0.02	ND
4175323	ND	−0.21	ND	0.11	−0.42	−0.66	−0.21	−0.30	ND	−0.25
4380064	ND	−0.33	ND	−0.09	−0.05	−0.68	−0.22	ND	−0.18	−0.06
4413637	ND	0.23	ND	0.33	0.44	−0.69	0.19	−0.08	0.31	0.49
4543123	ND	−0.43	ND	−0.01	−0.30	−0.64	−0.16	ND	−0.42	−0.04
4571104	ND	−0.31	ND	0.10	−0.45	−0.58	−0.39	−0.27	−0.39	−0.11
4853673	ND	−0.11	ND	0.15	−0.03	−0.66	−0.12	0.07	−0.02	ND
5271674	ND	−0.19	ND	−0.14	−0.07	−0.68	−0.08	ND	−0.13	−0.03
5293028	ND	−0.31	ND	0.22	0.07	−0.69	0.32	ND	−0.21	0.47
5509328	−0.17	0.48	0.09	0.32	−0.54	−0.81	−0.22	ND	−0.21	−0.37
5682290	−0.03	0.12	0.32	0.05	−0.03	−0.97	0.11	ND	0.30	0.19
5903108	−0.25	−0.30	−0.43	−0.11	−0.26	−0.62	−0.29	ND	−0.30	−0.20
2717435	ND	−0.46	ND	−0.25	−0.60	0.29	−0.10	0.29	−0.05	−0.09
5013541	ND	−0.06	ND	0.01	−0.67	−0.01	−0.01	ND	−0.03	−0.04
1405844	ND	0.64	ND	−0.31	−0.62	−0.44	−0.40	−0.58	−0.41	−0.38
1624102	−0.05	−0.18	−0.25	−0.01	−0.63	0.22	−0.29	ND	−0.15	−0.17
1889028	ND	−0.40	ND	−0.34	−0.74	−0.04	−0.27	−0.24	−0.29	−0.22
2175008	ND	−0.47	ND	−0.50	−0.94	0.13	−0.28	−0.08	ND	−0.45
4252862	ND	−0.11	ND	−0.21	−0.75	−0.08	−0.21	ND	−0.06	−0.08
4521868	ND	−0.18	ND	−0.20	−0.69	−0.06	−0.32	ND	−0.26	−0.16
682741	ND	0.25	ND	0.01	−0.77	−0.35	−0.26	−0.30	ND	−0.40
2042056	ND	0.07	ND	−0.04	−0.58	−0.20	−0.31	−0.42	−0.42	−0.38
2416447	ND	−0.16	ND	−0.24	−0.76	−0.53	0.00	−0.26	0.18	−0.06
2462908	ND	−0.26	ND	−0.22	−0.58	0.28	−0.23	−0.08	−0.35	−0.11
2727104	0.09	0.11	0.36	0.01	−0.85	0.06	−0.07	ND	0.14	0.11
2739076	ND	−0.44	ND	−0.30	−0.61	−0.71	−0.52	−0.35	ND	−0.46
3426776	ND	ND	ND	ND	−0.67	−0.43	ND	ND	ND	ND
3961930	ND	0.04	ND	−0.07	−0.96	−0.02	0.05	ND	0.00	0.05
3970808	ND	−0.06	ND	−0.04	−0.86	0.10	0.03	ND	0.10	0.02
3982273	ND	0.06	ND	0.05	−0.67	0.05	0.17	ND	0.11	0.14
4090358	ND	0.10	ND	0.11	−0.68	0.05	0.16	ND	0.07	0.00
4146967	−0.04	0.06	0.14	0.12	−0.78	0.07	−0.14	ND	ND	0.10
4185527	ND	−0.09	ND	0.02	−1.39	0.03	−0.05	ND	−0.10	−0.06
4219295	ND	−0.22	ND	−0.27	−1.35	−0.12	−0.28	ND	−0.09	−0.16
4306221	ND	−0.04	ND	−0.20	−0.59	−0.02	−0.09	ND	−0.06	−0.05
4334474	ND	0.14	ND	0.27	−1.63	−0.18	0.15	ND	0.06	0.22
4383888	ND	−0.06	ND	−0.12	−1.27	0.23	−0.01	ND	0.06	−0.04
4420064	ND	−0.04	ND	−0.03	−0.60	0.18	0.05	ND	0.02	0.07
4422135	ND	−0.01	ND	0.01	−0.60	0.17	−0.02	ND	−0.01	0.07
4508211	ND	−0.05	ND	−0.07	−0.97	0.14	−0.10	ND	0.09	0.00
4533406	ND	−0.14	ND	0.00	−0.73	0.05	−0.13	ND	0.07	0.12
4561939	ND	−0.10	ND	0.06	−1.49	0.21	−0.07	ND	−0.08	0.06
4606503	ND	0.00	ND	−0.01	−1.48	0.07	0.04	ND	0.05	0.06
4633555	ND	0.17	ND	0.15	−1.42	0.01	0.09	ND	0.08	0.03
4636285	ND	−0.06	ND	0.03	−0.97	0.12	0.08	ND	0.08	0.09
4639612	ND	0.08	ND	0.02	−1.25	0.06	0.13	ND	0.12	0.13
4704961	ND	0.02	ND	0.11	−1.14	0.07	−0.04	ND	0.02	0.01
4710591	ND	−0.03	ND	0.05	−1.53	0.13	−0.05	ND	−0.17	0.00
4718317	ND	0.08	ND	0.06	−1.04	−0.08	0.12	ND	0.05	−0.02
4766304	ND	−0.04	ND	−0.03	−0.62	0.14	0.11	ND	−0.02	−0.04
4771265	ND	−0.11	ND	0.07	−0.69	0.07	0.03	ND	−0.03	−0.02
4860482	ND	−0.02	ND	0.02	−0.73	0.07	−0.06	ND	−0.03	0.00
4875773	ND	−0.03	ND	0.14	−0.92	0.04	0.02	ND	0.09	−0.01
4886749	ND	0.09	ND	0.04	−1.02	0.07	0.15	ND	0.07	0.08
5024011	ND	−0.10	ND	0.02	−0.79	0.05	−0.18	ND	−0.09	−0.06
5069809	ND	−0.01	ND	−0.03	−1.10	−0.01	0.00	ND	0.12	0.08
5094869	ND	−0.12	ND	0.03	−0.73	0.18	−0.04	ND	0.11	−0.01
5099959	ND	−0.04	ND	−0.21	−0.73	−0.03	−0.17	ND	−0.04	−0.13
5219277	ND	0.17	ND	0.06	−1.10	0.18	0.07	ND	0.13	0.16
5262750	ND	0.13	ND	0.12	−0.83	−0.09	0.08	ND	0.06	−0.03
5265974	ND	−0.12	ND	−0.07	−1.67	0.04	−0.03	ND	0.01	0.06
5407110	ND	ND	ND	ND	−1.24	ND	0.12	ND	ND	0.20
5508135	ND	0.13	ND	0.05	−1.15	−0.17	0.20	ND	−0.09	−0.02
5639879	−0.42	−0.47	−0.28	−0.43	−0.64	0.07	−0.18	ND	−0.30	−0.23
5680271	ND	−0.11	ND	−0.10	−1.78	0.04	−0.16	ND	−0.11	−0.08

[0205]

	TABLE 2
		avg log2 ratio	avg log2 ratio	Δ MV and
	Clone ID	MV	non-MV	non-MV
	1624543	−1.36	0.88	−2.25
	2527665	−1.17	0.29	−1.46
	2069806	0.22	1.63	−1.41
	5407110	−1.24	0.16	−1.40
	3180888	−0.74	0.55	−1.29
	2862539	−0.84	0.39	−1.22
	1558081	−0.96	0.24	−1.20
	3428945	−0.77	0.37	−1.14
	4175376	−0.51	0.57	−1.08
	4334474	−0.90	0.14	−1.05
	1555545	−0.50	0.46	−0.96
	392380	−0.10	0.80	−0.90
	302934	−0.16	0.73	−0.89
	1921574	−0.54	0.35	−0.88
	1963963	−0.48	0.39	−0.87
	4737158	−0.36	0.51	−0.86
	2656707	−0.76	0.08	−0.85
	211389	−0.06	0.77	−0.82
	2009822	0.03	0.85	−0.81
	3328081	−0.34	0.47	−0.81
	4633555	−0.71	0.09	−0.80
	1938951	−0.10	0.70	−0.80
	2955269	−0.68	0.11	−0.80
	5265974	−0.81	−0.02	−0.79
	3172586	−0.69	0.08	−0.78
	275587	0.00	0.77	−0.78
	3205003	−0.48	0.28	−0.76
	148825	0.00	0.76	−0.76
	1959179	−0.52	0.24	−0.76
	3735389	−0.69	0.06	−0.76
	4514944	−0.65	0.10	−0.75
	3172265	−0.21	0.54	−0.75
	5680271	−0.87	−0.12	−0.75
	311197	−0.64	0.11	−0.75
	4606503	−0.70	0.04	−0.74
	263340	0.00	0.74	−0.74
	1887959	−0.88	−0.15	−0.73
	289407	0.06	0.79	−0.73
	318182	−0.12	0.61	−0.73
	1909038	−0.44	0.29	−0.73
	2005968	−0.04	0.69	−0.73
	5026408	−0.65	0.08	−0.72
	341884	−0.08	0.65	−0.72
	351475	0.02	0.74	−0.72
	680	0.01	0.73	−0.72
	2007151	0.04	0.76	−0.72
	1938947	0.01	0.73	−0.72
	82954	−0.03	0.69	−0.72
	58000	−0.05	0.67	−0.72
	5508135	−0.66	0.06	−0.71
	44877	−0.10	0.61	−0.71
	4639612	−0.60	0.11	−0.71
	3182642	−0.35	0.35	−0.70
	508113	0.03	0.72	−0.69
	387305	−0.15	0.54	−0.69
	1447055	−0.34	0.34	−0.69
	3519816	−0.31	0.38	−0.68
	419316	0.08	0.75	−0.67
	5047895	−0.83	−0.16	−0.67
	320332	−0.21	0.46	−0.67
	499848	−0.04	0.63	−0.67
	5501816	−0.58	0.10	−0.67
	5682290	−0.50	0.17	−0.67
	1358185	−0.35	0.32	−0.67
	219488	−0.04	0.63	−0.67
	2137464	−0.36	0.31	−0.67
	2137041	−0.37	0.29	−0.66
	96425	−0.60	0.06	−0.66
	3249851	−0.63	0.02	−0.65
	345378	0.06	0.71	−0.65
	385550	−0.04	0.61	−0.65
	349854	0.07	0.71	−0.65
	42920	−0.20	0.44	−0.65
	2776	−0.02	0.63	−0.64
	238793	−0.07	0.57	−0.64
	1599036	−0.33	0.31	−0.64
	2924347	−0.39	0.24	−0.64
	465496	0.07	0.71	−0.64
	284124	−0.02	0.62	−0.64
	464323	0.01	0.65	−0.64
	4710591	−0.70	−0.06	−0.63
	388931	0.09	0.72	−0.63
	3086929	−0.50	0.13	−0.63
	2011839	0.04	0.67	−0.63
	1987738	−0.43	0.19	−0.62
	75549	0.02	0.65	−0.62
	4718317	−0.56	0.06	−0.62
	1995380	−0.26	0.36	−0.62
	4386789	−0.11	0.50	−0.61
	408522	0.04	0.65	−0.61
	5509328	−0.67	−0.07	−0.61
	274627	0.03	0.64	−0.61
	4185527	−0.68	−0.07	−0.61
	414891	0.00	0.61	−0.61
	5069809	−0.55	0.05	−0.60
	278282	−0.02	0.58	−0.60
	9079	0.00	0.61	−0.60
	241643	−0.02	0.58	−0.60
	44525	0.01	0.60	−0.60
	4561939	−0.64	−0.05	−0.60

[0206]

TABLE 3
								1368	1497	1569
CloneID	HAEC	HCAEC	HPAEC	HIAEC	UtMVEC	HMVECD	HUAEC	HUVEC	HUVEC	HUVEC
4398915	−0.82	−0.51	−0.23	−0.52	0.09	−0.42	0.23	ND	−0.02	0.38
2947513	−0.58	−0.43	−0.30	−0.13	−0.15	−0.29	−0.20	ND	−0.46	−0.15
2152363	−0.71	−0.42	−0.21	−0.33	−0.13	−0.44	−0.43	ND	−0.27	−0.23
5680843	−0.65	−0.35	−0.24	−0.26	0.05	−0.17	−0.23	ND	−0.32	0.01
3099768	−0.74	−0.30	0.14	−0.08	−0.11	−0.29	−0.19	ND	−0.21	−0.16
5911570	−0.63	−0.24	−0.23	−0.29	0.15	−0.01	−0.11	ND	−0.20	0.07
5902267	−1.00	−0.22	−0.37	−0.12	−0.15	−0.51	−0.65	ND	−0.41	−0.44
4557571	−0.73	−0.22	−0.38	−0.29	−0.17	−0.44	−0.32	ND	−0.27	−0.52
5911592	−0.87	−0.16	−0.47	−0.17	−0.36	−0.45	−0.86	ND	−0.32	−0.30
5519430	−0.62	−0.03	−0.29	−0.21	0.01	0.07	−0.02	ND	−0.22	−0.07
1969756	−0.60	−0.02	0.51	0.16	0.04	−0.18	0.10	ND	0.04	0.14
1512990	−0.60	0.02	0.72	0.15	−0.01	−0.01	0.16	ND	−0.04	−0.03
4739765	−0.71	0.07	−0.22	0.15	0.03	−0.33	−0.23	ND	−0.24	−0.14
5046165	−0.70	0.14	−0.10	0.16	0.20	−0.08	0.31	ND	−0.30	−0.04
2580580	−0.62	0.20	0.66	0.15	0.12	−0.02	0.10	ND	0.08	0.13
760553	−0.90	0.23	−0.55	0.34	−0.56	−0.20	−0.35	ND	−0.43	−0.20
4295711	−0.68	0.28	1.07	0.11	0.12	0.02	0.11	ND	−0.08	0.15
431455	−0.58	0.42	1.01	0.29	0.07	0.18	0.27	ND	0.35	0.37
3142396	−0.85	0.55	−0.13	0.64	−0.21	−0.11	−0.07	ND	−0.04	−0.21
3129691	−0.96	0.66	−0.24	0.74	−0.33	0.10	0.04	ND	0.10	−0.26
2962167	ND	−0.83	ND	−0.41	−0.21	0.04	−0.30	0.36	ND	0.56
5587406	ND	−0.79	ND	−0.10	0.35	0.10	−0.72	0.10	−0.03	0.07
2966620	ND	−0.76	ND	−0.40	−0.17	−0.35	0.60	−0.26	ND	−0.19
2895245	ND	−0.70	ND	−0.43	−0.41	−0.13	−0.54	0.28	ND	0.15
1648251	ND	−0.70	ND	−0.58	−0.11	−0.30	−0.67	−0.43	ND	−0.54
1601323	ND	−0.70	ND	−0.41	−0.45	−0.04	−0.55	−0.55	ND	−0.48
2047549	ND	−0.68	ND	−0.54	0.07	−0.32	−0.38	0.00	−0.01	0.08
1624459	ND	−0.68	ND	−0.36	−0.49	−0.17	−0.53	0.22	0.00	0.29
2235515	ND	−0.68	ND	−0.42	0.27	0.13	−0.16	0.00	0.27	0.29
2771046	ND	−0.68	ND	−0.24	0.18	0.01	−0.05	0.89	ND	0.32
3658034	ND	−0.67	ND	−0.51	−0.06	0.01	0.13	0.15	0.38	0.71
2722128	ND	−0.66	ND	−0.50	−0.17	−0.45	−0.14	0.34	ND	0.08
2845102	ND	−0.66	ND	0.01	−0.21	−0.28	0.06	0.27	ND	0.57
1271606	ND	−0.66	ND	−0.42	−0.19	−0.45	−0.53	−0.13	ND	−0.56
2151054	ND	−0.65	ND	ND	ND	ND	ND	ND	ND	ND
1600586	ND	−0.63	ND	−0.51	−0.27	−0.25	−0.42	−0.23	ND	−0.28
2740187	ND	−0.61	ND	−0.04	0.05	−0.21	−0.17	0.25	ND	−0.02
4462162	ND	−0.61	ND	−0.56	0.18	−0.02	−0.78	−0.26	ND	−0.52
1510714	ND	−0.61	ND	0.08	0.21	−0.09	−0.30	−0.54	ND	−0.48
2134968	ND	−0.60	ND	−0.42	−0.42	−0.04	−0.34	−0.17	ND	−0.20
2200604	ND	−0.60	ND	−0.44	−0.25	−0.10	−0.31	0.02	ND	−0.02
2657738	ND	−0.60	ND	−0.31	−0.38	−0.20	−0.34	−0.26	ND	−0.30
1559731	ND	−0.60	ND	−0.48	0.06	−0.22	−0.31	0.18	ND	0.09
3136864	ND	−0.59	ND	−0.45	−0.32	−0.39	−0.51	−0.19	ND	−0.15
5198974	ND	−0.59	ND	0.07	−0.10	−0.06	−0.10	−0.11	−0.19	0.03
2765165	ND	−0.59	ND	−0.44	−0.25	−0.15	−0.49	ND	−0.27	−0.33
1481225	ND	−0.59	ND	−0.25	−0.29	0.10	−0.17	0.08	ND	0.27
3088261	ND	−0.59	ND	−0.47	0.02	−0.18	−0.19	−0.04	−0.26	−0.16
1518328	ND	−0.59	ND	−0.50	0.68	0.06	−0.45	0.31	ND	0.23
1619865	ND	−0.58	ND	−0.18	0.02	−0.57	0.81	−0.21	0.21	0.11
1551767	ND	−0.58	ND	−0.43	−0.28	0.47	−0.58	−0.05	ND	−0.43
1498947	ND	−1.11	ND	−0.63	0.24	0.16	0.03	0.27	0.44	0.35
3316684	ND	−0.95	ND	−0.71	0.33	−0.25	0.67	−0.24	−0.15	−0.28
1617218	ND	−0.91	ND	−0.87	1.22	−0.21	0.15	ND	0.35	0.25
4144001	ND	−0.83	ND	−0.83	0.95	−0.19	−0.01	0.13	0.23	0.14
2657149	ND	−0.75	ND	−0.60	0.28	0.13	−0.03	0.24	ND	0.49
2455118	ND	−0.72	ND	−0.59	−0.29	−0.44	−0.31	−0.11	ND	−0.38
2174441	ND	−0.69	ND	−0.69	0.70	−0.10	−0.61	−0.47	ND	−0.23
1997915	ND	−0.68	ND	−0.65	−0.18	−0.48	−0.31	0.27	−0.02	−0.07
1357231	ND	−0.65	ND	−0.70	−0.04	−0.07	−0.35	ND	0.41	0.15
698665	ND	−0.65	ND	−0.76	−0.52	0.53	−0.87	−0.51	ND	−0.42
4295364	ND	−0.62	ND	−0.63	−0.29	−0.29	−0.49	0.04	ND	−0.28
1909266	ND	−0.59	ND	−0.62	−0.12	−0.28	−0.44	−0.23	ND	−0.32
3132987	ND	−0.56	ND	−0.70	0.18	−0.31	−0.25	0.30	ND	−0.23
1886842	ND	−0.55	ND	−0.65	−0.33	−0.47	−0.55	−0.48	ND	−0.36
4144001	ND	−0.54	ND	−0.61	0.97	−0.01	0.05	0.14	ND	0.28
3596853	ND	−0.53	ND	−0.60	−0.01	−0.21	−0.29	0.30	ND	0.24
3393606	ND	−0.53	ND	−0.67	0.22	0.36	0.08	0.67	0.63	0.44
693783	ND	−0.52	ND	−0.58	0.64	−0.52	−0.04	ND	−0.40	−0.56
2470485	ND	−0.46	ND	−0.77	0.09	−0.01	−0.10	0.06	ND	0.22
3081067	ND	−0.45	ND	−0.62	0.21	0.18	−0.11	0.55	ND	0.34
1313183	ND	−0.43	ND	−0.63	0.48	−0.20	0.82	−0.17	ND	−0.29
1979792	ND	−0.37	ND	−0.66	−0.28	−0.50	−0.56	−0.46	ND	−0.33
494970	ND	−0.33	ND	−0.60	0.03	0.16	−0.21	ND	0.06	0.07
3030988	ND	−0.28	ND	−0.62	−0.19	−0.22	−0.47	−0.28	ND	−0.26
2453436	ND	−0.06	ND	−0.60	−0.29	0.07	−0.58	−0.25	ND	−0.12
129009	ND	0.05	ND	−0.60	−0.05	−0.13	−0.34	0.64	ND	0.12
2220338	ND	0.05	ND	−0.75	−0.20	−0.12	−0.52	−0.26	ND	0.05
1581451	−0.51	−0.29	−0.79	0.05	0.03	−0.05	0.41	ND	0.38	0.21
3598343	0.04	−0.06	−0.68	−0.14	−0.34	−0.14	−0.18	ND	−0.20	−0.26

[0207]

TABLE 4
SEQ ID NO	Template ID	Clone ID	Start	Stop
1	1383156.23	311197	36	607
2	1097334.8	2586533	533	940
3	1330210.32	2539263	1	521
4	1330210.33	2539263	1043	1568
5	978840.1	2228990	15	1740
6	1850670CB1	3126622	115	2046
7	427830.26	1612384	371	802
8	002838.45	924936	113	534
8	002838.45	3891867	74	536
9	002838.44	924936	226	924
10	1451265CB1	1655416	250	1459
11	347284.6	1746640	2375	2776
12	255966.9	2325661	1472	1734
13	406387.7	2373263	5565	6689
14	062199.7	2510835	630	3421
15	1383059.61	3206154	737	1305
16	332595.5	3249851	160	733
17	332595.12	3249851	4134	4685
18	5401223CB1	3600367	4817	5226
19	002838.40	3891867	346	659
20	378186.30	3972234	109	638
20	378186.30	5903108	231	506
21	348196.83	3973887	1	321
22	348196.89	3973887	5446	5720
23	1136856.23	4514944	564	1065
24	1136856.25	4514944	1198	1615
25	101575.1	797406	853	1279
26	276310.1	1655734	1	275
27	014284CB1	1817646	1360	1854
28	272599.1	2278772	1	278
29	1383712.24	2739076	183	657
30	1820861CB1	2828159	18	439
31	330902.2	2828159	723	1137
32	211881.1	3068978	1	548
33	399161.1	3172265	473	1102
34	138206.1	3639751	1	806
35	1241528.18	3734094	228	801
36	1091353.11	3804843	−132	287
37	405058.1	4092750	2916	3391
38	406573.9	4175323	60	247
39	3365683CB1	4380064	132	462
40	406992.1	4413637	875	1787
41	399626.1	4543123	−1	813
42	474725.3	4571104	1	257
43	402460.13	4853673	1164	1687
44	038033.1	5271674	28	619
45	208075.1	5293028	417	737
46	3479061CB1	5509328	395	1113
47	1454465.1	5682290	88	600
48	378186.29	5903108	51	592
49	330836.4	2717435	2752	3160
50	330836.5	2717435	1929	2391
51	410882.6	5013541	1082	1848
52	1405844CB1	1405844	72	424
53	330933.15	1624102	4491	4895
54	979182.3	1889028	1751	2236
55	1382838.129	2175008	4210	4704
56	898864.15	4252862	1	300
57	033527.1	4521868	896	1523
58	351122.2	682741	879	1348
59	522433CB1	2042056	4	384
60	081187.1	2416447	1	64
61	2462908CB1	2462908	0	1028
62	350416.3	2462908	1453	1650
63	1061366.15	2727104	29	462
64	026200.1	3426776	1	388
65	214744.1	3961930	1	291
66	214811.1	3970808	297	516
67	107572.1	3982273	1	614
68	215003.1	4090358	−82	473
69	106250.1	4146967	1	544
70	403668.1	4185527	144	694
71	011664.1	4219295	54	201
72	059849.1	4306221	858	1191
73	1197030.23	4334474	766	1514
74	042195.1	4383888	1	256
75	983907.1	4420064	1420	1861
76	210826.1	4422135	1778	2267
77	343356.1	4508211	97	461
78	1439948.1	4533406	1	546
79	984996.3	4561939	13	552
80	102564.1	4606503	121	520
81	146768.1	4633555	241	747
82	340291.1	4636285	1	405
83	134460.1	4639612	499	924
84	053411.1	4704961	80	571
85	1448208.1	4710591	256	742
86	070655.1	4718317	97	490
87	406389.1	4766304	697	1495
88	167601.1	4771265	172	449
89	341084.1	4860482	1	231
90	404224.1	4875773	807	1490
91	026596.1	4886749	1	499
92	1041038.1	5024011	1	601
93	080588.1	5069809	1	206
94	014408.2	5094869	633	989
95	351752.1	5099959	853	1138
96	002661.1	5219277	38	833
97	399162.11	5262750	502	746
98	174086.1	5265974	219	542
99	116731.1	5407110	96	365
100	1453971.4	5508135	1819	2037
101	347635.1	5639879	258	785
102	1401326.2	5680271	−2	1079
103	1330204.51	1624543	750	1807
104	1327351.31	2527665	1	345
105	1327351.35	2527665	943	1530
106	332723.6	2069806	1883	2175
107	1184503.1	3180888	45	567
108	410610.1	2862539	502	771
109	986073.1	1558081	1571	3089
110	1102234.29	3428945	1	1199
111	1102234.28	3428945	698	1194
112	029061.1	4175376	176	1158
113	233180.39	1555545	889	1940
114	010317.1	392380	252	635
115	2007614CB1	302934	2163	2527
116	065582.1	1921574	1	492
117	1188409.1	1963963	5567	6043
117	1188409.15	1963963	1936	2307
119	1102234.75	4737158	1	237
120	403534.6	2656707	3273	4071
121	238349.4	211389	3334	3488
122	017058.1	2009822	216	477
123	190188CB1	3328081	6981	7353
124	1383106.7	1938951	2878	3117
125	1510895.1	2955269	843	1348
126	1454873.5	3172586	683	1217
127	222836.2	275587	21	410
128	3098081CB1	3205003	687	1188
129	1383398.6	148825	67	612
130	1383398.7	148825	173	979
131	198862.10	1959179	669	1046
132	2515360CB1	3735389	26	474
132	2515360CB1	1887959	4110	4667
133	334180.6	263340	1556	2800
134	982535.2	289407	748	1100
135	1501794.11	318182	3742	4129
136	983910CB1	1909038	399	849
137	999097.1	2005968	902	1069
138	293858.32	5026408	1	561
139	1269631CB1	341884	5433	6307
140	337208.11	351475	1327	2388
141	337800.3	680	1875	2134
142	1361343CB1	2007151	3511	3706
143	1504867.1	1938947	290	440
144	410917.13	82954	1480	1625
145	2080904CB1	58000	676	927
146	2520680CB1	44877	1312	1482
147	1274560.12	3182642	20	1177
148	313149.1	508113	1567	1968
149	268514.25	387305	1522	1901
150	2100016CB1	1447055	17	735
151	2906265CB1	3519816	1	464
152	189155.2	419316	1042	1239
153	336733.9	5047895	1	419
154	1273136.1	320332	1083	1471
155	253888.1	499848	1444	1851
156	407570.3	5501816	1094	1274
157	977136.8	1358185	1	425
158	977136.1	1358185	2274	2803
159	1500085.3	219488	203	564
160	1439712.1	2137464	1	630
161	015508.1	2137041	23	414
162	111552CB1	96425	1391	1645
163	1641902CB1	345378	1011	1676
164	252999.1	385550	874	1379
165	977391.15	349854	1740	2103
166	1363208CB1	42920	2573	2827
167	002776CB1	2776	1604	1896
168	140557.23	238793	2888	3029
169	140557.22	238793	4820	4961
170	1286746CB1	1599036	391	1072
171	1102234.13	2924347	286	808
172	1401226.4	465496	321	558
173	331677.2	284124	4201	4705
174	234870.4	464323	2255	2659
175	346051.5	3086929	474	954
176	232055.1	2011839	720	859
177	070248.106	1987738	38	901
178	028253.6	75549	329	508
179	989381.1	1995380	86	951
180	984009.1	4386789	130	1807
181	404758.7	408522	469	653
182	208350.1	274627	2871	3129
183	334193.1	414891	1242	1602
184	335046.8	278282	63	429
185	198947.2	9079	1153	1406
186	014669.1	241643	994	1143
187	2951807CB1	44525	451	683
188	482452.13	388931	1	434
189	2513610CB1	4398915	39	675
190	1376812.4	2947513	7	338
191	1446355.3	2152363	345	805
192	1446355.2	2152363	757	1138
193	365121.1	5680843	708	841
194	1399068.1	3099768	1	367
195	474395.1	5911570	0	588
196	001300.10	5902267	19	429
197	482193.2	4557571	1	294
198	450887.1	5911592	−30	569
199	1281959CB1	5519430	31	316
200	197856.4	1969756	1514	1888
201	197856.9	1969756	756	966
202	1088680.3	1512990	2186	2790
203	246020.6	4739765	529	882
204	1446842.1	5046165	1	502
205	2580580CB1	2580580	520	1281
206	1503093.1	760553	503	965
207	338662.1	4295711	1	1043
208	324037.1	431455	610	1245
209	989010.3	3142396	1	780
210	470784CB1	3142396	16	704
211	989010.5	3129691	279	1000
212	473040.1	2962167	953	4477
213	400203.1	5587406	1234	1598
214	474437.17	2966620	744	934
215	1345561CB1	2895245	486	1988
216	1513913CB1	1648251	761	1296
217	1453827.5	1601323	1028	1456
218	199471.2	2047549	149	1452
219	000278CB1	1624459	39	1688
220	233942.1	2235515	128	1984
221	1136923.19	2771046	1585	2068
222	1136923.18	2771046	3424	3644
223	3658034CB1	3658034	33	1112
224	1450005.5	2722128	708	1186
225	1450005.6	2722128	1771	2258
226	464689.64	2845102	2180	4636
227	334388.7	1271606	227	714
228	994660.1	2151054	1090	1518
229	3147649CB1	1600586	3059	4037
230	2652949CB1	2740187	1	1692
231	350300.25	4462162	6221	6833
232	1273213CB1	1510714	416	1968
233	222242.35	2134968	1	1553
234	621792CB1	2200604	379	1464
235	148820CB1	2657738	1534	1980
236	1812221CB1	1559731	2850	4201
237	1504168.4	3136864	338	408
238	109925.11	5198974	171	442
239	1073168.8	2765165	786	4417
240	2239072CB1	1481225	730	2171
241	4767318CB1	3088261	1282	1562
242	1339068CB1	1518328	1597	1933
243	2966620CB1	1619865	84	275
244	1347119.1	1551767	771	3446
245	816792CB1	1498947	136	514
245	816792CB1	2657149	1379	1843
246	1092427.6	3316684	388	642
246	1092427.6	1313183	4362	5823
247	251793.1	1617218	16	651
248	1622869CB1	4144001	722	1109
249	2581158CB1	2455118	144	2070
250	2174441CB1	2174441	471	946
251	235191.3	1997915	12	473
252	235191.4	1997915	213	782
253	1867417CB1	1357231	3075	3752
254	1285632CB1	698665	1377	4119
255	336992.5	4295364	1431	3446
256	1909266CB1	1909266	23	556
257	1214953.16	3132987	−20	1965
258	1850092CB1	1886842	1589	2576
259	1555752CB1	3596853	27	3636
260	441283.5	3393606	5296	8423
261	4349106CB1	693783	344	1030
262	2700132CB1	2470485	412	985
263	441283.4	3081067	713	1208
264	2613976CB1	1979792	961	1906
265	337894.8	494970	1	458
266	1452351.17	3030988	586	1971
267	1754695CB1	2453436	10	1489
268	232888.4	129009	3844	5648
269	247817.4	2220338	3585	5794
270	365801.2	1581451	210	1205
271	1449816.11	3598343	1	535

[0208]

TABLE 5
SEQ ID
NO	Template ID	GenBank ID	E-value	Annotation
1	1383156.23	g262770	9.00E−47	type II surfactant protein C, type II SP-C [Oryctolagus cuniculus]
2	1097334.8	g4321787	1.00E−12	6-pyruvoyl-tetrahydropterin synthase
3	1330210.32	g10439446	0	FLJ22911 fis, clone KAT05860, highly similar to stimulatory regulatory component Gs of adenylyl
				cyclase
4	1330210.33	g31914	0	Human mRNA for coupling protein G(s) alpha subunit (alpha-S2)
5	978840.1	g3882333	5.00E−82	KIAA0806 protein
6	1850670CB1	g182606	0	Human factor I (C3b/C4b inactivator) mRNA, complete cds.
7	427830.26	g182967	0	Human GTP-binding protein superfamily, G-protein alpha-olf subunit (olfactory) mRNA, complete
				cds.
8	002838.45	g2326174	0	Human selenoprotein W (selW) mRNA, complete cds.
9	002838.44	g2326174	0	Human selenoprotein W (selW) mRNA, complete cds.
10	1451265CB1	g3025445	0	R32184_1
11	347284.6	g1575562	0	Human hematopoietic progenitor kinase (HPK1) mRNA, complete cds.
12	255966.9	g7020703	0	Human cDNA FLJ20533 fis, clone KAT10931.
13	406387.7	g5689439	0	KIAA1051 protein [Homo sapiens]
14	062199.7	g7320864	0	Human mRNA for putative integral membrane transporter protein (LC27 gene).
15	1383059.61	g186840	0	Human colin carcinoma laminin-binding protein mRNA, complete cds.
16	332595.5	g2979417	0	Human mRNA for PCDH7 (BH-Pcdh)a, complete cds.
17	332595.12	g2979419	0	Human mRNA for PCDH7 (BH-Pcdh)b, complete cds.
18	5401223CB1	g3021700	0	Human BAI3 mRNA, complete cds.
19	002838.40	g2384720	0	Human selenoprotein W (hSelW) mRNA, complete cds.
20	378186.30	g338446	0	Human ribosomal protein S16 mRNA, complete cds.
21	348196.83	g177208	0	Human 4F2 glycosylated heavy chain (4F2HC) antigen gene, exon 1 and 2.
22	348196.89	g1200185	0	Human U22 snoRNA host gene (UHG) gene, complete sequence.
23	1136856.23	g183994	0	Human heregulin-beta1 gene, complete cds.
24	1136856.25	g183998	0	Human heregulin-beta3 gene, complete cds.
25	101575.1	g34328	0	Human mRNA for lactate dehydrogenase B (LDH-B).
26	276310.1	g488074	4.00E−57	Human insulinoma-associated (IA-1) gene, partial cds.
27	014284CB1	g1006656	0	Human mRNA for cathepsin C.
28	272599.1			Incyte Unique
29	1383712.24	g292442	0	Human ribosomal protein S20 (RPS2O) mRNA, complete cds.
30	1820861CB1	g2580587	0	Human platelet activating receptor homolog (H963) mRNA, complete cds.
31	330902.2	g2580588	0	platelet activating receptor homolog [Homo sapiens]
32	211881.1	g340088	7.00E−15	Human small nuclear rna pseudogene (clone pul-1) and flanks.
33	399161.1	g36076	2.00E−37	Human gene possibly encoding U1 RNA.
34	138206.1			Incyte Unique
35	1241528.18	g388031	0	Human ribosomal protein L11 mRNA, complete cds.
36	1091353.11	g506338	5.00E−75	flavoprotein subunit of complex II [Homo sapiens]
37	405058.1	g30398	0	Human mRNA for D-1 dopamine receptor.
38	406573.9	g10433269	3.00E−60	Human cDNA FLJ11898 fis, clone HEMBA1007322.
39	3365683CB1	g34328	0	Human mRNA for lactate dehydrogenase B (LDH-B).
40	406992.1	g11493651	0	Human calcium channel blocker resistance protein CCBR1 mRNA, complete cds.
41	399626.1	g5668857	2.00E−53	Homo sapiens glutathione transferase A4 gene, exon 1.
42	474725.3	g10440217	1.00E−16	Human cDNA: FLJ23506 fis, clone LNG03055.
43	402460.13	g10438153	0	Human cDNA: FLJ21937 fis, clone HEP04432.
44	038033.1	g6807879	0	Human mRNA; cDNA DKFZp434C0326 (from clone DKFZp434C0326).
45	208075.1	g23914	1.00E−117	Human 7SK45 DNA.
46	3479061CB1	g1103903	0	Human spermidine/spermine N1-acetyltransferase (SSAT) gene, complete cds.
47	1454465.1	g5419854	0	Human mRNA; cDNA DKFZp566N043 (from clone DKFZp566N043).
48	378186.29	g338446	0	Human ribosomal protein S16 mRNA, complete cds.
49	330836.4	g11275977	0	Human NOTCH 2 (N2) mRNA, complete cds.
50	330836.5	g11527996	0	Human NOTCH2 protein (NOTCH2) mRNA, complete cds.
51	410882.6	g1674386	0	diabetes mellitus type I autoantigen
52	1405844CB1	g180925	0	Human CO-029.
53	330933.15	g6330032	0	Human mRNA for KIAA1147 protein, partial cds.
54	979182.3	g424133	0	Human connexin45 gene, complete cds.
55	1382838.129	g189208	0	Human nidogen mRNA, complete cds.
56	898864.15	g11691653	7.00E−10	Human PDX1 gene for lipoyl-containing component X, exons 1-11.
57	033527.1	g10434733	3.00E−86	FLJ12953 fis, clone NT2RP2005490, moderately similar to Mouse D3Mm3e mRNA.
58	351122.2	g183581	0	Human glycoprotein IIIa (ITGB3) gene, exon 15 and partial cds.
59	522433CB1	g1813326	0	Human mRNA for TGF-beta superfamily protein, complete cds.
60	081187.1	g219928	1.00E−19	Human midkine gene, complete cds.
61	2462908CB1	g7861556	0	Human mRNA for SIRP-b2, complete cds.
62	350416.3	g7861556	0	Human mRNA for SIRP-b2, complete cds.
63	1061366.15			Incyte Unique
64	026200.1			Incyte Unique
65	214744.1	g3978547	0	Human chromosome 3 subtelomeric region.
66	214811.1	g10433865	0	Human cDNA FLJ12399 fis, clone MAMMA1002780.
67	107572.1			Incyte Unique
68	215003.1			Incyte Unique
69	106250.1	g7768719	4.00E−10	Homo sapiens genomic DNA, chromosome 21q, section 63/105.
70	403668.1			Incyte Unique
71	011664.1	g35504	0	Human M gene for M1-type and M2-type pyruvate kinase.
72	059849.1			Incyte Unique
73	1197030.23	g36139	0	Human mRNA for ribosomal protein L7.
74	042195.1			Incyte Unique
75	983907.1			Incyte Unique
76	210826.1	g7768770	2.00E−12	Homo sapiens genomic DNA, chromosome 21q, section 97/105.
77	343356.1			Incyte Unique
78	1439948.1			Incyte Unique
79	984996.3			Incyte Unique
80	102564.1			Incyte Unique
81	146768.1			Incyte Unique
82	340291.1	g3387951	3.00E−10	Homo sapiens clone 24487 mRNA sequence.
83	134460.1	g5823146	3.00E−80	testis specific protein
84	053411.1			Incyte Unique
85	1448208.1			Incyte Unique
86	070655.1	g5926700	0	Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 12/20.
87	406389.1	g4150939	2.00E−33	GSG1 [Mus musculus]
88	167601.1			Incyte Unique
89	341084.1			Incyte Unique
90	404224.1			Incyte Unique
91	026596.1			Incyte Unique
92	1041038.1	g5042384	0	Human KVLQT1 gene.
93	080588.1			Incyte Unique
94	014408.2			Incyte Unique
95	351752.1	g10432680	0	Homo sapiens cDNA FLJ11425 fis, clone HEMBA1001051.
96	002661.1			Incyte Unique
97	399162.11	g7770237	5.00E−11	PRO2822 [Homo sapiens]
98	174086.1			Incyte Unique
99	116731.1			Incyte Unique
100	1453971.4	g5106994	9.00E−38	HSPC038 protein [Homo sapiens]
101	347635.1	g11275977	0	Human NOTCH 2 (N2) mRNA, complete cds.
102	1401326.2	g5926687	9.00E−09	Human genomic DNA, chromosome 3p21.3, clone: 603 to 320, anti-oncogene region, section 3/3.
103	1330204.51	g2554613	0	Human mRNA for RGS5, complete cds.
104	1327351.31	g34613	0	Human mRNA for matrix Gla protein.
105	1327351.35	g187590	0	Human matrix Gla protein (MGP) gene, complete cds.
106	332723.6	g10434712	0	Human cDNA FLJ12937 fis, clone NT2RP2005020.
107	1184503.1	g3002924	0	Human T-cell receptor beta chain (TCRBV13S1-TCRBJ2S1) mRNA, complete cds.
108	410610.1	g187590	0	Human matrix Gla protein (MGP) gene, complete cds.
109	986073.1	g180670	0	Human collagenase type IV mRNA, 3’ end.
110	1102234.29	g36760	0	Human mRNA fragment for T-cell receptor beta chain D-J-C (JUR-beta 2).
111	1102234.28	g36760	0	Human mRNA fragment for T-cell receptor beta chain D-J-C (JUR-beta 2).
112	029061.1			Incyte Unique
113	233180.39	g7770154	0	Human PRO1873 mRNA, complete cds.
114	010317.1			Incyte Unique
115	2007614CB1	g4151118	0	Human fertilin beta mRNA, complete cds.
116	065582.1			Incyte Unique
117	1188409.1	g818	0	protein-glutamine gamma-glutamyltransferase [Bos taurus]
118	1188409.15	g53667	0	biglycan (PGI) [Mus musculus]
119	1102234.75	g33543	0	Human mRNA for T-cell receptor V beta gene segment V-beta-6, clone IGRb11.
120	403534.6	g7243180	0	Human mRNA for KIAA1400 protein, partial cds.
121	238349.4	g6624095	1.00E−07	similar to Coch-5B2
122	017058.1	g8515813	0.0004	RSD-6
123	190188CB1	g29469	0	Human mRNA for basement membrane heparan sulfate proteoglycan.
124	1383106.7	g10437335	0	Human cDNA: FLJ21267 fis, clone COL01717.
125	1510895.1	g189519	5.00E−66	Human pro-alpha-1 (V) collagen mRNA, complete cds.
126	1454873.5	g339561	0	Human transforming growth factor-beta (tgf-beta) mRNA, complete cds.
127	222836.2	g35685	0	Human testis specific mRNA for protamine 1 (P1).
128	3098081CB1	g3970845	0	Human mRNA for RALDH2-T, complete cds.
129	1383398.6	g1165211	0	Human microfibril-associated glycoprotein-2 MAGP-2 mRNA, complete cds.
130	1383398.7	g1165211	0	Human microfibril-associated glycoprotein-2 MAGP-2 mRNA, complete cds.
131	198862.10	g1907077	0	Human mRNA for Pirin, isolate 17.
132	2515360CB1	g2370201	0	Human mRNA for procollagen alpha 2(V).
133	334180.6	g2224696	0	Human mRNA for KIAA0378 gene, partial cds.
134	982535.2	g2612787	0	Human P2X7 gene, exon 9-11.
135	1501794.11	g7672348	0	Human unknown mRNA.
136	983910CB1	g36760	0	Human mRNA fragment for T-cell receptor beta chain D-J-C (JUR-beta 2).
137	999097.1	g2781413	0	Human clone DT2P1G7 mRNA, CAG repeat region.
138	293858.32	g10439538	0	Human cDNA: FLJ22988 fis, clone KAT11812.
139	1269631CB1	g2654025	0	gp250 precursor [Mus musculus]
140	337208.11	g10439667	0	FLJ23092 fis, clone LNG07245
141	337800.3	g7959737	1.00E−177	PRO1037 [Homo sapiens]
142	1361343CB1	g3882220	0	Human mRNA for KIAA0750 protein, complete cds.
143	1504867.1	g10436845	0	Human cDNA: FLJ20887 fis, clone ADKA03276.
144	410917.13	g506431	0	lysosomal acid lipase [Homo sapiens]
145	2080904CB1	g10437138	0	Human cDNA: FLJ21110 fis, clone CAS05377.
146	2520680CB1	g2058395	0	Human adenovirus protein E3-14.7 k interacting protein 1 (FIP-1) mRNA, complete cds.
147	1274560.12	g36748	0	Human mRNA for T-cell specific protein.
148	313149.1	g10435386	0	FLJ13386 fis, clone PLACE1001104, weakly similar to MYOSIN HEAVY CHAIN,
				NON-MUSCLE.
149	268514.25	g1552523	0	Human ionotropic ATP receptor P2X5b mRNA, complete cds.
150	2100016CB1	g2565275	4.00E−48	Dim1p homolog [Homo sapiens]
151	2906265CB1	g338928	0	Human T-cell receptor active beta-chain V-D-J-beta-1.2-C-beta-1 (TCRB) mRNA, partial cds.
152	189155.2	g5931525	3.00E−12	Human genomic DNA, chromosome 22q11.2, Cat Eye Syndrome region
153	336733.9	g6049603	0	Human dickkopf-1 (DKK-1) mRNA, complete cds.
154	1273136.1	g9956031	0	Human clone CDABP0045 mRNA sequence.
155	253888.1	g6682959	0	nuclear receptor binding factor-2
156	407570.3	g11907922	0	Human enhancer of polycomb mRNA, complete cds.
157	977136.8	g1314305	0	Human channel-like integral membrane protein (AQP-1) mRNA, clone AQP-1-2344, partial cds.
158	977136.1	g1314305	0	Human channel-like integral membrane protein (AQP-1) mRNA, clone AQP-1-2344, partial cds.
159	1500085.3	g5410279	0	Human HSPCO34 protein mRNA, complete cds.
160	1439712.1	g10434784	0	FLJ12987 fis, clone NT2RP3000068, weakly similar to SON OF SEVENLESS PROTEIN
				HOMOLOG 1.
161	015508.1	g7243180	0	Human mRNA for KIAA1400 protein, partial cds.
162	111552CB1	g10439538	0	Human cDNA: FLJ22988 fis, clone KAT11812.
163	1641902CB1	g6093424	0	Human connexin 37 (GJA4) mRNA, complete cds.
164	252999.1	g10047170	0	Human mRNA for KIAA1553 protein, partial cds.
165	977391.15	g7019914	0	Human cDNA FLJ20061 fis, clone COL01383.
166	1363208CB1	g3283351	0	Human lens epithelium-derived growth factor mRNA, complete cds.
167	002776CB1	g5868895	0	Human BAG-family molecular chaperone regulator-4 mRNA, complete cds.
168	140557.23	g7020279	0	Human cDNA FLJ20287 fis, clone HEP04390.
169	140557.22	g3925386	0	Human inversin protein mRNA, complete cds.
170	1286746CB1	g35439	0	Human mRNA for protein gene product (PGP) 9.5.
171	1102234.13	g36172	0	Human rearranged T-cell receptor beta chain mRNA.
172	1401226.4	g7768753	3.00E−12	Homo sapiens genomic DNA, chromosome 21q, section 95/105.
173	331677.2	g190220	0	protein phosphatase 2A 130 kDa regulatory subunit [Homo sapiens]
174	234870.4	g11320875	3.00E−79	cdk-binding protein
175	346051.5	g532677	0	Human mRNA for ribonuclease A (RNase A), complete cds.
176	232055.1			Incyte Unique
177	070248.106	g7959920	0	Human PRO2646 mRNA, complete cds.
178	028253.6	g4096056	8.00E−84	R28379_1
179	989381.1	g183655	0	Human glutathione S-transferase mRNA, complete cds.
180	984009.1	g863029	0	Human high-mobility group phosphoprotein isoform I-C (HMGIC) gene, exon 5.
181	404758.7	g7684246	0	Human mRNA for organic anion transporter OATP-D, complete cds.
182	208350.1	g10434511	0	Homo sapiens cDNA FLJ12807 fis, clone NT2RP2002316.
183	334193.1			Incyte Unique
184	335046.8	g940201	0	Human clone LS637A M73 hypoxanthine phosphoribosyltransferase (hprt) deletion mutant mRNA,
				partial cd
185	198947.2	g10435130	0	Human cDNA FLJ13209 fis, clone NT2RP4000424.
186	014669.1			Incyte Unique
187	2951807CB1	g4589683	0	Human mRNA for KIAA1017 protein, complete cds.
188	482452.13	g180055	0	Human thymocyte antigen CD1b mRNA, complete cds.
189	2513610CB1	g2088550	0	Human hereditary haemochromatosis region
190	1376812.4	g339840	0	Human triosephosphate isomerase mRNA, complete cds.
191	1446355.3	g8919392	0	Human mRNA full length insert cDNA clone EUROIMAGE 781354.
192	1446355.2	g8919392	0	Human mRNA full length insert cDNA clone EUROIMAGE 781354.
193	365121.1	g10439528	0	FLJ22979 fis, clone KAT11379, highly similar to HUMTUBAK Human alpha-tubulin mRNA.
194	1399068.1	g23925	2.00E−41	Human mRNA for 7SL RNA pseudogene.
195	474395.1	g496886	8.00E−60	Human mRNA for beta tubulin, clone nuk_278.
196	001300.10	g6457274	7.00E−12	putative E1-E2 ATPase
197	482193.2	g5926699	2.00E−86	Human genomic DNA, chromosome 6p21.3, HLA Class I region, section 11/20.
198	450887.1	g7629994	4.00E−33	60S RIBOSOMAL PROTEIN L36 homolog
199	1281959CB1	g1805279	0	Human alpha II spectrin mRNA, complete cds.
200	197856.4	g3947687	0	Human mRNA for Sec24 protein (Sec24A isoform), partial.
201	197856.9	g6807899	0	Human mRNA; cDNA DKFZp434C1526 (from clone DKFZp434C1526).
202	1088680.3	g10434855	0	Human cDNA FLJ13032 fis, clone NT2RP3001120, moderately similar to ZINC FINGER
				PROTEIN 136.
203	246020.6	g5689566	0	Human mRNA for KIAA1115 protein, complete cds.
204	1446842.1	g7293054	3.00E−26	Top1 gene product
205	2580580CB1	g9957753	0	Human kidney-specific membrane protein NX-17 mRNA, complete cds.
206	1503093.1	g207500	4.00E−07	alpha-tropomyosin 2 [Rattus norvegicus]
207	338662.1	g804810	1.00E−08	unknown protein [Rattus norvegicus]
208	324037.1	g5689412	0	Human mRNA for KIAA1038 protein, partial cds.
209	989010.3	g34513	0	Human mRNA for monocyte chemoattractant protein 1 (MCP-1).
210	470784CB1	g240867	0	monocyte chemoattractant protein-1 [Human, mRNA, 739 nt].
211	989010.5	g240867	0	monocyte chemoattractant protein-1 [Human, mRNA, 739 nt].
212	473040.1	g10442025	0	seladin-1 [Homo sapiens]
213	400203.1	g4836418	0	Human endothelial lipase mRNA, complete cds.
214	474437.17	g339972	0	Human TRPM-2 mRNA, complete cds.
215	1345561CB1	g1698395	0	Human lanosterol 14-demethylase cytochrome P450 (CYP51) mRNA, complete cds.
216	1513913CB1	g1665722	0	Human mRNA for RPD3 protein, complete cds.
217	1453827.5	g1350551	0	Human mRNA for ceramide glucosyltransferase, complete cds.
218	199471.2	g2463195	0	Human mRNA for MAD2 protein.
219	000278CB1	g435676	0	Human mRNA for squalene synthase.
220	233942.1	g11934947	0	Human neuropilin 2 (NRP2) gene, complete cds, alternatively spliced.
221	1136923.19	g178535	0	Human aminopeptidase N/CD13 mRNA encoding aminopeptidase N, complete cds.
222	1136923.18	g178535	0	Human aminopeptidase N/CD13 mRNA encoding aminopeptidase N, complete cds.
223	3658034CB1	g3881654	2.00E−50	similarity to Pfam domain: PF00207 (Alpha-2-macroglobulin family), Score = 377.3,
				E-value = 1.1e−113, N = 1
224	1450005.5	g7271464	0	Human soluble neuropilin-1 mRNA,
				complete cds.
225	1450005.6	g2978559	0	Human vascular endothelial cell growth factor 165 receptor/neuropilin (VEGF165) mRNA,
				complete cds.
226	464689.64	g7415720	0	Human Scd mRNA for stearoyl-CoA desaturase, complete cds.
227	334388.7	g285944	0	Human mRNA for KIAA0104 gene, complete cds.
228	994660.1	g2326205	0	Human Rac3 (RAC3) mRNA, complete cds.
229	3147649CB1	g6006514	0	Human mRNA for SAP 130 spliceosomal protein.
230	2652949CB1	g1827451	0	Human mRNA for VRK2, complete cds.
231	350300.25	g3600027	0	Human dysferlin mRNA, complete cds.
232	1273213CB1	g34458	0	Human DNA for monoamine oxidase type A (14) (partial).
233	222242.35	g2228774	0	Human paraoxonase (PON2) mRNA, complete cds.
234	621792CB1	g10439430	0	FLJ22896 fis, clone KAT04996
235	148820CB1	g3417295	0	KIAA0220 [Homo sapiens]
236	1812221CB1	g307382	0	Human RNA helicase A mRNA, complete cds.
237	1504168.4	g403455	0	Human 26S protease (S4) regulatory subunit mRNA, complete cds.
238	109925.11	g188542	0	Human MIC2 mRNA, complete cds.
239	1073168.8	g5689450	0	Human mRNA for KIAA1057 protein, partial cds.
240	2239072CB1	g2696053	0	Human ADDL mRNA for adducin-like protein, complete cds.
241	4767318CB1	g7299015	0	CG9615 gene product
242	1339068CB1	g33943	0	Human mRNA for integrin alpha 6.
243	2966620CB1	g339972	0	Human TRPM-2 mRNA, complete cds.
244	1347119.1	g2370125	0	Human mRNA for ZNF185 gene.
245	816792CB1	g11934948	0	neuropilin-2a(17) [Homo sapiens]
246	1092427.6	g339547	0	Human transforming growth factor-beta 1 binding protein mRNA, complete cds.
247	251793.1	g5852061	0	Human partial mRNA for Claudin-11 protein (CLDN11 gene).
248	1622869CB1	g3283414	0	Human oligodendrocyte-specific protein (OSP) mRNA, complete cds.
249	2581158CB1	g35070	0	Human mRNA for NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase
				(EC 1.5.1.15).
250	2174441CB1	g2257932	0	Human angiopoietin-2 mRNA, complete cds.
251	235191.3	g6706799	6.00E−46	dJ447F3.2 (ubiquitin-conjugating enzyme E2 H10) [Homo sapiens]
252	235191.4	g2062372	0	Human cyclin-selective ubiquitin carrier protein mRNA, complete cds.
253	1867417CB1	g292829	0	Human DNA topoisomerase II (top2) mRNA, complete cds.
254	1285632CB1	g3342264	0	Na-K-Cl cotransporter [Rattus norvegicus]
255	336992.5	g339805	0	Human topoisomerase I mRNA, complete cds.
256	1909266CB1	g184564	0	Human transformation-sensitive protein (IEF SSP 3521) mRNA, complete cds.
257	1214953.16	g190126	0	Human mitochondrial matrix protein P1 (nuclear encoded) mRNA, complete cds.
258	1850092CB1	g339679	0	Human threonyl-tRNA synthetase mRNA, complete cds.
259	1555752CB1	g2992633	0	Human protein kinase mRNA, complete cds.
260	441283.5	g29864	0	Human CENP-E mRNA.
261	4349106CB1	g10439047	0	FLJ22600 fis, clone HSI04447, highly similar to zinc finger transcription factor GKLF mRNA.
262	2700132CB1	g415818	0	Human mki67a mRNA (long type) for antigen of monoclonal antibody Ki-67.
263	441283.4	g29864	0	Human CENP-E mRNA.
264	2613976CB1	g603954	0	Human mRNA for KIAA0098 protein, partial cds.
265	337894.8	g8489880	0	Human actin binding protein anillin mRNA, complete cds.
266	1452351.17	g5809677	0	Human sperm membrane protein BS-63 mRNA, complete cds.
267	1754695CB1	g35564	0	Human mRNA for purine nucleoside phosphorylase (PNP; EC 2.4.2.1).
268	232888.4	g292829	0	Human DNA topoisomerase II (top2) mRNA, complete cds.
269	247817.4	g2655411	0	Human KDR/flk-1 protein mRNA, complete cds.
270	365801.2	g32368	0	Human Hox1.8 gene for homeobox protein.
271	1449816.11	g1203968	0	Human chromosome X region from filamin (FLN) gene to glucose-6-phosphate dehydrogenase
				(G6PD) gene

[0209]

Claims

1. A combination comprising a plurality of cDNAs wherein the cDNAs are SEQ ID NOs:1-292 that are differentially expressed in vascular endothelium and the complements of SEQ ID NOs:1-292.

2. The combination of claim 1 wherein the cDNAs are SEQ ID NOs:1-209 that are expressed at a higher level in vascular endothelium from microvasculature and the complements of SEQ ID NOs:1-209.

3. The combination of claim 1, wherein the cDNAs are SEQ ID NOs:210-292 that are expressed at a higher level in vascular endothelium from non-microvasculature and the complements of SEQ ID NOs:210-292.

4. The combination of claim 1, wherein the vascular endothelium is from a subject with a vascular disorder.

5. The combination of claim 4, wherein the vascular disorder is selected from atherosclerosis, hemangioma, hemangioendothelioma, diabetic retinopathy, warts, pyogenic granulomas, Kaposi's sarcoma, scar keloids, allergic oedema, neoplasms, psoriasis, ulcers, follicular cysts, endometriosis, peritoneal sclerosis, and obesity.

6. The combination of claim 1, wherein the cDNAs are immobilized on a substrate.

7. A method for detecting differential expression of one or more cDNAs in a sample containing nucleic acids, the method comprising: a) hybridizing the substrate of claim 6 with nucleic acids of the sample, thereby forming one or more hybridization complexes; b) detecting the hybridization complexes; and c) comparing the hybridization complexes with those of a standard, wherein differences between the standard and sample hybridization complexes indicate differential expression of cDNAs in the sample.

8. The method of claim 7, where in the nucleic acids of the sample are amplified prior to hybridization.

9. The method of claim 7, wherein the sample is from a subject with a vascular disorder and comparison with a standard defines an early, mid, or late stage of that disease or monitors efficacy of a therapeutic regime.

10. A method of screening a plurality of molecules or compounds to identify a molecule or compound which specifically binds a cDNA, the method comprising: a) combining the combination of claim 1 with the plurality of molecules or compounds under conditions to allow specific binding; and b) detecting specific binding between each cDNA and at least one molecule or compound, thereby identifying a molecule or compound that specifically binds to each cDNA.

11. The method of claim 10 wherein the plurality of molecules or compounds are selected from DNA molecules, enhancers, mimetics, peptide nucleic acids, proteins, repressors, regulatory proteins, RNA molecules, and transcription factors.

12. An isolated cDNA selected from SEQ ID NOs:36, 51, 61, 83, 87, 110, 111, 136, 159, 198, and 244.

13. A vector containing the cDNA of claim 12.

14. A host cell containing the vector of claim 13.

15. A method for producing a protein, the method comprising the steps of: a) culturing the host cell of claim 14 under conditions for expression of protein; and b) recovering the protein from the host cell culture.

16. A protein or a portion thereof produced by the method of claim 15.

17. A method for using a protein to screen a plurality of molecules or compounds to identify at least one ligand which specifically binds the protein, the method comprising: a) combining the protein of claim 16 with the plurality of molecules or compounds under conditions to allow specific binding; and b) detecting specific binding between the protein and a molecule or compound, thereby identifying a ligand which specifically binds the protein.

18. The method of claim 17 wherein the plurality of molecules or compounds is selected from agonists, antagonists, antibodies, DNA molecules, small molecule drugs, immunoglobulins, inhibitors, mimetics, peptide nucleic acids, peptides, pharmaceutical agents, proteins, RNA molecules, and ribozymes.

19. A method of using a protein to produce an antibody which specifically binds the protein, the method comprising: a) immunizing an animal with the protein of claim 16 under conditions to elicit an antibody response; b) isolating animal antibodies; and c) screening the isolated antibodies with the protein, thereby identifying an antibody which specifically binds the protein.

20. An antibody produced by the method of claim 19.