Genes expressed in foam cell differentiation

TECHNICAL FIELD

[0001] The present invention relates to a plurality of polynucleotides which may be used in detecting genes modulated in human foam cells. In particular, the present invention provides for the use of these polynucleotides in the diagnosis of conditions, disorders, and diseases associated with atherosclerosis.

BACKGROUND OF THE INVENTION

[0002] Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common cause of death in industrialized nations. Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and provide care for this complex disease has not been achieved. Molecular characterization of growth and regression of atherosclerotic vascular lesions requires identification of the genes that contribute to features of the lesion including growth, stability, dissolution, rupture and, most lethally, induction of occlusive vessel thrombus.

[0003] An early step in the development of atherosclerosis is formation of the “fatty streak”. Lipoproteins, such as the cholesterol-rich low-density lipoprotein (LDL), accumulate in the extracellular space of the vascular intima, and undergo modification. Oxidation of LDL occurs most avidly in the sub-endothelial space where circulating antioxidant defenses are less effective. The degree of LDL oxidation affects its interaction with target cells. “Minimally oxidized” LDL (MM-LDL) is able to bind to LDL receptor but not to the oxidized LDL (Ox-LDL) or “scavenger” receptors that have been identified, including scavenger receptor types A and B, CD36, CD68/macrosialin and LOX-1 (Navab et al. (1994) Arterioscler Thromb Vasc Biol 16:831-842; Kodama et al. (1990) Nature 343:531-535; Acton et al. (1994) J Biol Chem 269:21003-21009; Endemann et al. (1993) J Biol Chem 268:11811-11816; Ramprasad et al. (1996) Proc Natl Acad Sci 92:14833-14838; Kataoka et al. (1999) Circulation 99:3110-3117). MM-LDL can increase the adherence and penetration of monocytes, stimulate the release of monocyte chemotactic protein 1 (MCP-1) by endothelial cells, and induce scavenger receptor A (SRA) and CD36 expression in macrophages (Cushing et al. (1990) Proc Natl Acad Sci 87:5134-5138; Yoshida et al. (1998) Arterioscler Thromb Vasc Biol 18:794-802; Steinberg (1997) J Biol Chem 272:20963-20966). SRA and the other scavenger receptors can bind Ox-LDL and enhance uptake of lipoprotein particles.

[0004] Mononuclear phagocytes enter the intima, differentiate into macrophages, and ingest modified bpids including Ox-LDL. In most cell types, cholesterol content is tightly controlled by feedback regulation of LDL receptors and biosynthetic enzymes (Brown and Goldstein (1986) Science 232:34-47). In imacrophages, however, the additional scavenger receptors lead to unregulated uptake of cholesterol (Brown and Goldstein (1983) Annu Rev Biochem 52:223-261) and accumulation of multiple intracellular lipid droplets producing “foam cell” phenotype. Cholesterol-engorged and dead macrophages contribute most of the mass of early “fatty streak” plaques and typical “advanced” lesions of diseased arteries. Numerous studies have described a variety of foam cell responses that contribute to growth and rupture of atherosclerotic vessel wall plaques. These responses include production of multiple growth factors and cytokie, which promote proliferation and adherence of neighboring cells; chemokines, which further attract circulating monocytes into the growing plaque; proteins, which cause remodeling of the extracellular matrix; and tissue factor, which can trigger thrombosis (Ross (1993) Nature 362:801-809; Quin et al. (1987) Proc Natl Acad Sci 84:2995-2998). Thus, cholesterol-loaded macrophages which occur in abundance in most stages of the atherosclerotic plaque formation contribute to inception of the atheroscerotic process and to eventual plaque rupture and occlusive thrombus.

[0005] During Ox-LDL uptake, macrophages produce cytokines and growth factors that elicit further cellular events that modulate atherogenesis such as smooth muscle cell proliferation and production of extracellular matrix. Additionally, these macrophages may activate genes involved in inflammation including inducible nitric oxide synthase. Thus, genes differentially expressed during foam cell formation may reasonably be expected to be markers of the atherosclerotic process.

[0006] The present invention provides a method of high-throughput screening using a plurality of probes and purified polynucleotides in a diagnostic context as markers of atherosclerosis and other cardiovascular disorders.

SUMMARY OF THE INVENTION

[0007] The present invention provides a composition comprising a plurality of polynucleotides differentially expressed in foam cell development selected from SEQ ID NOs:1-276 as presented in the Sequence Listing. In one embodiment, each polynucleotide is an early marker of foam cell formation and is either unregulated, SEQ ID NOs:1-55, or downregulated, SEQ ID NOs:171-196. In a second embodiment, each polynucleotide is differentially expressed greater than 3-fold and is either upregulated, SEQ ID NOs:47-67, or downregulated, SEQ ID NOs:194-213. Further, the invention encompasses complements of the polynucleotides and immobilization of the polynucleotides on a substrate.

[0008] The invention provides a high throughput method for detecting altered expression of one or more polynucleotides in a sample. The method comprises hybridizing the polynucleotide composition with the sample, thereby forming one or more hybridization complexes; detecting the hybridization complexes; and comparing the hybridization complexes with those of a standard, wherein each difference in the size and intensity of a hybridization complex indicates altered expression of a polynucleotide in the sample. The sample can be from a subject with atherosclerosis and comparison with a standard defines early, mid, and late stages of that disease.

[0009] The invention also provides a high throughput method of screening a library of molecules or compounds to identify a ligand. The method comprises combining the polynucleotide composition with a library of molecules or compounds under conditions to allow specific binding; and detecting specific binding, thereby identifying a ligand. Libraries of molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids (PNAs), mimetics, peptides, and proteins. The invention additionally provides a method for purifying a ligand, the method comprising combining a polynucleotide of the invention with a sample under conditions which allow specific binding, recovering the bound polynucleotide, and separating the polynucleotide from the ligand, thereby obtaining purified ligand.

[0010] The invention also provides a method of obtaining an extended or full length gene from a library of expressed or genomic nucleic acid sequences. The method comprises arranging individual library sequences on a substrate; hybridizing a polynucleotide selected from the Sequence Listing with the library sequences under conditions which allow specific binding; detecting hybridization between the polynucleotide and a sequence; and isolating the library sequence, thereby obtaining the extended or full length gene.

[0011] The present invention further provides a substantially purified polynucleotide selected from SEQ ID NOs:35-48, 68-80, 192,193, 214-224 as presented in the Sequence Listing. The invention also provides an expression vector containing the polynucleotide, a host cell containing the expression vector, and a method for producing a protein comprising culturing the host cell under conditions for the expression of protein and recovering the protein from the host cell culture.

[0012] The present invention further provides a protein encoded by a polynucleotide of the invention. The invention also provides a high-throughput method for screening a library of molecules or compounds to identify at least one ligand which specifically binds the protein. The method comprises combining the protein or a portion thereof with the library of molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. Libraries of molecules or compounds are selected from DNA molecules, RNA molecules, PNAs, mimetic, peptides, proteins, agonists, antagonists, antibodies or their fragments, immunoglobulins, inhibitors, drug compounds, and pharmaceutical agents. The invention further provides for using a protein to purify a ligand. The method comprises combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and separating the protein from the ligand, thereby obtaining purified ligand. The invention also provides a pharmaceutical composition comprising the protein in conjunction with a pharmaceutical carrier and a purified antibody that specifically binds to the protein.

DESCRIPTION OF THE TABLES

[0013] A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimilereproduction 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.

[0014] The Sequence Listing is a compilation of polynucleotides obtained by sequencing clone inserts (isolates) of different cDNAs and identified by hybrid complex formation using the cDNAs as probes on a microarray. Each sequence is identified by a sequence identification number (SEQ ID NO) and by an Incyte ID number. The Incyte ID number represents the gene sequence that contains the clone insert.

[0015] Table 1 shows the differentially expressed genes associated with foam cell development identified by cluster analysis. Column 1 shows the SEQ ID NO, column 2 shows the Incyte ID number, and column 3 shows the gene annotation. Columns 4 through 10 show the normalized differential expression, and column 11 shows the cluster to which the gene was assigned.

[0016]
FIGS. 1A and 1B show graphs of the average normalized expression pattern over the time points for genes in each cluster. Clusters 1 through 4 contain genes which are up-regulated at days 1, 2, or 4. Clusters 5 and 6 contain genes that are down-regulated at later time points, and cluster 7 contains genes that are up-regulated at 8 hours.

[0017] Table 2 shows an identification map for each sequence. Column 1 shows the SEQ ID NO, and column 2 shows the Incyte ID number. Column 3 shows the Clone number of the Incyte clone represented on the UNIGEM V 2.0 microarray. Columns 4 and 5 show the START and STOP sites for the clone insert sequence relative to the gene sequence identified in column 2 and shown in the Sequence Listing.

[0018] Table 3 is a list of the genes that show differential expression early in foam cell differentiation. Column 1 shows the SEQ ID NO, column 2 shows the Incyte ID number, and column 3 shows the gene annotation. Columns 4 through 10 show the differential expression values for each time point. Columns 11 and 12 show the maximum change in expression up or down, respectively, over the time course. Column 12 shows the maximum difference seen over the time course.

[0019] Table 4 is a list of the genes that show greater than 3-fold differential expression during foam cell differentiation. Column 1 shows the SEQ ID NO, column 2 shows the Incyte ID number, and column 3 shows the gene annotation. Columns 4 through 10 show the differential expression values for each time point. Columns 11 and 12 show the maximum change in expression up or down, respectively, over the time course. Column 12 shows the maximum difference seen over the time course.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Before the nucleic acid sequences and methods are presented, it is to be understood that this invention is not limited to the particular machines, methods, and materials described. Although particular embodiments are described, machines, methods, and materials similar or equivalent to these embodiments may be used to practice the invention. The preferred machines, methods, and materials set forth are not intended to limit the scope of the invention which is limited only by the appended claims.

[0021] The singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. All technical and scientific terms have the meanings commonly understood by one of ordinary skill in the art. All publications are incorporated by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are presented and which might be used in connection with the invention. Nothing in the specification is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0022] Definitions

[0023] “Amplification” refers to the production of additional copies of a nucleotide sequence and is carried out using polymerase chain reaction (PCR) technologies well known in the art.

[0024] “Complementary” describes the relationship between two single-stranded nucleotide sequences that anneal by base-pairing (5′-A-G-T-3′ pairs with its complement 3′-T-C-A-5′).

[0025] “E-value” refers to the statistical probability that a match between two sequences occurred by chance.

[0026] “Derivative” refers to a polynucleotide or a polypeptide that has been subjected to a chemical modification. Illustrative of such modifications would be replacement of a hydrogen by, for example, an acetyl, acyl, alkyl, amino, formyl, or morpholino group. Derivative polynucleotides may encode polypeptides that retain the essential biological characteristics (such as catalytic and regulatory domains) of naturally occurring polypeptides.

[0027] “Fragment” refers to at least 18 consecutive nucleotides of a polynucleotide of the Sequence Listing or its complement. A “unique” fragment refers to at least 18 consecutive nucleotides of a particular polynucleotide or its complement that is specific to a polynucleotide of the Sequence Listing and that under hybridization conditions would not detect related polynucleotides in which it does not appear.

[0028] “Homology” refers to sequence similarity between a reference sequence and at least a fragment of a polynucleotide or a portion of a polypeptide.

[0029] “Hybridization complex” refers to a complex between two polynucleotides by virtue of the formation of hydrogen bonds between purines and pyrimidines.

[0030] “Immunological activity” is the capability of the natural, recombinant, or synthetic polypeptide or portion thereof to induce in an animal a specific immune response that results in the production of antibodies.

[0031] “Ligand” refers to any molecule, agent, or compound which will bind specifically to a complementary site on a polynucleotide or protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins of the invention and may be composed of at least one of the following: inorganic and organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids.

[0032] “Microarray” refers to an ordered arrangement of hybridizable elements on a substrate. The elements are arranged so that there are a “plurality” of elements, preferably more than one element, more preferably at least 100 elements, and even more preferably at least 1,000 elements, and most preferably at least 10,000 on a 1 cm2 substrate. The maximum number of elements is unlimited, but is at least 100,000 elements. Furthermore, the hybridization signal from each of the elements is individually distinguishable. In the present and preferred embodiment, the elements comprise polynucleotide probes.

[0033] “Modulates” refers to any change in activity (increased or decreased; biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule and a polynucleotide or polypeptide of the invention.

[0034] “Oligonucleotide” or “oligomer” refers to a nucleotide sequence of at least about 15 nucleotides to as many as about 60 nucleotides, preferably about 18 to 30 nucleotides, and most preferably about to 25 nucleotides that are used as a “primer” or “amplimer” in the polymerase chain reaction (PCR) or as an array element.

[0035] “Peptide nucleic acid” (PNA) refers to a DNA mimic in which nucleotide bases are attached to a pseudopeptide backbone to increase stability. PNAs, also designated antigene agents, can prevent gene expression by hybridizing to complementary messenger RNA.

[0036] “Polynucleotide” refers to an oligonucleotide, nucleotide sequence, nucleic acid molecule, DNA molecule, or any fragment or complement thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, coding and/or noncoding, an exon or an intron of a genomic DNA molecule, or combined with carbohydrate, lipids, protein or inorganic elements or substances.

[0037] “portion” refers to at least six contiguous amino acids of a polypeptide encoded by a polynucleotide of the Sequence Listing. A portion may represent an amino acid sequence that is conserved among related proteins (e.g., a catalytic domain such as a kinase domain).

[0038] “Post-translational modification” of a polypeptide may involve lipidation, glycosylation, phosphorylation, acetylation, racenlization, 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.

[0039] “Probe” refers to a polynucleotide or a fragment thereof that hybridizes to a nucleic acid molecule in a sample or on a substrate. A probe is used to detect, amplify, or quantify cDNAs, endogenous genes, or transcript mRNAs by employing conventional, molecular biology techniques. As used herein, probes are the reporter molecule of hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies.

[0040] “Protein” refers to a protein or any portion thereof including a polypeptide or an oligopeptide. A portion of a polypeptide generally retains biological or immunogenic characteristics of a native protein. An “oligopeptide” is an amino acid sequence of at least about 5 residues, more preferably 10 residues and most preferably about 15 residues that are immunogenic and are used as part of a fusion protein to produce an antibody.

[0041] “Purified” refers to polynucleotides, polypeptides, antibodies, and the like, that are isolated from at least one other component with which they are naturally associated.

[0042] “Sample” is used herein in its broadest sense. A sample containing polynucleotides, polypeptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.

[0043] “Specific binding” or “specifically binding” refers to the interaction between two molecules. In the case of a polynucleotide, specific binding may involve hydrogen bonding between sense and antisense strands or between one stand and a protein which affects its replication or transcription, intercalation of a molecule or compound into the major or minor groove of the DNA molecule, or interaction with at least one molecule which functions as a transcription factor, enhancer, repressor, and the like. In the case of a polypeptide, specific binding may involve interactions with polynucleotides, as described above or with molecules or compounds such as agonists, antibodies, antagonists, and the like. Specific binding is dependent upon the presence of structural features that allow appropriate chemical or molecular interactions between molecules.

[0044] “Substrate” refers to any rigid or semi-rigid support to which molecules or compounds 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.

[0045] The Invention

[0046] The present invention provides a composition comprising a plurality of polynucleotides, wherein each polynucleotide is differentially expressed in macrophages as they differentiate into foam cells. The plurality of polynucleotides comprise at least a fragment of the identified sequences, SEQ ID NOs:1-276, as presented in the Sequence Listing. Additionally, the invention provides a subset of polynucleotides whose expression is upregulated, SEQ ID NOs:1-55, or downregulated, SEQ ID NOs:171-196, early in foam cell formation. The invention also provides a subset of polynucleotides whose expression is upregulated, SEQ ID NOs:47-67, or downregulated, SEQ ID NOs:194-213, greater than 3-fold during foam cell formation. The invention also provides novel polynucleotides whose expression is upregulated, SEQ ID NOs:35-48 and 68-80, or downregulated, SEQ ID NOs:192, 193, and 214-222, during foam cell development.

[0047] Method for Selecting Polynucleotides

[0048] Human THP-1 cells (American Type Culture Collection, Manassas Va.) were grown in serum-containing medium and differentiated with 12-0-tetradecanoyl-phorbol-13-acetate (Research Biochemical International, Natick Mass.) for 24 hours. Cells were then cultured either in the presence or absence of Ox-LDL from time points ranging from 30 minutes to 4 days. Poly (A) RNA from cultured cells was prepared for expression profiling after 0, 0.5, 2.5, 8, 24, 48, and 96 hours exposure to Ox-LDL. Poly(A) RNA from experimental and control cells was labeled with separate fluorescent dyes and hybridized in time-matched pairs on UNIGEM V 2.0 arrays (Incyte Pharmaceuticals, Palo Alto Calif.).

[0049] Agglomerative cluster analysis was used to identify response patterns and to establish relationships between different gene expression profiles. Each gene measurement was normalized by dividing the expression ratios by the maximum value for each time series. The clustering process defined a hierarchical tree with the number of branches intersecting at each branch level of the tree equal to the number of clusters at that level. Division of the tree at branch level 5 divided the genes into 7 clusters of gene expression containing 276 differentially expressed genes and splice variants, SEQ ID NOs:1-276.

[0050] Table 1shows the differentially expressed genes and splice variants associated with foam cell development identified by cluster analysis. Column 1 shows the SEQ ID NO, column 2 shows the Incyte ID number, and column 3 shows the gene annotation. Columns 4 through 10 show the normalized differential expression; each gene has a maximum value of 1.0. The background shading indicates the relative expression in response to Ox-LDL; white represents relative expression ranging from 0-25% of maximum for that particular gene; light gray from 26-50%; dark gray from 51-75%; black from 76-100%. Column 11 shows the cluster to which the gene was assigned.

[0051]
FIG. 1 shows a graph of the average normalized expression pattern over the time points for all the genes in each cluster. Clusters 1 through 4 contain genes which are up-regulated at days 1, 2, or 4. Clusters 5 and 6 contain genes that are down-regulated at later time points, and cluster 7 contains genes that are up-regulated at 8 hours.

[0052] Table 2 shows an ID map for each SEQ ID NO. Column 1 shows the SEQ ID NO and column 2 shows the Incyte ID number. Column 3 shows the Clone number of the Incyte clone represented on the UNIGEM V 2.0 microarray. Columns 4 and 5 show the START and STOP sites for the clone insert sequence relative to the gene sequence identified in column 2.

[0053] Table 3 is a list of the genes that show differential expression early in foam cell differentiation. Column 1 shows the SEQ ID NO, column 2 shows the Incyte ID number, and column 3 shows the gene annotation. Columns 4 through 10 show the differential expression values for each time point. Values represent treated sample divided by time matched untreated sample. Columns 11 and 12 show the maximum change in expression up or down, respectively, over the time course. Column 12 shows the maximum difference seen over the time course.

[0054] Table 4 is a list of the genes that show greater than 3-fold differential expression during foam cell differentiation. Column 1 shows the SEQ ID NO, column 2. shows the Incyte ID number, and column 3 shows the gene annotation. Columns 4 through 10 show the differential expression values for each time point. Values represent treated sample divided by time matched untreated sample. Columns 11 and 12 show the maximum change in expression up or down, respectively, over the time course. Column 12 shows the maximum difference seen over the time course.

[0055] The polynucleotides of the invention can be genomic DNA, cDNA, mRNA, or any RNA-like or DNA-like material such as peptide nucleic acids, branched DNAs and the like. Polynucleotide probes can be sense or antisense strand. Where targets are double stranded, probes may be either sense or antisense strands. Where targets are single stranded, probes are complementary single strands. In one embodiment, polynucleotides are cDNAs. In another embodiment, polynucleotides are plasmids. In the case of plasmids, the sequence of interest is the cDNA insert.

[0056] Polynucleotides can be prepared by a variety of synthetic or enzymatic methods well known in the art. Polynucleotides 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, polynucleotides can be produced enzymatically or recombinantly, by in vitro or in vivo transcription.

[0057] Nucleotide analogs can be incorporated into polynucleotide probes by methods well known in the art. The only requirement is that the incorporated nucleotide analogs of the probe must base pair with target nucleotides. For example, certain guanine nucleotides can be substituted with hypoxanthine which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine. Alternatively, adenine nucleotides can be substituted with 2,6-diaminopurine which can form stronger base pairs with thymidine than those between adenine and thymidine. Additionally, polynucleotides can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups.

[0058] Polynucleotides 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 WO95/25 1116). Alternatively, the polynucleotides 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; incorporated herein by reference).

[0059] Complementary DNA (cDNA) can be arranged and then immobilized on a substrate. Polynucleotides can be immobilized by covalent means such as by chemical bonding procedures or UV irradiation. In one such method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another case, a cDNA probe is placed on a polylysine coated surface and then UV cross-linked as described by Shalon et al. (WO95/35505). In yet another method, a DNA is actively transported from a solution to a given position on a substrate by electrical means (Heller et al., supra). Alternatively, polynucleotides, clones, 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.

[0060] Furthermore, polynucleotides 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 probe. 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 polynucleotide.

[0061] Polynucleotides can be attached to a substrate by sequentially dispensing reagents for probe synthesis on the substrate surface or by dispensing preformed DNA fragments to the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions efficiently.

[0062] Use of the Polynucleotides

[0063] The polynucleotide of the present invention may be used for a variety of purposes. For example, the composition of the invention may be used as elements on a nucroarray. The microarray can be used in high-throughput methods such as for detecting a related polynucleotide in a sample, screening libraries of molecules or compounds to identify a ligand, or diagnosing a particular cardiovascular condition, disease, or disorder such as atherosclerosis. Alternatively, a polynucleotide complementary to a given sequence of the sequence listing can inhibit or inactivate a therapeutically relevant gene related to the polynucleotide.

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

[0065] Hybridization

[0066] The polynucleotides or fragments or complements thereof of the present invention may be used in various hybridization technologies. The polynucleotides may be naturally occurring, recombinant, or chemically synthesized; based on genomic or cDNA sequences; and labeled using a variety of reporter molecules by either PCR or enzymatic techniques. Commercial kits are available for labeling and cleanup of such polynucleotides or probes. Radioactive (Amersham Pharmacia Biotech), fluorescent (Operon Technologies, Alameda Calif.), and chemiluminescent labeling (Promega, Madison Wis.), are well known in the art. Alternatively, a polynucleotide is cloned into a commercially available vector, and probes are produced by transcription. The probe is synthesized and labeled by addition of an appropriate polymerase, such as T7 or SP6 polymerase, and at least one labeled nucleotide.

[0067] A probe may be designed or derived from unique regions of the polynucleotide, such as the 3′ untranslated region or from a conserved motif, and used in protocols to identify naturally occurring molecules encoding the same polypeptide, allelic variants, or related molecules. The probe may be DNA or RNA, is usually single stranded and should have at least 50% sequence identity to any of the nucleic acid sequences. The probe may comprise at least 18 contiguous nucleotides of a polynucleotide. Such a probe may be used under hybridization conditions that allow binding only to an identical sequence or under conditions that allow binding to a related sequence with at least one nucleotide substitution or deletion. Discovery of related sequences may also be accomplished using a pool of degenerate probes and appropriate hybridization conditions. Generally, a probe for use in Southern or northern hybridizations may be from about 400 to about 4000 nucleotides long. Such probes may be single-stranded or double-stranded and may have high binding specificity in solution-based or substrate-based hybridizations. A probe may also be an oligonucleotide that is used to detect a polynucleotide of the invention in a sample by PCR.

[0068] The stringency of hybridization is determined by G+C content of the probe, 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 permits 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 polynucleotides 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 (supra, pp. 6.11-6.19, 14.11-14.36, and A1-43).

[0069] Dot-blot, slot-blot, low density and high density arrays are prepared and analyzed using methods known in the art. Probes or array elements from about 18 consecutive nucleotides to about 5000 consecutive nucleotides are contemplated by the invention and used in array technologies. The preferred number of probes or array elements is at least about 40,000; a more preferred number is at least about 18,000; 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; PCT application WO95/11995; PCT application WO95/35505; U.S. Pat. Nos. 5,605,662; and 5,958,342.)

[0070] Screening Assays

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

[0072] In one embodiment, the polynucleotide of the invention may be incubated with a library of isolated and purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gel-retardation assay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional assay. In another embodiment, the polynucleotide may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the polynucleotide and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay.

[0073] In another embodiment, the polynucleotide may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the polynucleotide 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 polynucleotide. The molecule or compound which is bound to the polynucleotide may be released from the polynucleotide by increasing the salt concentration of the flow-through medium and collected.

[0074] Purification of Ligand

[0075] The polynucleotide or a fragment thereof may be used to purify a ligand from a sample. A method for using a mammalian polynucleotide or a fragment thereof to purify a ligand would involve combining the polynucleotide or a fragment thereof with a sample under conditions to allow specific binding, recovering the bound polynucleotide, and using an appropriate agent to separate the polynucleotide from the purified ligand.

[0076] Protein Production and Uses

[0077] The polynucleotides of this application or their full length cDNAs may be used to produce purified polypeptides using recombinant DNA technologies described herein and taught in Ausubel (supra; pp. 16.1-16.62). One of the advantages of producing polypeptides by these procedures is the ability to obtain highly-enriched sources of the polypeptides thereby simplifying purification procedures. The present invention also encompasses amino acid substitutions, deletions or insertions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Such substitutions may be conservative in nature when the substituted residue has structural or chemical properties similar to the original residue (e.g., replacement of leucine with isoleucine or valine) or they may be nonconservative when the replacement residue is radically different (e.g., a glycine replaced by a tryptophan). Computer programs included in LASERGENE software (DNASTAR, Madison Wis.), MACVECTOR software (Genetics Computer Group, Madison Wis.) and RasMol software (www.umass.edu/microbio/rasmol) may be used to help determine which and how many amino acid residues in a particular portion of the polypeptide may be substituted, inserted, or deleted without abolishing biological or immunological activity.

[0078] Expression of Encoded Proteins

[0079] Expression of a particular cDNA may be accomplished by cloning the cDNA into an appropriate vector and transforming this vector into an appropriate host cell. The cloning vector used for the construction of the human and rat cDNA libraries 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 an appropriate host strain of E. coli. Induction of the isolated bacterial strain with isopropyltliogalactoside (IPTG) using standard methods will produce a fusion protein that contains an N terminal metbionine, the first seven residues of β-galactosidase, about 15 residues of linker, and the polypeptide encoded by the cDNA.

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

[0081] Signal sequences that dictate secretion of soluble proteins are particularly desirable as component parts of a recombinant sequence. For example, a chimeric protein may be expressed that includes one or more additional purification-facilitating domains. Such domains include, but are not limited to, metal-chelating domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized inumunoglobulin, 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 polypeptide and the purification domain may also be used to recover the polypeptide.

[0082] Suitable expression hosts 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, yeast cells such as Saccharomyces cerevisiae, and bacteria such as E, coli. For each of these cell systems, a useful expression vector may also include an origin of replication and one or two selectable markers to allow selection in bacteria as well as in a transfected eukaryotic host. Vectors for use in eukaryotic expression hosts may require the addition of 3′ poly(A) tail if the polynucleotide lacks poly(A).

[0083] Additionally, the vector may contain promoters or enhancers that increase gene expression. Most promoters are host specific, and they include MMTV, SV40 or metallothionein promoters for CHO cells; trp, lac, tac or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase or PGH promoters for yeast. Adenoviral vectors with enhancers such as the rous sarcoma virus (RSV) enhancer or retroviral vectors with promoters such as the long terminal repeat (LTR) promoter may be used to drive protein expression in mammalian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of a secreted soluble polypeptide may be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art. An alternative method for the production of large amounts of secreted protein involves the transformation of mammalian embryos and the recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep, and the like.

[0084] In addition to recombinant production, polypeptides or portions thereof may be produced 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), manually, or using machines such as the ABI 431A Peptide synthesizer (PE Biosystems, Norwalk Conn.). Polypeptides produced by any of the above methods may be used as pharmaceutical compositions to treat disorders associated with underexpression.

[0085] Screening Assays

[0086] A protein or a portion thereof encoded by the polynucleotide may be used to screen libraries 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 polypeptide 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 polypeptide on their cell surface can be used in screening assays. The cells are screened against libraries or a plurality of ligands and the specificity of binding or formation of complexes between the expressed polypeptide and the ligand may be measured. The ligands may be DNA, RNA, or PNA molecules, agonists, antagonists, antibodies, immunoglobulin, inhibitors, peptides, pharmaceutical agents, proteins, drugs, or any other test molecule or compound that specifically binds the polypeptide. An exemplary assay involves combining the mammalian polypeptide or a portion thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound polypeptide to identify at least one ligand that specifically binds the polypeptide.

[0087] This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the polypeptide specifically compete with a test compound capable of binding to the polypeptide 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 mammalian model system to evaluate their toxicity, diagnostic, or therapeutic potential.

[0088] Purification of a Ligand

[0089] The polypeptide or a portion thereof may be used to purify a ligand from a sample. A method for using a mammalian polypeptide or a portion thereof to purify a ligand would involve combining the polypeptide or a portion thereof with a sample under conditions to allow specific binding, recovering the bound polypeptide, and using an appropriate chaotropic agent to separate the polypeptide from the purified ligand.

[0090] Production of Antibodies

[0091] A polypeptide encoded by a polynucleotide of the invention may be used to produce specific antibodies. Antibodies may be produced using an oligopeptide or a portion of the polypeptide with inherent immunological activity. Methods for producing antibodies include: 1) injecting an animal (usually goats, rabbits, or mice) with the polypeptide, or a portion or an oligopeptide thereof, to induce an immune response; 2) engineering hybridomas to produce monoclonal antibodies; 3) inducing in vivo production in the lymphocyte population; or 4) screening libraries of recombinant immunoglobulins. Recombinant immunoglobunns may be produced as taught in U.S. Pat. No. 4,816,567.

[0092] Antibodies produced using the polypeptides 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 polypeptide. A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies specific for polypeptides are well known in the art. immunoassays typically involve the formation of complexes between a polypeptide and its specific binding molecule or compound and the measurement of complex formation. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific polypeptide is preferred, but a competitive binding assay may also be employed

[0093] Immunoassay procedures may be used to quantify expression of the polypeptide in cell cultures, in subjects with a particular disorder or in model animal systems under various conditions. Increased or decreased production of polypeptides 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 polypeptide 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 polypeptide.

[0094] Labeling of Molecules for Assay

[0095] A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various polynucleotide, polypeptide or antibody arrays or assays. Synthesis of labeled molecules may be achieved using Promega or Amersham Pharmacia Biotech kits for incorporation of a labeled nucleotide such as 32p-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as 35S-methionine. Polynucleotides, polypeptides, 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.).

[0096] The polypeptides 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.

[0097] Diagnostics

[0098] The polynucleotides, or fragments thereof, may be used to detect and quantify altered gene expression; absence, presence, or excess expression of mRNAs; or to monitor mRNA levels during therapeutic intervention. Conditions, diseases or disorders associated with altered expression include atherosclerosis and associated complications. These polynucleotides can also be utilized as markers of treatment efficacy against the diseases noted above and other cardiovascular 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.

[0099] For example, the polynucleotide may be labeled by standard methods and added to a biological sample from a patient under conditions for the formation of hybridization complexes. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. If 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.

[0100] In order to provide a basis for the diagnosis of a condition, disease or disorder associated with gene expression, a normal or standard expression profile is established This may be accomplished by combining a biological sample taken from normal subjects, either animal or human, with a probe under conditions for hybridization or amplification Standard hybridization may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a substantially purified target sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular condition is used to diagnose that condition.

[0101] 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.

[0102] Gene Expression Profiles

[0103] A gene expression profile comprises a plurality of polynucleotides and a plurality of detectable hybridization complexes, wherein each complex is formed by hybridization of one or more probes to one or more complementary sequences in a sample. The polynucleotide composition of the invention is used as elements on a microarray to analyze gene expression profiles. In one embodiment, the microarray is used to monitor the progression of disease. Researchers can assess and catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic. The invention can be used to formulate a prognosis and to design a treatment regimen. The invention can also be used to monitor the efficacy of treatment. For treatments with known side effects, the microarray is employed to improve the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.

[0104] In another embodiment, animal models which mimic a human disease can be used to characterize expression profiles associated with a particular condition, disorder or disease or treatment of the condition, disorder or disease. Novel treatment regimens may be tested in these animal models using microarrays to establish and then follow expression profiles over time. In addition, microarrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug.

[0105] Assays Using Antibodies

[0106] Antibodies directed against epitopes on a protein encoded by a polynucleotide of the invention may be used in assays to quantify the amount of protein found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions. The antibodies may be used with or without modification, and labeled by joining them, either covalently or noncovalently, with a labeling moiety.

[0107] Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include ELISA, RIA, and fluorescent activated cell sorting (FACS). Such immunoassays typically involve the formation of complexes between the protein and its specific antibody and the measurement of such complexes. These and other assays are described in Pound (supra). The method may employ a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes, or a competitive binding assay. (See, e.g., Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience, New York N.Y.; Pound, supra)

[0108] Therapeutics

[0109] The polynucleotides of the present invention and fragments thereof can be used in gene therapy. Polynucleotides of the invention can be delivered to a target tissue, such as mononuclear phagocytes. Expression of the protein encoded by the polynucleotide may correct a disease state associated with reduction or loss of endogenous target protein. Polynucleotides may be delivered to specific cells in vitro. Transformed cells are transferred in vivo to various tissues. Alternatively, polynucleotides may be delivered in vivo. Polynucleotides are delivered to cells or tissues 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, artifical 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; August et al. (1997) Gene Theraov (Advances in Pharmacolog Vol. 40), Academic Press, San Diego Calif.).

[0110] In addition, expression of a particular protein can be modulated through the specific binding of an antisense polynucleotide sequence to a nucleic acid sequence which either encodes the protein or directs its expression. The antisense polynucleotide can be DNA, RNA, or nucleic acid mimics and analogs. The nucleic acid sequence can be cellular niRNA and/or genomic DNA and binding of the antisense sequence can affect translation and/or transcription, respectively. Antisense sequences can be delivered intracellularly using viral vectors or non-viral vectors as described above (Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics, Humana Press Inc., Totowa N.J.).

[0111] Both polynucleotides and antisense sequences can be produced ex vivo by using any of the ABI nucleic acid synthesizers or other automated systems known in the art. Polynucleotides and antisense sequences can also be produced biologically by transforming an appropriate host cell with an expression vector containing the sequence of interest.

[0112] Molecules which modulate the expression of a polynucleotide of the invention or activity of the encoded protein are useful as therapeutics for conditions and disorders associated with an immune response. 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 targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express the protein.

[0113] Additionally, any of the proteins or their ligands, or complementary nucleic acid sequences may be administered 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 may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton Pa.).

[0114] Model Systems

[0115] 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. Mannmals 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.

[0116] Transgenic Animal Models

[0117] 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. Nos. 5,175,383 and 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.

[0118] Embryonic Stem Cells

[0119] Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene. The vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.

[0120] ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types that differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes.

[0121] Knockout Analysis

[0122] 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. Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines that lack a functional copy of the mammalian gene.

[0123] Knockin Analysis

[0124] ES cells can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastulae and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of the analogous human condition These methods have been used to model several human diseases.

[0125] As described herein, the uses of the polynucleotides, provided in the Sequence Listing of this application, and their encoded polypeptides are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art. Furthermore, the polynucleotides 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.

[0126] It is to be understood that the 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

[0127] I. Construction of cDNA Libraries

[0128] RNA was purchased from Clontech Laboratories, Inc. (Palo Alto Calif.) or isolated from various tissues. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL reagent (Life Technologies, Rockville Md.). The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated with either isopropanol or ethanol and sodium acetate, or by other routine methods.

[0129] 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.).

[0130] 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 plasrid system (Life Technologies) using the recommended procedures or similar methods known in the art (See Ausubel, supra, Units 5.1 through 6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech, Piscataway N.J.) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of the PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), or PINCY plasmid (Incyte Pharmaceuticals). Recombinant plasmids were transformed into XL1-Blue, XL1-BlueMRF, or SOLR competent E. coli cells (Stratagene) or DH5α, DH10B, or ELECTROMAX DH10B competent E, coli cells (Life Technologies).

[0131] 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, Nucl Acids Res 19:1954), and Bonaldo et al. (1996, Genome Research 6:791-806). The modified Soares normalization procedure was utilized to reduce the repetitive cloning of highly expressed high abundance cDNAs while maintaining the overall sequence complexity of the library. Modification included significantly longer hybridization times which allowed for increased gene discovery rates by biasing the normalized libraries toward those infrequently expressed low-abundance cDNAs which are poorly represented in a standard transcript image (Soares et al., supra).

[0132] II. Isolation and Sequencing of cDNA Clones

[0133] 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 R.E.A.L. 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.

[0134] 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 singlereaction mixture. Samples wereprocessed 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 (absystems Oy, Helsinki, Finland).

[0135] cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 thermal cycler (PE Biosystems) or the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.) in conjunction with the HYDRA microdispenser (Robbins Scientific, Sunnyvale Calif.) or the MICROLAB 2200 system (Hamilton, Reno Nev.). cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE cycle sequencing kit (PE Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Pharmacia Biotech); the ABI PRISM 373 or 377 sequencing system (PE Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, supra, Unit 7.7).

[0136] III. Extension of cDNA Sequences

[0137] Nucleic acid sequences were extended using Incyte cDNA clones and oligonucleotide primers. One primer was synthesized to initiate 5′ extension of the known fragment, and the other, to initiate 3′ extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided

[0138] 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.

[0139] High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg+2, (NH4)2SO4, and β-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Pharmaceuticals): 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 mm; Step 7: storage at 4° C.

[0140] 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 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.

[0141] 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 (Amersham Pharmacia Biotech). 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 pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected 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.

[0142] The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) 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 (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE terminator cycle sequencing kit (PE Biosystems).

[0143] IV. Assembly and Analysis of Sequences

[0144] Component nucleotide sequences from chromatograms were subjected to PHRED analysis (Phil's Revised Editing Program; 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 Pharmaceuticals), 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).

[0145] 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 the Basic Local Alignment Search Tool (BLAST; Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol. 215:403-410; Karlin et al. (1988) Proc. Natl. Acad. Sci. 85:841-845), BLASTn (v.1.4, WashU), and CROSSMATCH software (Phil Green, supra). Candidate pairs were identified as all BLAST hits having a quality score greater than or equal to 150. Alignments of at least 82% local identity were accepted into the bin. The component sequences from each bin were assembled using PHRAP (Phil's Revised Alignment Program; Phil Green, supra). Bins with several overlapping component sequences were assembled using DEEP PHRAP (Phil Green, supra).

[0146] 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 Pharmaceuticals).

[0147] The assembled templates were annotated using the following procedure. Template sequences were analyzed using BLASTn (v2.0, NCBI) versus GBpri (GenBank version 109). “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 of 1×10−8. 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 “Database and System for Storing, Comparing and Displaying Related Biomolecular Sequence Information,” U.S. Ser. No. 09/276,534, filed Mar. 25, 1999, incorporated by reference herein, and the LIFESEQ GOLD user manual (Incyte Pharmaceuticals).

[0148] Following assembly, template sequences were subjected to motif, BLAST, Hidden Markov Model (HMM; Pearson and Lipman (1988) Proc Natl Acad Sci 85:2444-2448; Smith and Waterman (1981) J Mol Biol 147:195-197), and functional analyses, and categorized in protein hierarchies using methods described in “Database System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence Data,” U.S. Ser. No. 08/812,290, filed Mar. 6, 1997; “Relational Database for Storing Biomolecule Information,” U.S. Ser. No. 08/947,845, filed October 9, 1997; “Project-Based Full-Length Biomolecular Sequence Database,” U.S. Pat. No. 5,953,727;; and “Relational Database and System for Storing Information Relating to Biomolecular Sequences,” U.S. Ser. No. 09/034,807, filed Mar. 4, 1998, all of which are incorporated by reference herein. Template sequences may be further queried against public databases such as the GenBank rodent, mammalian, vertebrate, eukaryote, prokaryote, and human EST databases.

[0149] V. Preparation of Microarrays

[0150] The polynucleotides present on the human UNIGEM V 2.0 microarray (Incyte Pharmaceuticals) represent template sequences derived from the LIFESEQ GOLD assembled human sequence database (incyte Pharmaceuticals) based on a non-redundant set of gene-oriented clusters derived from IMAGE (integrated molecular analysis of genomes and their expression) cDNA library clones and derived ESTs in the gbEST database (National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md.). A single clone representing each particular template was used on the microarray. Polynucleotides were amplified from bacterial cells using primers complementary to vector sequences flanking the cDNA insert. Thirty cycles of PCR increased the initial quantity of polynucleotide from 1-2 ng to a final quantity greater than 5 μg. Amplified polynucleotides were then purified using SEPHACRYL400 columns (Amersham Pharmacia Biotech).

[0151] Purified polynucleotides 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 Corporation, West Chester Pa.), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma Aldrich, St. Louis Mo.) in 95% ethanol. Coated slides were cured in a 110° C. oven. polynucleotides were applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522, incorporated hereinby reference. One microliter of the polynucleotide 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 polynucleotide per slide.

[0152] 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.

[0153] VI. Preparation of Target Polynucleotides

[0154] Human THP-1 cells (American Type Culture Collection, Manassas Va.) were grown in RPMI1640 medium containing 10% fetal serum (v/v), 0.45% glucose (w/v), 10 mM Hepes, 1 mM sodium pyruvate, 1×10-−5 M β-mercaptoethanol, penicillin (100 units/ml) and streptomycin (100 mg/ml). For oxidized-LDL loading experiments, cells were seeded at a density of 1×106 cells/il in medium containing 12-0-tetradecanoyl-phorbol-13-acetate (Research Biochemical International, Natick Mass.) at 1×10−7 M for 24 hr. The medium was then replaced by culture medium with or without 100 μg/ml of CuSO4 “fully” oxidized LDL (Intracel, Rockville Md.) according to the method of Hammer et al. (1995; Arterio Thromb Vasc Biol 15:704-713). Medium was replaced every two days during the time of culture. Cells were treated with Ox-LDL over time points ranging from 30 minutes to 4 days. During this period, cells remained adherent and had a typical speckled Nile red staining pattern. RNA was prepared for expression profiling at 0, 0.5, 2.5, and 8 hours, and 1, 2, and 4 days of Ox-LDL exposure.

[0155] Total RNA was extracted using the RNA STAT-60 kit (Tel-Test, Friendswood Tex.). Poly(A) RNA was purified using the POLYATRACT mRNA isolation system (Promega). Each poly(A) RNA sample was reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oligo-dT 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 (Amersham Pharmacia Biotech). The reverse transcription reaction was performed in a 25 ml volume containing 200 ng poly(A) RNA using the GEMBRIGHT kit (Incyte Pharmaceuticals). Specific control poly(A) RNAs (YCFR06, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, control mRNAs (YCFR06, YCFR45, YCFR67, and YCFR85) at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse transcription reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100 (w/w) to sample mRNA, respectively. To sample differential expression patterns, control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were diluted into reverse transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA. Reactions were incubated at 37° C. for 2 hr, treated with 2.5 ml of 0.5M sodium hydroxide, and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA.

[0156] Probes were purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech). Cy3- and Cy5-labeled reaction samples were combined as described below and ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The probe was then dried to completion using a SpeedVAC system (Savant Instruments, Holbrook N.Y.) and resuspended in 14 μl 5×SSC/0.2% SDS.

[0157] VII. Hybridization and Detection

[0158] Hybridization reactions contained 9 μl of probe mixture consisting of 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products from pairs of matched time point experimental and control cells in 5×SSC, 0.2% SDS hybridization buffer. The target 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] The output of the photomultiplier tube was digitized using a 12-bit RTI-835H analog-to-digital (AID) 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.

[0163] 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 Pharmaceuticals).

[0164] VIII. Data Analysis and Results

[0165] An agglomerative cluster analysis was used to identify the typical response patterns and establish the relationships between the different gene expression profiles. Each gene measurement was first normalized by dividing the expression ratios by the maximum value for each time series. To emphasize the variation from one time point to the next, slopes were added to the expression vectors by taking the expression differences between consecutive time points. The Euclidean distance was used as a similarity measure for the expression responses.

[0166] The agglomerative algorithm employed constructs a dendrogram. Starting with N clusters each containing a single gene, at each step in the iteration the two closest clusters were merged into a larger cluster. The distance between clusters was defined as the distance between their average expression patterns. After N-1 steps all the data points were merged together. The clustering process defines a hierarchical tree. Genes were automatically assigned to a cluster by cutting the tree between the root and each gene branch with a set of 10 lines (“branch levels”) separated by fixed distances. The branch level cut-off forms a cluster. The tree was first ‘normalized’ so that each branch was at the same distance from the root. In order to preserve the distance between the closest genes, the tree was distorted at the branch furthest from the leaf. The number of branches intersecting at each branch level of the tree equals the number of clusters at that level.

[0167] Division of the tree at branch level 5 divides the genes into 7 clusters of gene expression which include 276 differentially expressed genes and splice variants. In tables 1, columns 4through 10 show the level of gene expression at each time point in response to Ox-LDL exposure vs. no Ox-LDL. Differential regulation has been normalized to a maximum value of 1.0 for each gene. White represents relative expression in response to Ox-LDL ranging from 0-25% of maximum for that particular gene; light gray from 26-50%; dark gray from 51-75%; black from 76-100%.

[0168] IX. Complementary Nucleic Acid Molecules

[0169] Molecules complementary to the polynucleotide, or a fragment thereof, are used to detect, decrease, or inhibit gene expression Although use of oligonucleotides comprising from about 15 to about 30 base pairs is described, the same procedure is used with larger or smaller fragments or their derivatives (PNAs). Oligonucleotides are selected using OLIGO 4.06 software (National Biosciences) and SEQ ED NOs:1-278. To inhibit transcription by preventing promoter binding, a complementary oligonucleotide is designed to bind to the most unique 5′ sequence, most preferably about 10 nucleotides before the initiation codon of the open reading frame. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the mRNA encoding the protein.

[0170] In addition to using antisense molecules constructed to interrupt transcription or translation, modifications of gene expression can be obtained by designing antisense molecules to genomic sequences (such as enhancers or introns) or even to trans-acting regulatory genes. Similarly, antisense inhibition can be achieved using Hogeboom base-pairing methodology, also known as “triple helix” base pairing. Antisense molecules involved in triple helix pairing compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.

[0171] Such antisense molecules are placed in expression vectors and used to transform preferred cells or tissues. This may include introduction of the expression vector into a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell or other reproducing lineage for long term or stable gene therapy. Transient expression may last for a month or more with a non-replicating vector and for three months or more if appropriate elements for inducing vector replication are used in the transformation/expression system.

[0172] Stable transformation of appropriate dividing cells with a vector encoding the antisense molecule can produce a transgenic cell line, tissue, or organism (U.S. Pat. No. 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough antisense molecules to compromise or entirely eliminate activity of the polynucleotide.

[0173] X. Hybridization Technologies and Analyses

[0174] Hybridization technology utilizes a variety of substrates such as polymer coated glass slides and nylon membranes. Arranging elements on polymer coated slides is described in Example V; probe preparation and hybridization and analysis using polymer coated slides is described in examples VI and VII, respectively.

[0175] Polynucleotides are applied to a membrane substrate by one of the following methods. A mixture of polynucleotides is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the polynucleotides are individually ligated to a vector and inserted into bacterial host cells to form a library. The polynucleotides 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 (carbenicimin, kanamycin, ampicillin, or cnloramphenicol 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).

[0176] In the second method, polynucleotides 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 SEPHACRYL400 beads (Amersham Pharmacia Biotech). 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.

[0177] Hybridization probes derived from polynucleotides of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the polynucleotides 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 polynucleotide is then added to a REDIPRIME tube (Amersham Pharmacia Biotech), 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 (Amersham Pharmacia Biotech). The purified probe is heated to 100° C. for five min, snap cooled for two min on ice.

[0178] 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 visually.

[0179] XI. Expression of the Encoded Protein

[0180] Expression and purification of a protein encoded by a polynucleotide of the invention is achieved using bacterial or virus-based expression systems. For expression in bacteria, cDNA is subcloned into a vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into bacterial hosts, such as BL21(DE3). Antibiotic resistant bacteria express the protein upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression in eukaryotic cells is achieved by infecting Spodoptera frugiperda (Sf9) insect cells with recombinant baculovirus, Autogaphica californica nuclear polyhedrosis virus. The polyhedrin gene of baculovirus is replaced with the polynucleotide by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of polynucleotide transcription.

[0181] For ease of purification, the protein is synthesized as a fusion protein with glutathione-S-transferase (GST; Amersham Pharmacia Biotech) 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.).

[0182] XII. Production of Specific Antibodies

[0183] A denatured polypeptide 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 polypeptide is radioiodinated and incubated with murine B-cell hybridomas to screen for monoclonal antibodies. About 20 mg of polypeptide is sufficient for labeling and screening several thousand clones.

[0184] In another approach, the amino acid sequence translated from a polynucleotide of the invention is analyzed using PROTEAN software (DNASTAR) to determine regions of high inmunogenicity. The optimal sequences for immunization are usually at the C-terminus, the N-terminus, and those intervening, hydrophilic regions of the polypeptide that are likely to be exposed to the external environment when the polypeptide is in its natural conformation. Typically, oligopeptides about 15 residues in length are synthesized using an ABI 431 Peptide synthesizer (PE Biosystems) using Fmoc-chemistry and then coupled to keyhole limpet hemocyanin (KLH; Sigma Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester. If necessary, a cysteine may be introduced at the N-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.

[0185] Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with radioiodinated polypeptide to identify those fusions producing a monoclonal antibody specific for the polypeptide. 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 polypeptide at 1 mg/ml. Clones producing antibodies bind a quantity of labeled polypeptide that is detectable above background.

[0186] 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 (Amersham Pharmacia Biotech). Monoclonal antibodies with affinities of at least 108 M−1, preferably 109 to 1010 M−1or stronger, are made by procedures well known in the art.

[0187] XIH. Purification of Naturally Occurring Protein Using Specific Antibodies

[0188] Naturally occurring or recombinant protein is substantially purified by immunoaffinity chromatography using antibodies specific for the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (Amersham Pharmacia Biotech). 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.

[0189] XIV. Screening Molecules for Specific Binding

[0190] The polynucleotide or fragments thereof are labeled with 32P-dCTP, Cy3-dCTP, Cy5-dCTP (Amersham Pharmacia Biotech), or the protein or portions thereof are labeled with BIODIPY or FITC (Molecular Probes). A library or a plurality of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled polynucleotide or protein. After incubation under conditions for a polynucleotide or protein, the substrate is washed. Any position on the substrate retaining label, that indicates specific binding or complex formation, identifies a ligand. Data obtained using different concentrations of the polynucleotide or polypeptide are used to calculate affinity between the labeled polynucleotide or protein and the bound ligand.

[0191] All publications and patents mentioned in the above specification are herein incorporated 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 above-described modes for carrying out the invention which 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.

1TABLE 1SEQ ID NOIncyte IDGene Annotation0 h0.5 h2.5 h8 h1 d2 d4 dCluster1440295.1Human SBC2 mRNA for sodium bicarbonate transporter 2, complete cds.

12g34387annexin 1 (lipocortin I)

23247178.2sperm surface protein

24567938integrin, alpha X (antigen CD11C (p150), alpha polypeptide)

25351122.2integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)

26481379.9paired basic amino acid cleaving enzyme (furin, membrane associated receptor protein)

27215391.7phosphogluconate dehydrogenase

28243812.1protein kinase mitogen-activated 13

291085755.1folate receptor 1 (adult)

210347809.3solute carrier family 6 (neurotransmitter transporter, taurine), member 6

211331734.4prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)

212116840.38interferon regulatory factor 3

0.4

213903565.11proprotein convertase subtilisin/kexin type 4

214903565.8Human mRNA for PACE4E-I, complete cds.

0.5

15474310.13transglutaminase 2 (C polypeptide, protein-glutamine-gamma-glutamyltransferase)

0.2

100

101

102

216413006.13differentiated Embryo Chondrocyte expressed gene 1

103

104

105

106

107

108

109

21776460.2pyridoxal (pyridoxine, vitamin B6) kinase

110

111

112

113

114

115

116

218474374.4pim-1 oncogene

117

118

119

120

121

122

123

219427792.8cathepsin B

124

125

126

127

128

129

130

220364482.3carnitine palmitoyltransferase I, liver

131

132

133

134

135

136

137

221978487.1carnitine palmitoyltranserase I, liver

138

139

140

141

142

143

144

222410626.2Human retinoid X receptor-gamma mRNA, complete cds

145

146

147

148

149

150

151

223234480.6glutaredoxin (thioltransferase)

152

0.20.20.2

153

154

155

324253542.2dual specificity phosphatase 5

156

0.20.2

157

158

159

160

325234202.24microsomal glutathione S-transferase 1

161

162

163

164

165

166

167

326253946.4interleukin 6 signal transducer (gp130, oncostatin M receptor)

168

169

170

171

172

173

174

327348801.1pro-platelet basic protein

175

176

177

178

179

180

181

328980611.1matrilin 1, cartilage matrix protein

182

183

184

185

186

187

188

329283885.8dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 4

189

190

191

192

193

194

195

30348196.33antigen identified by monoclonal antibodies 4F2, TRA1.10, TROP4, and T43

196

197

198

199

200

201

202

331256009.4AHNAK nucleoprotein (desmoyokin)

203

204

205

206

207

208

209

332481594.12Human RACH1 (RACH1) mRNA, complete cds

210

211

212

213

214

215

216

333978788.1Human RACH1 (RACH1) mRNA, complete cds

217

218

219

220

221

222

223

334335171.1integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)

224

225

226

227

228

229

230

335998433.2ESTs, Highly similar to DIAMINE ACETYLTRANSFERASE ([H. sapiens]

231

232

233

234

235

236

237

236221928.9ESTs0.30.3

238

239

240

241

242

237331291.3Homo sapiens mRNA for KIAA0291 gene, partial cds0.4

243

244

245

246

247

248

238233331.3Homo sapiens KIAA0439 mRNA, partial cds0.4

249

250

251

252

253

254

239474682.2ESTs, Weakly similar to W01A11.2 gene product [C. elegans]

255

256

257

258

259

260

261

2403161.7ESTs, Weakly similar to (define not available 4529890) [H. sapiens]

262

263

264

265

266

267

268

341984248.1ESTs

269

270

271

272

273

274

275

342196590.2ESTs

276

277

278

279

280

281

282

343255109.1ESTs

283

284

285

286

287

288

289

344238622.2Human clone 46690 brain expressed mRNA from Chromosome X

290

291

292

293

294

295

296

345334385.3Homo sapiens mRNA for KIAA0284 gene, partial cds

297

298

299

300

301

302

303

346998997.1ESTs

304

305

306

307

308

309

310

747200578.1ESTs0.20.20.2

311

312

313

314

348208134.1ESTs0.2

315

0.20.2

316

317

318

349153659.2interleukin 1 receptor antagonist0.2

319

320

321

322

323

324

150241930.15liver X receptor, alpha0.2

325

326

327

0.4

328

329

151413466.5adipose differentiation-related protein; adipophilin0.10.10.2

330

331

332

333

1523249239colony stimulating factor 1 (macrophage)0.2

334

0.2

335

336

337

338

153337518.18CD36 antigen (collagen type I receptor, thrombospondin receptor)0.2

339

340

341

342

343

344

254g3116213SH3 binding protein0.2

345

346

347

348

349

350

355g5912216SH3 binding protein0.2

351

352

353

354

355

356

356992917.1ferritin, heavy polypeptide 10.20.20.20.2

357

358

359

157411424.12LIM and senescent cell antigen-like domains 10.2

360

361

362

363

364

365

158995600.17Homo sapiens clone 24649 mRNA sequence0.20.20.20.20.20.2

366

159441292.7epithelial membrane protein 10.20.20.20.20.3

367

368

16042176.5Down syndrome dandidate region 10.10.20.2

369

0.2

370

371

161234537.35′ nucleotidase (CD73)0.20.20.20.2

372

373

374

1624704568.21uridine phosphorylase0.20.20.20.2

375

376

377

163240120.3diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth factor)0.10.10.10.1

378

379

380

16428779.3small inducible cytokine subfamily A (Cys-Cys), member 200.10.10.10.20.10.2

381

165238627.2BCL2-related protein A1

382

383

384

385

0.1

386

387

166254107.1thrombomodulin0.20.20.10.1

388

389

390

467330908.2leukemia inhibitory factor (cholinergic differentiation factor)

391

392

393

0.2

394

395

396

468g687589Human (AFlq) mRNA, complete cds

397

398

399

400

401

402

403

169197975.11KIAA0763 gene product

404

405

406

407

408

409

410

170227928.2KIAA0429 gene product

411

412

413

414

415

416

417

171248785.7ESTs

418

419

420

0.2

421

0.2

422

172977757.3KIAA0237 gene product

423

424

425

426

427

428

429

173232773.2ESTs

430

431

432

433

434

435

436

174g6634024Human mRNA for KIAA0379 gene, partial cds

437

438

439

440

441

442

443

175g4589571ESTs, Weakly similar to DAP-1 beta [H. sapiens]

444

445

446

447

448

449

450

176334370.3KIAA0024 gene product

451

452

453

454

455

456

457

177980461.1ESTs

458

459

460

461

462

463

464

178422969.5KIAA0598 gene product

465

466

467

468

469

470

471

279244150.4Human mRNA for KIAA0194 gene, partial cds

472

473

474

475

476

477

478

280410257.11ESTs

479

480

481

482

483

484

485

28128253.3Homo sapiens chromosome 19, cosmid R28379

486

487

488

489

0.2

490

491

182g31670guanylate cyclase 1, soluble, alpha 2

492

493

494

495

496

497

498

183977552.1musculin (activated B-cell factor-1)

499

500

501

502

503

504

505

184977552.2Human activated B-cell factor-1 (ABF-1) mRNA, complete cds.

506

507

508

509

510

511

512

185347829.6yes-associated protein 65 kDa

513

514

515

516

517

518

519

186251776.11integrin, beta 5

520

521

522

523

524

525

526

187343674.9GTP-binding protein overexpressed in skeletal muscle

527

528

529

530

531

532

533

188479136.1core-binding factor, runt domain, alpha subunit 3

534

535

536

537

0.4

538

539

1891078147.1early development regulator 2 (homolog of polyhomeotic 2)

540

541

542

543

544

545

546

190474275.1podocalyxin-like

547

548

549

550

551

552

553

1911320658fibulin 1

554

555

556

557

0.4

558

559

192242114.16PTK2 protein tyrosine kinase 2

560

561

0.5

562

0.4

563

564

193445186.7LIM domain only 4

565

566

567

568

569

570

571

194474496.2toll-like receptor 2

572

573

574

575

576

577

578

195257114.7solute carrier family 31 (copper transporters), member 2

579

580

0.5

581

582

583

584

196984005.1high-mobility group (nonhistone chromosomal) protein isoform I-C

585

586

587

588

0.3

589

590

197977667.1complement component 5 receptor 1 (C5a ligand)

591

592

593

594

595

596

597

198996862.4TG-interacting factor (TALE family homeobox)

598

599

0.5

600

601

602

603

199364940.19sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican)

604

605

606

607

0.1

608

609

11001041140.4Fc fragment of IgG, low affinity IIIa, receptor for (CD16)

610

611

612

613

614

615

616

1101408246.2leupaxin

617

618

619

620

621

622

623

1102902740.4aminolevulinate, delta-, dehydratase

624

625

626

627

628

629

630

1103475486.9peptidylprolyl isomerase F (cyclophilin F)

631

632

633

634

635

636

637

1104233778.9acid sphingomyelinase-like phosphodiesterase

638

639

640

641

642

643

644

1105350392.3myosin IC

645

646

647

648

649

650

651

1106458045.4integrin, alpha 5 (fibronectin receptor, alpha polypeptide)

652

653

654

655

656

657

658

1107471362.17Homo sapiens myosin light chain kinase (MLCK) mRNA, complete cds

659

660

661

662

663

664

665

1108336716.3cytochrome P450, subfamily XXVIIB (25-hydroxyvitamin D-1-alpha-hydroxylase), polypeptide 1

666

667

668

669

670

671

672

1109995211.5syndecan 2 (heparan sulfate proteoglycan 1, cell surface-associated, fibroglycan)

673

674

675

676

677

678

679

1110238824.23-prime-phosphoadenosine 5-prime-phosphosulfate synthase 1

680

681

682

683

0.2

684

685

1111474592.3Human leukemia virus receptor 1 (GLVR1) mRNA, complete cds

686

687

688

689

690

691

692

1112431338.2regulator of G-protein signaling 16

693

694

695

696

0.2

697

698

1113412631.5plectin 1, intermediate filament binding protein, 500 kD

699

700

701

702

0.5

703

704

1114350480.6Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog

705

706

707

708

709

710

711

1115350521.15tumor necrosis factor receptor superfamily, member 10b

712

713

714

715

716

717

718

1116445076.9plasminogen activator, urokinase receptor

719

720

721

722

723

724

725

1117995028.4fibroblast activation protein, alpha

726

727

728

729

730

731

732

1118245008.4phosphodiesterase 8A

733

734

735

736

737

0.5

738

1119350895.1twist (Drosophila) homolog

739

740

741

742

0.2

743

744

1120434265.5ribosomal protein S6 kinase, 90 kD, polypeptide 2

745

0.5

746

747

748

749

750

1121427813.14fibronectin 1

751

752

753

0.20.20.2

754

112214704.3activin A receptor, type II

755

756

757

0.5

758

759

760

1123344240.2macrophage scavenger receptor 1

761

762

763

764

765

766

767

1124239694.6a disintegrin and metalloproteinase domain 17 (tumor necrosis factor, alph, converting enzyme)

768

769

770

771

0.3

772

773

1125255772.2activin A receptor, type I0.5

774

775

776

777

778

779

2126232066.3integrin, beta 70.5

780

781

782

783

784

785

2127246504.1activating transcription factor 1

786

787

788

789

790

791

792

2128986123.22vimentin

793

794

795

796

797

798

799

2129898945.14kynurenine 3-monooxygenase (kynurenine 3-hydroxylase)

800

801

802

803

804

805

806

2130236208.16peptidylglycine alpha-amidating monooxygenase

807

808

809

0.4

810

811

812

2131246531.2hippocalcin-like 1

813

814

815

0.4

816

817

818

2132238586.2matrix metalloproteinase 7 (matrilysin, uterin)

819

820

821

822

823

824

825

2133245532.7cyclin-dependent kinase inhibitor 1A (p21, Cip1)

826

827

828

829

830

831

832

2134200972.2Human putative cyclin G1 interacting protein mRNA, partial sequence

833

834

835

0.3

836

837

838

2135348061.1glucan (1,4-alpha-), branching enzyme 1

839

840

841

842

843

844

845

2136233711.7pyruvate dehydrogenase kinase, isoenzyme 4

846

847

848

849

850

851

852

2137256043.19cathepsin L

853

854

0.4

855

856

857

858

2138445012.6N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1

859

860

861

862

863

864

865

2139g463906syntaxin 4A (placental)

866

867

868

869

870

871

872

2140475621.1CD36 antigen (collagen type I receptor, thrombospondin receptor)-like 2

873

874

875

876

877

878

879

2141216063.17Human lysophospholipase homolog (HU-K5) mRNA, complete cds0.40.4

880

0.4

881

882

883

21421099498.9apoliproprotein C-I0.5

884

885

886

887

888

889

21431099076.1fatty acid binding protein 5 (psoriasis-associated)

890

891

892

893

894

895

896

2144902119.3CD63 antigen (melanoma 1 antigen)

897

898

899

0.5

900

901

902

2145g2982500neutropathy target esterase

903

0.5

904

905

906

907

908

21461097580.4ras homolog gene family, member C

909

910

911

912

913

914

915

2147391851.1ferritin, light polypeptide

916

917

918

919

920

921

922

214813105.9lectin, galactoside-binding, soluble, 3 (galectin 3)

923

924

925

0.5

926

927

928

2149356248.4inositol phosphate 5′-phosphatase 2 (synaptojanin 2)

929

930

931

0.3

932

933

934

2150331045.1phosphodiesterase 3B, cGMP-inhibited

935

936

937

938

939

940

941

215142480.3guanine nucleotide-releasing factor 2 (specific for crk proto-oncogene)

942

943

944

945

946

0.5

947

2152245099.8target of myb1 (chicken) homolog

948

949

950

951

952

953

954

2153245481.2ciliary neurotrophic factor receptor

955

956

957

958

959

960

961

2154225021.4Burkitt lymphoma receptor 1, GTP-binding protein

962

963

964

965

966

967

968

4155451767.28tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory)0.50.5

969

970

971

972

973

4156902142.11Homo sapiens leucocyte immunoglubulin-like receptor-5 (LIR-5) mRNA, complete cds0.5

974

975

976

977

978

979

4157291095.5cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1 (glaucoma 3, primary infantile)

980

981

982

983

984

985

986

4158332919.4H. sapiens mRNA for cytokine inducible nuclear protein

987

988

989

990

991

992

993

4159387130.26choline kinase-like

994

995

996

997

998

999

1000

4160410580.13plasminogen activator inhibitor, type I

1001

1002

1003

1004

1005

1006

1007

4161251715.1early growth response 1

1008

1009

1010

1011

1012

1013

1014

41621799017F6neuregulin 1

1015

1016

1017

1018

1019

1020

1021

4163348891.1BCL2/adenovirus E1B 19 kD-interacting protein 3-like

1022

1023

1024

1025

1026

1027

1028

4164903956.15numb (Drosophila) homolog

1029

1030

1031

1032

0.5

1033

1034

4165235184.1guanine nucleotide binding protein 11

1035

1036

1037

1038

1039

1040

1041

4166330948.3solute carrier family 9 (sodium/hydrogen exchanger), isoform 1

1042

1043

1044

1045

1046

1047

1048

4167994057.1thrombospondin 1

1049

1050

0.5

1051

1052

1053

1054

4168197301.4phosphoprotein regulated by mitogenic pathways

1055

1056

0.3

1057

1058

1059

1060

4169476016.17nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alphs

1061

1062

1063

1064

1065

1066

1067

41701098409.1early growth response 2 (Krox-20 (Drosophila) homolog)0.40.50.4

1068

1069

1070

1071

4197997377.1ribonuclease, RNase A family, 3 (eosinophil cationic protein)

1072

1073

1074

1075

1076

1077

0.2319842869.3cathepsin G

1078

1079

1080

1081

1082

1083

0.13199248306.1carbonic anhydrose II

1084

1085

1086

1087

1088

1089

0.13200247220.15thymidylate synthetase

1090

1091

1092

1093

1094

0.20.2520126662.3centromere protein F (350/400 kD, motosin)

1095

1096

1097

1098

1099

1100

0.25202977509.3v-myb avian myeloblastosis viral oncogene homolog-like 2

1101

1102

1103

1104

1105

0.20.25203221961.2myeloid cell nuclear differentiation antigen

1106

1107

1108

1109

1110

1111

0.15204246824.1ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin)

1112

1113

1114

1115

1116

1117

0.25205407557.2cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)

1118

1119

1120

1121

1122

1123

0.25206372981.2Homo sapiens ZW10 interactor Zwint mRNA, complete cds

1124

1125

1126

1127

1128

1129

0.25207201409.6Fc fragment of IgG, high affinity Ia, receptor for (CD64)

1130

1131

1132

1133

1134

1135

0.25208331025.1Homo sapiens mitotic centromere-associated kinesin mRNA, complete cds

1136

1137

1138

1139

1140

1141

0.25209247515.1elastase 2, neutrophil

1142

1143

1144

1145

1146

1147

1148

5210199471.2MAD2 (mitotic arrest deficient, yeast, homolog)-like 1

1149

1150

1151

1152

1153

1154

1155

52112916753high-mobility group (nonhistone chromosomal) protein 2

1156

1157

1158

1159

1160

1161

0.25212343899.2hyaluronan-mediated motility receptor (RHAMM)

1162

1163

1164

1165

1166

1167

0.25213335775.2lamin B1

1168

1169

1170

1171

1172

1173

0.25214232714.5ESTs

1174

1175

1176

1177

1178

1179

1180

5215305039.4ESTs

1181

1182

1183

1184

1185

1186

1187

5216233603.2ESTs

1188

1189

1190

1191

1192

1193

1194

5217330930.1ESTs

1195

1196

1197

1198

1199

1200

1201

5218247289.1Human clone 23815 mRNA sequence

1202

1203

1204

1205

1206

1207

1208

5219331033.1KIAA0008 gene product

1209

1210

1211

0.5

1212

1213

1214

52201098766.1ESTs

1215

1216

1217

1218

1219

1220

0.35221245632.3ESTs

1221

1222

1223

1224

1225

1226

1227

5222333461.2Human mRNA for KIAA0074 gene, partial cd

1228

1229

1230

1231

1232

1233

5223347876.6minichromosome maintenance deficient (S. cerevisiae) 4

1234

1235

1236

1237

1238

1239

1240

5224413842.1Human ECRP gene for eosinophil cationic related protein

1241

1242

1243

1244

1245

1246

1247

5225235867.2polo (Drosophia)-like kinase

1248

1249

1250

1251

1252

1253

1254

5226428665ribonucleotide reductase M1 polypeptide

1255

1256

1257

1258

1259

1260

1261

52272234.3Homo sapiens histone H2A.F/Z variant (H2AV) mRNA, complete cds

1262

1263

1264

1265

1266

1267

1268

52281000139.13insulin-like growth factor binding protein 7

1269

1270

1271

1272

1273

1274

1275

5229998534.1growth factor independent 1

1276

1277

1278

1279

1280

1281

1282

5230372377.6phosphorylase, glycogen; liver (Hers disease, glycogen storage disease type VI)

1283

1284

1285

1286

1287

1288

1289

52311101412.4trophinin-assisting protein (tastin)

1290

1291

1292

1293

1294

1295

1296

5232261567.5CDC28 protein kinase 2

1297

1298

1299

1300

1301

1302

1303

5233232713.2uracil-DNA glycosylase

1304

1305

1306

1307

1308

1309

1310

5234214335.13Homo sapiens E2F-related transcription factor (DP-1) mRNA, complete cds

1311

1312

1313

1314

1315

1316

1317

5235331022.33dihydropyrimidine dehydrogenase

1318

1319

1320

1321

1322

1323

1324

5236332259.3retinoblastoma-like 1 (p107)

1325

1326

1327

1328

1329

1330

1331

5237253570.8forkhead (Drosophila)-like 16

1332

1333

1334

1335

1336

1337

1338

5238995529.5cell division cycle 2, G1 to S and G2 to M

1339

1340

1341

1342

1343

1344

1345

5239474435.16Human MAC30 mRNA, 3′ end

1346

1347

1348

1349

1350

1351

1352

5240994861.1Human chondroitin sulfate proteoglycan core protein mRNA, 3′ end

1353

1354

1355

1356

1357

1358

1359

5241g545708natural kill cell group 7 sequence

1360

1361

1362

1363

1364

1365

1366

5242347965.2CD39 antigen

1367

1368

1369

1370

1371

1372

1373

5243202361.1small nuclear ribonucleoprotein polypeptide A

1374

1375

1376

1377

1378

1379

1380

5244369950.12DNA-damage-inducible transcript 1

1381

1382

1383

1384

1385

1386

0.25245331403.8minichromosome maintenance deficient (S. cerevisiae) 5 (cell division cycle 46)

1387

1388

1389

1390

1391

1392

1393

5246233889.3CDC28 proeint kinase 1

1394

1395

1396

1397

1398

1399

1400

524721148.4nucleobindin 2

1401

1402

1403

1404

1405

1406

0.25248976749.1replication factor C (activator 1) 4 (37 kD)

1407

1408

1409

1410

1411

1412

1413

5249252719.12Human beta 3-endonexin mRNA, long form and short form, complete cds

1414

1415

1416

1417

1418

1419

1420

5250g6063478G/T mismatch-binding protein

1421

1422

1423

1424

1425

1426

1427

5251347314.3serine/threonine kinase 15

1428

1429

1430

1431

1432

1433

1434

5252g3213196serine/threonine kinase 15

1435

1436

1437

1438

1439

1440

1441

5253245184.3transforming growth factor, beta-induced 68 kD

1442

1443

1444

1445

1446

1447

1448

5254243574.11cysteine-rich protein 1 (intestinal)

1449

1450

1451

1452

1453

1454

1455

5255474826.6nidogen (enactin)

1456

1457

1458

1459

1460

0.30.25256997347.6feline sarcoma viral (v-fes)/Fujinami avian sarcoma (PRCII) viral (v-fps) oncogene homolog

1461

1462

1463

1464

1465

1466

1467

5257222049.1H. sapiens mRNA for glutamine cyclotransferase

1468

1469

1470

1471

1472

0.4

1473

52589902659.8small nuclear ribonucleoprotein polypeptide G

1474

1475

1476

1477

1478

1479

1480

52592508261interferon, gamma-inducible protein 16

1481

1482

1483

1484

1485

1486

1487

5260232945.12RAD54 (S. cerevisiae)-like

1488

1489

1490

1491

1492

1493

1494

5261445101.8proliferating cell nuclear antigen

1495

1496

1497

1498

1499

1500

1501

5262255750.1metallothionein 3 (growth inhibitory factor (neurotrophic))

1502

1503

1504

1505

1506

1507

1508

5263988231.7interferon-induced protein 17

1509

1510

1511

1512

1513

1514

1515

5264444902.6interferon-inducible

1516

1517

1518

1519

1520

1521

1522

5265407546.8calreticulin

1523

1524

1525

1526

1527

1528

1529

5266346511.42′-5′oligoadenylate synthetase 2

1530

1531

1532

1533

1534

1535

1536

5267346411.52′-5′oligoadenylate synthetase 2

1537

1538

1539

1540

1541

1542

1543

52681098141.1breast cancer 1, early onset

1544

1545

1546

1547

1548

1549

1550

5269238089.2exonuclease 1

1551

1552

1553

1554

1555

1556

1557

52701100105.3CD74 antigen

1558

1559

1560

1561

1562

1563

1564

6271474729.2calponin 2

1565

1566

1567

1568

1569

1570

1571

627236300.3complement component 2

1572

1573

1574

1575

1576

1577

1578

6273395096.3minichromosome maintenance deficient (S. cerevisiae) 2 (mitotine)

1579

1580

1581

1582

1583

0.2

1584

7274374086.1high-mobility group (nonhistone chromosomal) protein 1

1585

1586

1587

1588

1589

1590

1591

727544495.4small nuclear ribonucleoprotein polypeptide F

1592

1593

1594

1595

1596

0.5

1597

7276474876.2Human mRNA for Sm protein F

1598

1599

1600

1601

1602

1603

1604

[0192]

2

TABLE 2

SEQ ID

NO
Incyte ID
Clone ID
Start
Stop

1
440295.1
3034487
2203
3330

2
g34387
79576
17
1395

3
247178.2
567292
3432
4661

4
567938
567938
669
1472

5
351122.2
682741
682
1151

6
481379.9
1219315
3730
4136

7
215391.7
1269046
861
1902

8
243812.1
1321761
698
1663

9
1085755.1
1376121
650
1291

10
347809.3
1516886
3615
4644

11
331734.4
1595081
334
876

12
116840.38
1606119
847
1284

13
903565.11
1672574
4016
4325

14
903565.8
1672574
1242
1787

15
474310.13
1672744
1281
3844

16
413006.13
1732479
1143
1904

17
76460.2
1749883
350
831

18
474374.4
2679117
1030
2542

19
427792.8
2806166
611
1994

20
364482.3
3178719
1331
1922

21
978487.1
3178719
54
526

22
410626.2
3602501
1153
1796

23
234480.6
1238577
298
1045

24
253542.2
1734561
1606
2355

25
234202.24
1995380
50
901

26
253946.4
2172334
1098
2397

27
348801.1
2203834
15
663

28
980611.1
2213735
1431
2249

29
283885.8
2415989
918
1576

30
348196.33
2852561
1095
1848

31
256009.4
3068454
4496
4936

32
481594.12
3211396
649
1098

33
978788.1
3211396
562
678

34
335171.1
3229778
5149
5670

35
998433.2
63038
2
1032

36
221928.9
674714
1199
1386

37
331291.3
1579487
3345
3833

38
233331.3
1712888
2259
2939

39
474682.2
1969044
1006
1509

40
3161.7
1484773
270
603

41
984248.1
1516047
968
1760

42
196590.2
1607510
723
1118

43
255109.1
1607510
304
429

44
238622.1
1669780
27
957

45
334385.3
1890138
6050
6479

46
998997.1
1640161
1104
1496

47
200578.1
1397926
1138
2288

48
208134.1
2293931
2596
2746

49
153659.2
519653
1355
1884

50
241930.15
1512213
1017
1540

51
413466.5
1985104
760
1861

52
3249239
3249239
740
2957

53
337518.18
3506985
151
500

54
g3116213
2170638
194
1738

55
g5912216
2170638
466
2010

56
992917.1
27775
386
910

57
411424.12
126888
88
599

58
995600.17
237730
799
1151

59
441292.7
1624024
1208
2738

60
42176.5
1650238
89
2297

61
234537.3
1718651
3061
3639

62
470468.21
1806435
800
1521

63
240120.3
1862257
472
2312

64
28779.3
2220923
8
785

65
238627.2
2555673
145
855

66
254107.1
2394637
3297
4186

67
330908.2
2987878
2395
3815

68
g687589
1403041
170
1592

69
197975.11
1560143
2995
4347

70
227928.2
1719657
1717
2098

71
258785.7
1738168
3345
3738

72
977757.3
1830303
4598
7208

73
232773.2
1958631
2317
2963

74
g6634024
2378601
697
1808

75
g4589571
2902846
3036
3495

76
334370.3
3335055
1195
2483

77
980461.1
4003857
293
702

78
422969.4
1369536
3174
4219

79
244150.4
1429306
1803
5218

80
410257.11
1965978
2763
3546

81
28253.3
75549
425
661

82
g31670
155892
1884
2388

83
977552.1
155904
993
1501

84
977552.2
155904
281
789

85
347829.6
185448
334
2046

86
251776.11
418731
2766
3414

87
343674.9
450618
919
1425

88
479136.1
885297
2474
3905

89
1078147.1
1000508
1289
2523

90
474275.1
1297562
4431
5815

91
g403532
1320658
1213
2771

92
242114.16
1361963
2792
4530

93
445186.7
1375107
243
1602

94
474496.2
1401002
1855
2387

95
257114.7
1424573
867
1703

96
984005.1
1446475
68
809

97
977667.1
1447909
1146
1705

98
996862.4
1449337
50
660

99
364940.19
1479437
2522
5308

100
1041140.4
2220025
448
2428

101
408246.2
1595756
890
1796

102
902740.4
1670773
373
845

103
475486.9
1694039
483
1534

104
233778.9
1695477
970
1500

105
350392.3
1719058
2417
4573

106
458045.4
1720114
2424
4196

107
471362.17
1720149
286
1089

108
336716.3
1749727
1431
2412

109
995211.5
1782172
1192
3936

110
238824.2
1841989
1082
2360

111
474592.3
1846463
2393
3281

112
431338.2
1890243
876
2359

113
412631.5
1907232
12440
12947

114
350480.6
1975575
1928
2274

115
350521.15
2078364
1075
1890

116
445076.9
2449986
356
1578

117
995028.4
2483605
132
606

118
245008.4
2900572
2225
3836

119
350895.1
2952864
440
1439

120
434265.5
3421442
740
1203

121
427813.14
3553729
6501
7091

122
14704.3
3742428
1000
2154

123
344240.2
3943651
2050
2530

124
239694.6
4144156
2287
3032

125
255772.2
433573
1321
2758

126
232066.3
514726
2248
2778

127
246504.1
570512
750
2361

128
986123.22
1522716
1264
1904

129
898945.14
1525829
830
1628

130
236208.16
1682642
2775
3010

131
246531.2
1692164
1374
1602

132
238586.2
1699587
427
910

133
245532.7
1804548
1196
1992

134
200972.2
1850135
1308
2138

135
348061.1
1867652
687
2825

136
233711.7
1902929
1146
2151

137
256043.19
1910469
1137
1625

138
445012.6
1911016
6908
7424

139
g463906
1959969
29
523

140
475621.1
1967160
1459
1932

141
216063.17
2174920
381
1030

142
1099498.9
2369312
463
601

143
1099076.1
2537805
299
664

144
902119.3
2594308
1
836

145
g2982500
2720693
3244
4316

146
1097580.4
2733928
126
1118

147
391851.1
2868138
490
851

148
13105.9
2921194
462
1362

149
356248.4
2967860
1184
5905

150
331045.1
3001809
2899
4165

151
482480.3
3003077
2356
2814

152
245099.8
3119252
1681
2281

153
245481.2
3606947
243
1980

154
225021.4
146667
1489
2773

155
451767.28
418041
66
864

156
902142.11
518094
1155
1925

157
291095.5
719318
4551
5099

158
332919.4
924319
781
1262

159
387130.26
1439677
133
3079

160
410580.13
1445767
645
2172

161
251715.1
1705208
1702
2383

162
1799017F6
1799017
1
459

163
348891.1
1877829
777
1288

164
903956.15
1879023
1328
3314

165
235184.1
1988432
660
979

166
330948.3
2054252
3800
4487

167
994057.1
2055534
4841
5856

168
197301.4
2591814
937
3287

169
476016.17
3142624
419
1641

170
1098409.1
3603037
1338
2945

171
202023.6
160822
2991
4412

172
350423.5
1624459
56
1711

173
1100023.1
2895245
512
2019

174
414196.8
1222317
1
476

175
331106.6
1518328
1675
2011

176
g180670
1558081
1074
2596

177
236574.12
1559730
2628
3663

178
1000033.6
1600726
1229
4571

179
37567.22
1672930
185
829

180
995610.1
1673876
1688
2958

181
1702374
1702374
1038
3139

182
427883.47
1881243
−14
435

183
93687.6
1907952
1259
1638

184
414100.4
1931275
323
1871

185
235148.4
1987127
199
873

186
430039.3
1988710
932
1388

187
348110.2
2158373
2064
2281

188
1098815.7
2831248
393
924

189
474491.18
3747901
190
1319

190
474491.19
3747901
267
1396

191
419031.5
1988019
1627
2079

192
399658.1
3967402
559
1697

193
474913.3
3138128
3685
4570

194
199898.3
1217764
271
932

195
253550.14
1447903
441
2458

196
331597.2
1975944
3277
4284

197
997377.1
1526665
256
788

198
42869.3
2016960
162
835

199
248306.1
2474163
198
1710

200
247220.15
39817
846
1550

201
26662.3
485111
7807
10242

202
977509.3
494905
166
2605

203
221961.2
633460
945
1672

204
246824.1
1488852
319
789

205
407557.2
1501556
1391
2055

206
372981.2
1576329
551
906

207
201409.6
1622987
791
1342

208
331025.1
2242674
1364
2791

209
247515.1
2399253
606
1044

210
199471.2
2414624
125
1464

211
2916753
2916753
114
1109

212
343899.2
3622417
97
897

213
335775.2
3771476
1324
2846

214
232714.5
277897
479
649

215
305039.4
522991
1009
1391

216
233603.2
1604056
1
190

217
330930.1
1740384
6110
6515

218
247289.1
1901271
2050
2558

219
331033.1
1970111
1079
2827

220
1098766.1
2113618
939
1345

221
245632.3
2396287
2506
2827

222
333461.2
4003342
1541
2067

223
347876.6
103669
290
2971

224
413842.1
173591
1
366

225
235867.2
343653
1519
2159

226
199636.2
428665
800
2432

227
2234.3
627654
124
682

228
1000139.13
690313
557
1118

229
998534.1
885129
1823
2762

230
372377.6
1315115
2445
2827

231
1101412.4
1340504
440
938

232
261567.5
1384823
32
576

233
232713.2
1405652
436
2078

234
214335.13
1439126
904
2629

235
331022.33
1485479
3584
4373

236
332259.3
1513664
2446
3297

237
253570.8
1516301
467
1240

238
995529.5
1525795
337
1781

239
474435.16
1610523
1323
2032

240
994861.1
1623237
9447
10862

241
g545708
1668794
39
798

242
347965.2
1672749
734
1895

243
202361.1
1700047
1121
1558

244
369950.12
1702350
840
1323

245
331403.8
1746529
2145
2537

246
233889.3
1758241
687
941

247
21148.4
1760517
462
1583

248
976749.1
1773638
1907
2394

249
252719.12
1809385
61
1008

250
g6063478
1926006
3426
4214

251
347314.3
2007691
1146
2115

252
g3213196
2007691
1240
2209

253
245184.3
2056395
946
2668

254
243574.11
2121863
581
836

255
474826.6
2175008
4211
4706

256
997347.6
2195430
2222
2834

257
222049.1
2365295
62
588

258
902659.8
2449837
403
806

259
2508261
2508261
541
2671

260
232945.12
2645840
968
2501

261
445101.8
2781405
911
1316

262
255750.1
2901811
129
471

263
988231.7
2902903
435
1058

264
444902.6
2949427
92
664

265
407546.8
2970280
564
1888

266
346511.4
3214930
17
588

267
346511.5
3214930
81
652

268
1098141.1
3563535
4062
4478

269
238089.2
4385292
1184
3103

270
1100105.3
1001730
91
1412

271
474729.2
1443061
319
2116

272
363000.3
1510424
965
2927

273
395096.3
1723834
2914
3253

274
374086.1
1813133
89
862

275
444495.4
2104530
743
1309

276
474876.2
2104530
148
472

[0193]

3

TABLE 3

SEQ

ID

max
max
max

NO
Incyte ID
Gene Annotation
0h
0.5h
2.5h
8h
1d
2d
4d
up
down
diff

1
3034487
solute carrier family 4, sodium bicarbonate
1.00
0.96
1.15
2.02
1.20
1.45
3.51
3.51
0.96
3.51
up-

cotransporter, member 6

regu-

lated

2
g34387
annexin I (lipocortin I)
1.00
1.23
1.33
1.23
2.83
2.54
1.86
2.83
1.00
2.83

3
247178.2
sperm surface protein
1.00
0.99
1.11
1.24
2.00
1.93
2.00
2.00
0.99
2.00

4
567938
integrin, alpha X (antigen CD11C (p150),
1.00
1.03
1.05
0.95
2.11
1.53
2.45
2.45
0.95
2.45

alpha polypeptide)

5
351122.2
integrin, beta 3 (platelet glycoprotein
1.00
1.05
1.07
0.87
3.20
2.22
2.61
3.20
0.87
3.20

IIIa, antigen CD61)

6
481379.9
paired basic amino acid cleaving enzyme
1.00
1.08
1.01
1.03
2.06
1.99
1.57
2.06
1.00
2.06

(furin, membrane associated receptor protein)

7
215391.7
phosphogluconate dehydrogenase
1.00
1.47
1.24
1.78
2.59
2.49
2.54
2.59
1.00
2.59

8
243812.1
protein kinase mitogen- activated 13
1.00
1.17
1.19
0.96
2.27
1.91
2.68
2.68
0.96
2.68

9
1085755.1
folate receptor 1 (adult)
1.00
0.80
1.00
1.07
2.28
1.89
2.16
2.28
0.80
2.28

10
347809.3
solute carrier family 6 (neurotransmitter
1.00
1.06
1.33
1.21
2.65
2.13
3.06
3.06
1.00
3.06

transporter, taurine), member 6

11
331734.4
prostaglandin-endoperoxide synthase 1
1.00
1.20
1.17
1.30
2.08
1.50
1.72
2.08
1.00
2.08

(prostaglandin G/H synthase and cyclooxygena

12
116840.38
interferon regulatory factor 3
1.00
1.15
1.02
0.94
2.31
2.37
1.85
2.37
0.94
2.37

13
903565.11
proprotein convertase subtilisin/kexin type 4
1.00
1.11
1.18
1.13
2.12
1.68
2.50
2.50
1.00
2.50

14
903565.8
Human mRNA for PACE4E-I, complete cds.
1.00
1.11
1.18
1.13
2.12
1.68
2.50
2.50
1.00
2.50

15
474310.13
transglutaminase 2 (C polypeptide,
1.00
1.20
1.09
0.78
2.17
3.20
3.29
3.29
0.78
3.29

protein-glutamine-gamma-glutamyltransferase)

16
413006.13
differentiated Embryo Chondrocyte
1.00
1.17
1.03
1.07
2.16
2.25
1.59
2.25
1.00
2.25

expressed gene 1

17
76460.2
pyridoxal (pyridoxine, vitamin B6) kinase
1.00
1.23
1.16
1.54
2.51
1.61
1.90
2.51
1.00
2.51

18
474374.4
pim-1 oncogene
1.00
1.16
1.09
1.13
3.56
2.75
3.02
3.56
1.00
3.56

19
427792.8
cathepsin B
1.00
1.25
1.01
1.51
2.13
1.78
1.37
2.13
1.00
2.13

20
364482.3
carnitine palmitoyltransferase I, liver
1.00
0.81
0.98
1.25
2.08
2.06
1.61
2.08
0.81
2.08

21
978487.1
carnitine palmitoyltransferase I, liver
1.00
0.81
0.98
1.25
2.08
2.06
1.61
2.08
0.81
2.08

22
410626.2
Human retinoid X receptor-gamma
1.00
1.08
1.19
1.66
2.48
2.51
3.09
3.09
1.00
3.09

mRNA, complete cds

23
234480.6
glutaredoxin (thioltransferase)
1.00
0.91
0.91
0.78
3.65
3.13
1.08
3.65
0.78
3.65

24
253542.2
dual specificity phosphatase 5
1.00
0.92
0.84
1.01
3.83
3.38
2.23
3.83
0.84
3.83

25
234202.24
microsomal glutathione S-transferase 1
1.00
0.93
1.02
1.28
3.07
2.33
1.68
3.07
0.93
3.07

26
253946.4
interleukin 6 signal transducer
1.00
1.01
1.29
1.07
2.04
0.94
2.24
2.24
0.94
2.24

(gp130, oncostatin M receptor)

27
348801.1
pro-platelet basic protein
1.00
1.01
1.03
1.00
3.77
3.69
1.81
3.77
1.00
3.77

28
980611.1
matrilin 1, cartilage matrix protein
1.00
0.94
0.92
0.90
2.86
0.73
1.25
2.86
0.73
2.86

29
283885.8
dual-specificity tyrosine-(Y)-phosphorylation
1.00
1.13
1.08
0.88
2.64
1.06
1.75
2.64
0.88
2.64

regulated kinase 4

30
348196.33
antigen identified by monoclonal antibodies
1.00
1.14
0.98
1.35
2.96
2.07
1.64
2.96
0.98
2.96

4F2, TRA1.10, TROP4, and T43

31
256009.4
AHNAK nucleoprotein (desmoyokin)
1.00
1.17
1.33
0.98
2.32
2.35
0.98
2.35
0.98
2.35

32
481594.12
Human RACH1 (RACH1) mRNA,
1.00
1.24
1.27
0.72
2.59
0.79
0.79
2.59
0.72
2.59

complete cds

33
978788.1
Human RACH1 (RACH1) mRNA,
1.00
1.24
1.27
0.72
2.59
0.79
0.79
2.59
0.72
2.59

complete cds

34
335171.1
integrin, alpha 2 (CD49B, alpha 2
1.00
1.13
1.16
0.68
2.13
1.10
2.34
2.34
0.68
2.34

subunit of VLA-2 receptor)

35
998433.2
ESTs, Highly similar to DIAMINE
1.00
1.22
1.15
1.38
2.81
2.41
2.27
2.81
1.00
2.81

ACETYLTRANSFERASE [H. sapiens]

36
221928.9
ESTs
1.00
0.94
1.02
1.09
3.25
2.26
2.72
3.25
0.94
3.25

37
331291.3

Homo sapiens
mRNA for KIAA0291
1.00
1.26
1.17
1.15
2.42
1.44
1.65
2.42
1.00
2.42

gene, partial cds

38
233331.3

Homo sapiens
KIAA0439 mRNA, partial cds
1.00
1.18
1.11
0.80
2.12
2.75
2.47
2.75
0.80
2.75

39
474682.2
ESTs, Weakly similar to W01A11.2
1.00
1.17
1.34
1.27
3.61
2.29
3.71
3.71
1.00
3.71

gene product [C.elegans]

40
3161.7
ESTs, Weakly similar to (defline not
1.00
1.06
1.02
0.84
2.84
1.13
1.03
2.84
0.84
2.84

available 4529890) [H. sapiens]

41
984248.1
ESTs
1.00
1.02
1.01
0.73
2.14
0.75
1.37
2.14
0.73
2.14

42
196590.2
ESTs
1.00
1.12
1.08
0.76
2.41
0.82
0.75
2.41
0.75
2.41

43
255109.1
ESTs
1.00
1.12
1.08
0.76
2.41
0.82
0.75
2.41
0.75
2.41

44
238622.1
Human clone 46690 brain expressed
1.00
1.06
0.93
0.77
2.11
0.95
2.04
2.11
0.77
2.11

mRNA from chromosome X

45
334385.3

Homo sapiens
mRNA for KIAA0284
1.00
1.20
1.13
0.87
2.00
0.83
1.39
2.00
0.83
2.00

gene, partial cds

46
998997.1
ESTs
1.00
1.05
1.01
2.37
0.84
0.93
1.18
2.37
0.84
2.37

47
200578.1
ESTs
1.00
1.18
1.25
1.75
5.54
5.02
3.11
5.54
1.00
5.54

48
208134.1
ESTs
1.00
1.17
1.09
0.81
3.53
2.33
4.59
4.59
0.81
4.59

49
153659.2
interleukin 1 receptor antagonist
1.00
1.33
1.29
1.26
2.00
3.16
4.88
4.88
1.00
4.88

50
241930.15
liver X receptor, alpha
1.00
1.13
1.28
2.29
2.15
2.27
4.34
4.34
1.00
4.34

51
413466.5
adipose differentiation-related
1.00
1.62
2.66
4.30
7.11
7.12
14.12
14.12
1.00
14.12

protein; adipophilin

52
3249239
colony stimulating factor 1 (macrophage)
1.00
1.20
1.18
1.24
2.42
2.41
4.73
4.73
1.00
4.73

53
337518.18
CD36 antigen (collagen type I receptor,
1.00
1.19
1.39
2.00
3.49
2.68
4.32
4.32
1.00
4.32

thrombospondin receptor)

54
g3116213
SH3 binding protein
1.00
1.25
1.10
1.50
4.20
3.19
2.44
4.20
1.00
4.20

55
g5912216
SH3 binding protein
1.00
1.25
1.10
1.50
4.20
3.19
2.44
4.20
1.00
4.20

171
202023.6
3-hydroxy-3-methylglutaryl-Coenzyme
1.00
0.96
0.87
0.50
0.52
0.68
0.61
1.00
0.50
2.01
down-

A reductase

regu-

lated

172
350423.5
farnesyl-diphosphate farnesyltransferase 1
1.00
1.19
1.05
0.49
0.47
0.55
0.54
1.19
0.47
2.14

173
1100023.1
cytochrome P450, 51 (lanosterol
1.00
1.31
1.33
0.62
0.41
0.59
0.70
1.33
0.41
2.44

14-alpha-demethylase)

174
414196.8
S100 calcium-binding protein A4
1.00
1.06
1.06
0.97
0.34
0.61
0.41
1.06
0.34
2.91

175
331106.6
integrin, alpha 6
1.00
0.94
1.05
1.08
0.45
0.76
0.81
1.08
0.45
2.23

176
g180670
matrix metalloproteinase 2 (gelatinase A, 72
1.00
1.22
1.13
0.92
0.48
0.72
0.67
1.22
0.48
2.10

kD gelatinase, 72 kD type IV collagenase)

177
236574.12
macrophage-associated antigen
1.00
1.17
1.20
0.95
0.40
0.54
1.04
1.20
0.40
2.48

178
1000033.6
alpha-2-macroglobulin
1.00
1.09
1.01
0.85
0.39
0.85
1.03
1.09
0.39
2.57

179
37567.22
RAN binding protein 1
1.00
0.90
0.98
0.99
0.49
0.56
0.52
1.00
0.49
2.04

180
995610.1
v-myc avian myelocytomatosis
1.00
1.17
1.16
0.91
0.46
0.81
0.86
1.17
0.46
2.19

viral oncogene homolog

181
1702374
v-myc avian myelocytomatosis viral oncogene
1.00
1.11
1.08
0.81
0.49
0.77
0.58
1.11
0.49
2.03

homolog 1, lung carcinoma derived

182
427883.47

Homo sapiens
LST1 mRNA, cLST1/E
1.00
1.15
0.98
1.00
0.40
0.76
0.45
1.15
0.40
2.49

splice variant, complete cds

183
93687.6
uncoupling protein 2 (mitochondrial,
1.00
1.09
1.12
1.05
0.50
0.51
0.46
1.12
0.46
2.18

proton carrier)

184
414100.4
leukocyte-associated Ig-like receptor 1
1.00
1.09
1.10
1.26
0.40
0.49
0.51
1.26
0.40
2.47

185
235148.4
arachidonate 5-lipoxygenase-activating protein
1.00
0.96
0.96
0.98
0.30
0.48
0.86
1.00
0.30
3.39

186
430039.3
CD14 antigen
1.00
1.17
1.10
0.99
0.48
0.63
1.10
1.17
0.48
2.07

187
2158373F6
platelet-derived growth factor
1.00
1.03
0.97
1.05
0.49
1.00
1.46
1.46
0.49
2.04

alpha polypeptide

188
1098815.7
Not mapped
1.00
0.93
0.96
1.13
0.50
0.78
0.53
1.13
0.50
2.00

189
474491.18
Human apurinic/apyrimidinic endonuclease
1.00
1.18
1.13
1.28
0.48
0.83
0.76
1.28
0.48
2.09

mRNA, complete cds.

190
474491.19
ref-1
1.00
1.18
1.13
1.28
0.48
0.83
0.76
1.28
0.48
2.09

191
419031.5
leukotriene A4 hydrolase
1.00
1.19
0.95
1.40
0.35
0.63
0.64
1.40
0.35
2.84

192
399658.1
Not mapped
1.00
1.11
1.00
0.77
0.46
0.53
1.47
1.47
0.46
2.18

193
474913.3
ESTs
1.00
1.01
0.98
0.77
0.35
1.00
0.81
1.01
0.35
2.84

194
199898.3
Human G0S2 protein gene, complete cds
1.00
1.07
1.10
0.87
0.21
0.41
0.56
1.10
0.21
4.84

195
253550.14
insulin-like growth factor binding protein 3
1.00
1.06
1.06
1.37
0.28
0.90
1.18
1.37
0.28
3.62

196
331597.2
cytochrome b-245, beta polypeptide
1.00
1.14
1.08
1.02
0.25
0.28
0.16
1.14
0.16
6.32

(chronic granulomatous disease)

[0194]

4

TABLE 4

SEQ

ID

max
max
max

NO
Incyte ID
Gene Annotation
0h
0.5h
2.5h
8h
1d
2d
4d
up
down
diff

47
200578.1
ESTs
1.00
1.18
1.25
1.75
5.54
5.02
3.11
5.54
1.00
5.54
up-

regu-

lated

48
208134.1
ESTs
1.00
1.17
1.09
0.81
3.53
2.33
4.59
4.59
0.81
4.59

49
153659.2
interleukin 1 receptor antagonist
1.00
1.33
1.29
1.26
2.00
3.16
4.88
4.88
1.00
4.88

50
241930.15
liver X receptor, alpha
1.00
1.13
1.28
2.29
2.15
2.27
4.34
4.34
1.00
4.34

51
413466.5
adipose differentiation-related
1.00
1.62
2.66
4.30
7.11
7.12
14.12
14.12
1.00
14.12

protein; adipophilin

52
3249239
colony stimulating factor 1 (macrophage)
1.00
1.20
1.18
1.24
2.42
2.41
4.73
4.73
1.00
4.73

53
337518.18
CD36 antigen (collagen type I
1.00
1.19
1.39
2.00
3.49
2.68
4.32
4.32
1.00
4.32

receptor, thrombospondin receptor)

54
g3116213
SH3 binding protein
1.00
1.25
1.10
1.50
4.20
3.19
2.44
4.20
1.00
4.20

55
g5912216
SH3 binding protein
1.00
1.25
1.10
1.50
4.20
3.19
2.44
4.20
1.00
4.20

56
992917.1
ferritin, heavy polypeptide 1
1.00
1.02
1.04
1.18
1.70
1.96
4.90
4.90
1.00
4.90

57
411424.12
LIM and senescent cell antigen-like domains 1
1.00
1.03
1.16
1.65
1.25
1.61
4.11
4.11
1.00
4.11

58
995600.17

Homo sapiens
clone 24649 mRNA sequence
1.00
1.00
0.99
1.07
1.04
1.03
5.85
5.85
0.99
5.85

59
441292.7
epithelial membrane protein 1
1.00
1.18
1.05
1.30
1.50
3.13
6.00
6.00
1.00
6.00

60
42176.5
Down syndrome candidate region 1
1.00
1.34
1.53
1.85
1.17
2.49
6.83
6.83
1.00
6.83

61
234537.3
5′ nucleotidase (CD73)
1.00
1.00
1.08
1.00
1.69
2.36
6.22
6.22
1.00
6.22

62
470468.21
uridine phosphorylase
1.00
1.30
1.03
1.18
1.93
2.46
5.37
5.37
1.00
5.37

63
240120.3
diphtheria toxin receptor (heparin-binding
1.00
1.03
0.95
0.98
1.82
2.95
7.00
7.00
0.95
7.00

epidermal growth factor-like growth fact

64
28779.3
small inducible cytokine subfamily A
1.00
0.97
1.00
1.26
0.56
2.02
8.18
8.18
0.56
8.18

(Cys-Cys), member 20

65
238627.2
BCL2-related protein A1
1.00
1.12
0.97
1.25
0.42
1.69
3.85
3.85
0.42
3.85

66
254107.1
thrombomodulin
1.00
1.05
0.93
0.89
2.12
6.24
2.12
6.24
0.89
6.24

67
330908.2
leukemia inhibitory factor (cholinergic
1.00
1.08
1.06
0.79
1.40
3.87
1.04
3.87
0.79
3.87

differentiation factor)

194
199898.3
Human G0S2 protein gene, complete cds
1.00
1.07
1.10
0.87
0.21
0.41
0.56
1.10
0.21
4.84
down-

regu-

lated

195
253550.14
insulin-like growth factor binding protein 3
1.00
1.06
1.06
1.37
0.28
0.90
1.18
1.37
0.28
3.62

196
331597.2
cytochrome b-245, beta polypeptide
1.00
1.14
1.08
1.02
0.25
0.28
0.16
1.14
0.16
6.32

(chronic granulomatous disease)

197
997377.1
ribonuclease, RNase A family, 3
1.00
0.99
0.99
0.75
1.25
0.77
0.28
1.25
0.28
3.55

(eosinophil cationic protein)

198
42869.3
cathepsin G
1.00
1.14
1.04
0.80
1.60
0.84
0.20
1.60
0.20
4.98

199
248306.1
carbonic anhydrase II
1.00
0.92
1.03
0.92
1.46
1.19
0.13
1.46
0.13
7.58

200
247220.15
thymidylate synthetase
1.00
1.14
1.12
1.12
0.77
0.23
0.25
1.14
0.23
4.34

201
26662.3
centromere protein F (350/400 kD, mitosin)
1.00
1.24
1.15
1.03
0.87
0.38
0.29
1.24
0.29
3.40

202
977509.3
v-myb avian myeloblastosis viral oncogene
1.00
1.21
1.10
0.81
0.77
0.25
0.27
1.21
0.25
3.95

homolog-like 2

203
221961.2
myeloid cell nuclear differentiation antigen
1.00
1.04
1.22
0.82
0.99
0.95
0.18
1.22
0.18
5.58

204
246824.1
ribonuclease, RNase A family, 2
1.00
1.15
1.03
0.97
1.17
0.71
0.19
1.17
0.19
5.33

(liver, eosinophil-derived neurotoxin)

205
407557.2
cyclin-dependent kinase inhibitor
1.00
1.21
1.03
0.80
1.09
0.43
0.24
1.21
0.24
4.17

2C (p18, inhibits CDK4)

206
372981.2

Homo sapiens
ZW10 interactor
1.00
1.10
1.07
1.17
0.62
0.30
0.29
1.17
0.29
3.48

Zwint mRNA, complete cds

207
201409.6
Fc fragment of IgG, high affinity
1.00
1.25
1.07
1.21
0.90
0.58
0.23
1.25
0.23
4.27

Ia, receptor for (CD64)

208
331025.1

Homo sapiens
mitotic centromere-associated
1.00
1.10
1.04
0.91
0.90
0.48
0.26
1.10
0.26
3.89

kinesin mRNA, complete cds

209
247515.1
elastase 2, neutrophil
1.00
0.96
1.11
0.95
1.11
1.08
0.29
1.11
0.29
3.48

210
199471.2
MAD2 (mitotic arrest deficient,
1.00
1.05
1.02
0.69
0.99
0.28
0.31
1.05
0.28
3.60

yeast, homolog)-like 1

211
2916753
high-mobility group (nonhistone
1.00
1.05
0.99
1.25
0.94
0.36
0.19
1.25
0.19
5.37

chromosomal) protein 2

212
343899.2
hyaluronan-mediated motility
1.00
1.08
1.14
0.94
0.88
1.02
0.26
1.14
0.26
3.81

receptor (RHAMM)

213
335775.2
lamin B1
1.00
1.05
1.09
1.07
0.52
0.32
0.18
1.09
0.18
5.44

[0195]

Genes expressed in foam cell differentiation

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Priority Claims (1)

PCT Information