WO2000077166A2 - Gene expression modulated in gastrointestinal inflammation - Google Patents

Abstract

Polynucleotides, polypeptides, kits and methods are provided related to regulated genes characteristic of gastrointestinal inflammation.

Description

GENE EXPRESSION MODULATED IN GASTROINTESTINAL INFLAMMATION

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application S.N. 60/138,487, filed June 10, 1999, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Chronic inflammatory bowel diseases (IBD) are as a persistent problem in medicine. The two major types of IBD are Crohn's disease (CD), which can affect the whole digestive tract from mouth to anus, and ulcerative colitis (UC), which affects only the large intestine.

Although CD and UC are both characterized by massive gut damage arising from intestinal inflammation, these diseases are quite distinct. Macroscopically, the best distinguishing features of these diseases are that UC is 1) a continuous inflammatory disorder, 2) primarily restricted to the mucosa of the colon and rectum, 3) usually primarily vascular, and 4) usually associated with a striking shortening of the colon. In contrast, inflammation in CD is nearly always discontinuous and may present anywhere in the large or small intestine. With CD the mucosa usually appears 'cobblestoned' and there is Assuring and a thickening of the bowel wall with associated stenosis of the lumen and strictures.

Microscopically, UC is essentially a superficial inflammation of the mucous membrane and even in chronic disease, the muscularis propria and serosa are free of inflammatory infiltration. In contrast, CD presents microscopically as a transmural inflammation, spreading through the mucosa and submucosa into the muscle layers. In UC, crypt abscesses and destruction of the epithelium are common, whereas in CD, a valuable diagnostic feature is the presence of sarcoid type granulomas.

The histological features of both of these diseases suggest that there is a dysregulation of the lymphoid tissue in IBD. Whether the immunological dysregulation is a primary cause of IBD or whether the inflammatory response is secondary to another mucosal insult, such as a breach in the epithelial barrier, remains unclear.

Despite advances in recent years, the precise etiology and pathogenesis of CD and UC remain undefined. In order to try and better understand the mechanistic basis of IBD, much effort has been directed towards the discovery and development of different animal models of inflammatory bowel disease. One of the best characterized systems for studying intestinal inflammation is the mouse model of dextran sodium sulfate (DSS)-induced colitis (I. Okayasu et al, Gastroenterology 98:694702, 1990; H.S. Cooper et al, Laboratory Investigation 69:238-249, 1993; L.A. Dielman et al, Gastroenterology 107:1643-1652, 1994; CO. Elson et al, Gastroenterology 109: 1344-1367, 1995).

DSS is thought to cause breaches in the epithelial tight junctions (J. Ni et al, Gut 39:234-241, 1996), thus permitting the huge antigenic load in the gut lumen to come into direct contact with the underlying tissues. As a result, a vigorous immune response directed against antigens located in the gut lumen is initiated in DSS-treated mice. The intestinal inflammation is primarily restricted to the large intestine.

The DSS-induced colitis model system is used to examine (i) the nature of the earliest response to epithelial damage, (ii) the mechanisms responsible for recruiting cells to the sites of inflammation, (iii) the nature of the protective immune response at the height of intestinal inflammation and (iv) the mechanisms that direct recovery and trigger the repair of damaged tissues. This model system can be used to examine the mechanisms of induction and recovery of IBD and should aid the identification of genes/proteins that may be able to modulate or prevent intestinal damage or stimulate recovery of the mucosa.

Given the diversity of factors that may contribute to these processes, a clear need is evident for the discovery, identification and elucidation of the roles of new proteins involved in the different stages of IBD.

Features of DSS-induced colitis

In the DSS-induced colitis model system, weight loss is apparent in mice beginning at day 4. Histological analysis of the intestine reveals the presence of early patchy lesions identifiable by the loss of epithelial cells and goblet cells. By day 8, weight loss is fairly severe (approximately 20% reduction) and the mice appear moribund. Histologically, the gut epithelium is almost totally destroyed at this stage. There is evidence of a large mixed inflammatory cell infiltration into the lamina propria and submucosa. The inflammatory cell infiltrate appears to be composed primarily of T cells, B cells and granulocytes. By day 12, weight gain is apparent as the mice recover. At this later stage, crypt recovery and epithelial regeneration provide histological evidence of the beginning of repair processes.

DSS-induced colitis and human IBD

The primary event in DSS-induced colitis is epithelial cell damage, with inflammation associated with immune activation a secondary event. Whether epithelial cell damage or immune dysregulation is the triggering event in human IBD is unclear. The model of DSS- colitis is therefore useful in providing an opportunity to study the development and consequences of both processes.

SUMMARY OF THE INVENTION

Molecules have been identified that correspond to genes that are regulated by the DSS treatment. Such molecules are useful in therapeutic, prognostic and diagnostic applications in the treatment of IBD and other gut pathologies. The present invention provides novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides. Also provided are diagnostic methods for detecting disorders related to the polypeptides and the polynucleotides encoding them, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying binding partners of the polypeptides.

The present invention provides novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides. Also provided are diagnostic methods for detecting disorders related to the polypeptides and the polynucleotides encoding them, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying binding partners of the polypeptides.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NOT 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ED NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ED NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62. Another embodiment comprises an isolated nucleic acid molecule at least 95% identical to the isolated nucleic acid molecule of SEQ ID NO: 1-62. A further embodiment comprises an isolated nucleic acid molecule at least ten bases in length that is hybridizable to the isolated nucleic acid molecule of SEQ ID NO: 1 -62 under stringent conditions.

In another embodiment, the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ED NO:l, SEQ ID NO:2, SEQ ED NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ED NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62. In another embodiment, the invention provides an isolated nucleic acid molecule encoding the polypeptide of the present invention.

In a further embodiment, the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in gastrointestinal inflammation, chosen from the group consisting of the DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in gastrointestinal inflammation.

The present invention also provides a system and method for detecting the presence of a gene regulated in gastrointestinal inflammation. In one embodiment, the present invention provides a kit for suitable for detecting the presence of a gene regulated in gastrointestinal inflammation, comprising at least one polynucleotide of the present invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay; label means; instructions for use; and suitable packaging material. In one embodiment, the polynucleotide is chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ED NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ED NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO:12, SEQ ED NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ED NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ED NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ED NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62. Another embodiment comprises a polynucleotide at least 95% identical to the isolated nucleic acid molecule of SEQ ED NO: 1-62. A further embodiment comprises a polynucleotide at least ten bases in length that is hybridizable to the isolated nucleic acid molecule of SEQ ED NO: 1-62 under stringent conditions. In yet another embodiment, the polynucleotide is chosen from the group consisting of the DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in gastrointestinal inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

Figure 1 is a graphical representation of the results of TOGA analysis (TOtal Gene expression Analysis) using a 5' PCR primer with parsing bases CGCG, showing PCR products produced from mRNA extracted from colons isolated from untreated mice (Figure 1A, "0 Days"), mRNA extracted from colons isolated from mice receiving DSS for four days (Figure IB, "4 Days"), mRNA extracted from colons isolated from mice receiving DSS for eight days (Figure IC, "8 Days"), and mRNA extracted from colons isolated from mice receiving DSS for twelve days (Figure ID, "12 Days"), where the vertical index line indicates a PCR product of about 458 b.p. that is up-regulated by DSS treatment, reaching a maximum at eight days, where the ordinate is in arbitrary units of fluorescence intensity and the abscissa is length of PCR product in nucleotides;

Figure 2 is a graphical representation of more detailed analysis of the 458 b.p. PCR product indicated in Figure 1 ; Figure 2A shows the PCR product obtained using an extended 5' primer as described in the text; Figure 2B shows the PCR products obtained using the original PCR primers, and in Figure 2C, the traces from Figure 2A and 2B are overlaid, demonstrating that the PCR product of the isolated and sequenced clone is the same length as the original PCR product, where the ordinate is in arbitrary units of fluorescence intensity and the abscissa is length of PCR product in nucleotides;

Figure 3 is a graphical representation of the results of Northern Blot analysis of clone

EMX 2_46, SEQ ID NO: 10, where an agarose gel containing poly A enriched mRNA from the four experimental samples (0, 4, 8 or 12 days DSS treatment) as well as size standards was blotted after electrophoresis and probed with radiolabelled IMX 2_46, SEQ ED NO: 10, imaged using a phosphorimager and quantified. Quantitative results showing the relative - 7 - expression levels of the 1.6 kb transcript were: 0 day, 64; 4 days, 53; 8 days, 223; and 12 days, 269. The amount of RNA loaded on the gel was determined by probing for cyclophilin ("eye").

Figure 4 is a graphical representation of the results of RT-PCR of clone IMX2_48.

Figure 5 is a graphical representation of the results of RT-PCR of clone IMX2_74.

Figure 6 is a graphical representation of the results of Northern blot analysis of clone EMX 2_17, SEQ ID NO: 3, where an agarose gel containing poly A enriched mRNA from the experimental samples and size standards was blotted after electrophoresis, probed, imaged using a phosphorimager and quantified. Figure 6A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knock- out mice without colitis and mdr knock-out mice with colitis. Figure 6B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8% DSS, and Balb/c mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment. The predicted transcript size for IMX2_17 CRP-ductin is 6.6 Kb; the actual transcript size found in this study was approximately 6.5 Kb.

Figure 7 is a graphical representation of the results of Northern blot analysis of clone EMX 2_22, SEQ ED NO: 4, where an agarose gel containing poly A enriched mRNA from the experimental samples and size standards was blotted after electrophoresis, probed, imaged using a phosphorimager and quantified. Figure 7 A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knockout mice without colitis, mdr knock-out mice with colitis and C57BL/6 spleen. Figure 7B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, and C57BL/6 normal lymphoid tissue samples (MLM, PP, spleen and thymus). The predicted transcript size for X2_22 HPK1 is 2.7 Kb; the actual transcript size found in this study was approximately 2.8 Kb. Figure 8 is a graphical representation of the results of Northern blot analysis of clone EMX 2_28, SEQ ID NO: 5, where an agarose gel containing poly A enriched mRNA from the experimental samples and size standards was blotted after electrophoresis, probed, imaged using a phosphorimager and quantified. Figure 8A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knockout mice without colitis and mdr knock-out mice with colitis. Figure 8B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8% DSS, and Balb/c mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment. The predicted transcript size for IMX2_28 DRA is 2.6 Kb; the actual transcript size found in this study was approximately 3 Kb.

Figure 9 is a graphical representation of the results of Northern blot analysis of clone EMX 2_33, SEQ ID NO:21, where an agarose gel containing poly A enriched mRNA from the experimental samples and size standards was blotted after electrophoresis, probed, imaged using a phosphorimager and quantified. Figure 9 A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knock-out mice without colitis and mdr knock-out mice with colitis. Figure 9B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8% DSS, and Balb/c mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment. The predicted transcript size for EMX2_33 SLPI is 1.1 Kb; the actual transcript size found in this study was 1.1 Kb.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.

In the present invention, a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein.

Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

As used herein, a "polynucleotide" refers to a molecule having a nucleic acid sequence contained in SEQ ED NO: 1-62. For example, the polynucleotide can contain all or part of the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO: 1-62, or the complement thereof, or the cDNA. "Stringent hybridization conditions" refers to an overnight incubation at 42° C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCI; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug ml salmon sperm blocking DNA; followed by washes at 50°C with lx SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5x SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al, Ann NY Acad Sci 663:48-62 (1992).)

"A polypeptide having biological activity" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.).

The translated amino acid sequence, beginning with the methionine, is identified although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by the translation of these alternative open reading frames are specifically contemplated by the present invention.

SEQ ED NO: 1-62 and the translations of SEQ ID NO: 1-62 are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from the translations of SEQ ID NO: 1-62 may be used to generate antibodies which bind specifically to the secreted proteins encoded by the cDNA clones identified.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

The present invention also relates to the genes corresponding to SEQ ID NO: 1-62, and translations of SEQ ID NO: 1-62. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material. Also provided in the present invention are species homologues. Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods which are well known in the art.

Signal Sequences

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues -13 to +2, where +1 indicates the amino terminus of the secreted protein. Therefore, from a deduced amino acid sequence, a signal sequence and mature sequence can be identified. Polvnucleotide and Polypeptide Variants

"Variant" refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

"Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, (1988); BIOCOMPUTENG: INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed., Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, (1991).) While there exists a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans. (Carillo, H., and Lipton, D., SIAM J Applied Math 48:1073 (1988).) Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers," Martin J. Bishop, ed., Academic Press, San Diego, (1994), and Carillo, H., and Lipton, D., SIAM J Applied Math 48: 1073 (1988). Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux, J., et al, Nucleic Acids Research (1984) 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J. Molec. Biol. 215:403 (1990), Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711 (using the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981).)

When using any of the sequence alignment programs to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters are set so that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.

A preferred method for determining the best overall match between a query sequence

(a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990).) The term "sequence" includes nucleotide and amino acid sequences. In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB search of a DNA sequence to calculate percent identity are: Matrix=Unitary, k- tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, and Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter. Preferred parameters employed to calculate percent identity and similarity of an amino acid alignment are: Matrix=PAM 150, k- tuple=2, Mismatch Penalty= 1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in amino acid residues, whichever is shorter.

As an illustration, a polynucleotide having a nucleotide sequence of at least 95%

"identity" to a sequence contained in SEQ D NO: 1-62 means that the polynucleotide is identical to a sequence contained in SEQ ID NO: 1-62 or the cDNA except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the total length (not just within a given 100 nucleotide stretch). In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to SEQ ID NO: 1-62, up to 5% of the nucleotides in the sequence contained in SEQ ID NO: 1-62 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.

Further embodiments of the present invention include polynucleotides having at least

80% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to a sequence contained in SEQ ID NO: 1-62. Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the polynucleotides having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%o identity will encode a polypeptide identical to an amino acid sequence contained in the translations of SEQ ID NO: 1-62.

Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide, is intended that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Further embodiments of the present invention include polypeptides having at least 80% identity, more preferably at least 85%o identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence contained in translations of SEQ ID NO: 1-62. Preferably, the above polypeptides should exhibit at least one biological activity of the protein.

In a preferred embodiment, polypeptides of the present invention include polypeptides having at least 90%> similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of SEQ ID NO: 1-62.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. colϊ).

Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al, J. Biol. Chem. 268: 2984-2988 (1993) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1 a. They used random mutagenesis to generate over 3,500 individual IL- 1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." (See Gayle et al., (1993), Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C- terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N- terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Tip, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al, Diabetes 36: 838- 845 (1987); Cleland et al, Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Polynucleotide and Polypeptide Fragments

In the present invention, a "polynucleotide fragment" refers to a short polynucleotide having a nucleic acid sequence contained in that shown in SEQ ED NO: 1-62. The short nucleotide fragments are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. A fragment "at least 20 nt in length," for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in SEQ ID NO: 1-62. These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, and more nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments having a sequence from about nucleotide number 1-50, 51- 100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, to the end of SEQ ID NO: 1-62. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity.

In the present invention, a "polypeptide fragment" refers to a short amino acid sequence contained in the translations of SEQ ID NO: 1-62. Protein fragments may be "freestanding," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50 or 60, amino acids in length. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.

Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of the translations of SEQ ID NO: 1-62 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotide fragments encoding these domains are also contemplated.

Other preferred fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Epitopes & Antibodies

In the present invention, "epitopes" refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human. A preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment. A region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." In contrast, an "immunogenic epitope" is defined as a part of a protein that elicits an antibody response. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81 :3998-4002 (1983).)

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,211.)

In the present invention, antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe, J. G. et al., Science 219:660-666 (1983).)

Similarly, immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al, Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).) A preferred immunogenic epitope includes the secreted protein. The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J, Nucl. Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.

Additional embodiments include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993).

One method for producing an antibody comprises immunizing a non-human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO: 1-62, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ ED NO: 1-62 are generated in said animal. Procedures have been developed for generating human antibodies in non-human animals. The antibodies may be partially human, or preferably completely human. Non-human animals (such as transgenic mice) into which genetic material encoding one or more human immunoglobulin chains has been introduced may be employed. Such transgenic mice may be genetically altered in a variety of ways. The genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization. Antibodies produced by immunizing transgenic animals with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO: 1-62 are provided herein.

Mice in which one or more endogenous immunoglobulin genes are inactivated by various means have been prepared. Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for production and use of such transgenic animals are described in U.S. Patents 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein. Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. The spleen cells may be fused with myeloma cells to produce hybridomas, by conventional procedures.

A method for producing a hybridoma cell line comprises immunizing such a transgenic animal with a immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ ED NO:l-

62; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO: 1-62. Such hybridoma cell lines, and monoclonal antibodies produced therefrom, are encompassed by the present invention. Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques.

Antibodies may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO: 1-62. Disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NO: 1-62.

Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ED NO: 1-62. Examples of such agents are well known, and include but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drugs. The conjugates find use in in vitro or in vivo procedures.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al, Nature 331 :84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0 232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, Bowes melanoma cells and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQΕ70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.

Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N- terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO: 1-62. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO: 1-62 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000- 4,000 bp are preferred. For a review of this technique, see Verma et al., "Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).) Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50- 500 potential causative genes.

Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. The polynucleotides of SEQ ID NO: 1-62 can be used for this analysis of individual humans.

First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix - see Lee et al, Nucl. Acids Res. 6:3073 (1979); Cooney et al, Science 241 :456 (1988); and Dervan et al., Science 251 : 1360 (1991) ) or to the mRNA itself (antisense - Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.

Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class H HLA gene.

Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip" or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response. Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101 :976-985 (1985); Jalkanen, M., et al, J. Cell . Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (^!4C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹I, ^U2In, ^99rnTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ^99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.

Moreover, polypeptides of the present invention can be used to treat disease.

For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B), to inhibit the activity of a polypeptide (e.g., an oncogene), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.

Biological Activities

The polynucleotides and polypeptides of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides could be used to treat the associated disease.

Immune Activitiv A polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

A polynucleotide or polypeptide of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells. A polypeptide or polynucleotide of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIN infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCEDs), Wiskott- Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polypeptide or polynucleotide of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotide or polypeptide of the present invention could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, a polynucleotide or polypeptide of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment of heart attacks (infarction), strokes, or scarring. A polynucleotide or polypeptide of the present invention may also be useful in treating or detecting autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,

Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polypeptide or polynucleotide of the present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

A polynucleotide or polypeptide of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly, a polypeptide or polynucleotide of the present invention may also be used to modulate inflammation. For example, the polypeptide or polynucleotide may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)

Hyperproliferative Disorders

A polypeptide or polynucleotide can be used to treat or detect hyperproliferative disorders, including neoplasms. A polypeptide or polynucleotide of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions.

Alternatively, a polypeptide or polynucleotide of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative disorders can also be treated or detected by a polynucleotide or polypeptide of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above. Infectious Disease

A polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, the polypeptide or polynucleotide of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention. Examples of viruses, include, but are not limited to the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalo virus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsielia, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal. These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

Moreover, parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

Preferably, treatment using a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997).) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. A polynucleotide or polypeptide of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stroke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease,

Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotide or polypeptide of the present invention.

Chemotaxis

A polynucleotide or polypeptide of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality. A polynucleotide or polypeptide of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.

It is also contemplated that a polynucleotide or polypeptide of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders.

Thus, a polynucleotide or polypeptide of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al, Current Protocols in Immunology 1(2), Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure polypeptide level or activity in a sample

(e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.

Other Activities

A polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

A polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

A polypeptide or polynucleotide of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

A polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, co factors or other nutritional components.

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 80%>, preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: 1-62.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO: 1-62 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the clone sequence and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO: 1-62 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the start codon and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NO: 1-62.

Similarly preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:l-62 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NO: 1-62.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: 1-62.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: 1-62.

A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ED NO: 1-62 beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NO: 1-62.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ED NO: 1-62.

Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.

A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: 1-62, which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95%> identical to said selected sequence. Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO: 1-62.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of a nucleotide sequence of SEQ ID NO: 1 -62.

The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ED NO: 1-62. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in an amino acid sequence translated from SEQ ID NO: 1-62. Also preferred is a polypeptide, wherein said sequence of contiguous amino acids is included in acids in an amino acid sequence translated from SEQ ID NO: 1-62, in the range of positions beginning with the residue at about the position of the first amino acid of the secreted portion and ending with the residue at about the last amino acid of the open reading frame.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95%) identical to a sequence of at least about 30 contiguous amino acids in an amino acid sequence translated from SEQ ID NO:l-62.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in an amino acid sequence translated from SEQ ID NO: 1-62.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95%o identical to acids in an amino acid sequence translated from SEQ ED NO: 1-62.

Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90%> identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NO: 1-62, which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90%> identical to said sequence of at least 10 contiguous amino acids.

Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90%> identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NO:l-62. Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90%> identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NO: 1-62.

Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NO: 1-62.

In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90%> identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NO: 1-62.

Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host. - 46 -

Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from SEQ ED NO: 1-62.

Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from SEQ ID NO: 1-62. The isolated polypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLE 1

Identification and Characterization of Polynucleotides Regulated by in vivo DSS Treatment For the induction of colitis, DSS (MW 40,000) is dissolved in the drinking water and given to mice ad libitum for a period of 7 days. The DSS water is then replaced with regular drinking water. Distinct mouse strains demonstrate differential susceptibility to DSS-colitis. Three percent DSS is sufficient for the induction of acute colitis in C57BL/6 mice, whereas 5% DSS is required for the induction of acute colitis in BALB/c mice.

For the purpose of this study, samples of colon were harvested from cohorts of C57BL/6 mice treated with 3% DSS. Tissues were harvested at day 0, day 4, day 8 or day 12 - a schedule designed to encompass the full range of induction of intestinal damage through recovery.

Damage to colonic tissue is a hallmark of IBD. The mouse model of DSS-induced colitis gives rise to damaged colonic tissue and as such it is one of the more useful models for studying IBD. Mice were treated with DSS in their drinking water as described above. At various intervals during DSS-treatment, cohorts of mice were sacrificed and the colons removed by dissection. Freshly dissected colonic tissue was immediately placed in GT buffer (4.5M guanidinium isothiocyanate, 50mM sodium citrate, 0.5%w/v sodium sarcosyl, 2%> 2-beta- mercaptoethanol) and homogenized. Homogenized lysates were spun briefly to remove large debris before being layered onto a CsCl gradient. RNA was extracted using conventional methods and was subsequently used for TOGA analysis.

Features of DSS-induced colitis

Weight loss is apparent in mice beginning at day 4. Histological analysis of the intestine reveals the presence of early patchy lesions identifiable by the loss of epithelial cells and goblet cells. By day 8, weight loss is fairly severe (approximately 20% reduction) and the mice appear moribund. Histologically, the gut epithelium is almost totally destroyed at this stage. There is evidence of a large mixed inflammatory cell infiltration into the lamina propria and submucosa. The inflammatory cell infiltrate appears to be composed primarily of T cells, B cells and granulocytes. By day 12, weight gain is apparent as the mice recover. At this later stage, crypt recovery and epithelial regeneration provide histological evidence of the beginning of repair processes.

Isolated RNA was analyzed using a method of simultaneous sequence-specific identification of mRNAs known as TOGA (TOtal Gene expression Analysis) described in Sutcliffe, J.G., et al Proc Natl Acad Sci USA 2000 Feb 29; 97(5): 1976-1981, International published application PCT US99/23655, U.S. Patent No. 5,459,037, U.S. Patent No.

5,807,680, and U.S. Patent No. 6,030,784, hereby incorporated herein by reference.

Preferably, prior to the application of the TOGA method or other methods, the isolated RNA was enriched to form a starting polyA-containing mRNA population by methods known in the art. In such a preferred embodiment, the TOGA method further comprises an additional Polymerase Chain Reaction ("PCR") step performed using one of four 5' PCR primers and cDNA templates prepared from a population of antisense complimentary RNA ("cRNAs"). A final PCR step using one of a possible 256 5' PCR primers and a universal 3' PCR primer produced as PCR products, cDNA fragments that corresponded to a 3'-region of the starting mRNA population. The produced PCR products were then identified by a) the sequence of at least the 5' seven base pairs, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences.

The method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags (ESTs) of the 3' end of mRNAs. DSTs that showed changes in relative levels following DSS treatment were selected for further study. The intensities of the laser-induced fluorescence of the labeled PCR products were compared across sample isolated at different time intervals after treatment.

In general, double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture of all 48 5'- biotinylated anchor primers of a set to initiate reverse transcription. One such suitable set is G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T- T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 63), where V is A, C or G and N is A, C, G or T. One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double stranded cDNA.

Each biotinylated double stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG. The 3' fragments of cDNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate. Suitable streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass particles. A preferred streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Lake Success, NY). After washing the streptavidin-coated substrate and captured biotinylated cDNA fragments, the cDNA fragment product was released by digestion with Notl, which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs. The 3' Mspl-Notl fragments, which are of uniform length for each mRNA species, were directionally ligated into Clal-, Notl-cleaved plasmid pBC SK⁺ (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli SURE cells (Stratagene). The ligation regenerates the Notl site, but not the Mspl site. Each library contained in excess of 5 x 10⁵ recombinants to ensure a high likelihood that the 3' ends of all mRNAs with concentrations of 0.001%> or greater were multiply represented. Plasmid preps (Qiagen) were made from the cDNA library of each sample under study.

An aliquot of each library was digested with Mspl, which effects linearization by cleavage at several sites within the parent vector while leaving the 3' cDNA inserts and their flanking sequences, including the T3 promoter, intact. The product was incubated with T3 RNA polymerase (MEGAscript kit, Ambion) to generate antisense cRNA transcripts of the cloned inserts containing known vector sequences abutting the Mspl and Notl sites from the original cDNAs.

At this stage, each of the cRNA preparations was processed in a three-step fashion. In step one, 250 ng of cRNA was converted to first-strand cDNA using the 5' RT primer (A-G- G-T-C-G-A-C-G-G-T-A-T-C-G-G, (SEQ ID NO: 64). In step two, 400 pg of cDNA product was used as PCR template in four separate reactions with each of the four 5' PCR primers of the form G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 65), each paired with a "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO: 66).

In step three, the product of each subpool was further divided into 64 subsubpools (2ng in 20μl) for the second PCR reaction, with 100 ng each of the fluoresceinated "universal" 3' PCR primer, the oligonucleotide (SEQ ID NO: 66) conjugated to 6-FAM and the appropriate 5' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 67), using a program that included an annealing step at a temperature X slightly above the T_m of each 5' PCR primer to minimize artifactual mispriming and promote high fidelity copying. Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).

The products from the final polymerase chain reaction step for each of the tissue samples were resolved on a series of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data were collected using the GeneScan software package (ABI) and normalized for amplitude and migration. Complete execution of this series of reactions generated 64 product subpools for each of the four pools established by the 5' PCR primers of the first PCR reaction, for a total of 256 product subpools for the entire 5' PCR primer set of the second PCR reaction.

The mRNA samples from each timepoint after DSS treatment as described above were analyzed. Table 1 is a summary of the expression levels of 414 mRNAs determined from cDNA. These cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule, as well as the relative amount of the molecule produced at different time intervals after treatment. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. The digital address length of the fragment was determined by interpolation on a standard curve and, as such, may vary + 1-2 b.p. from the actual length as determined by sequencing.

For example, the entry in Table 1 that describes a DNA molecule identified by the digital address Mspl AGTG 244, is further characterized as having a 5' terminus partial nucleotide sequence of CGGAGTG and a digital address length of 244 b.p. The DNA molecule identified as Mspl AGTG 244 is further described as being expressed at increasing levels at days 0, 4 and 8 with a moderate decline at day 12. However, the treatment results in a different pattern of expression of Mspl AGTA 187, which declines on days 4 and 8 from the relatively high level seen at day 0, but increases at day 12.

Similarly, the other 412 DNA molecules identified in Table 1 by their Mspl digital addresses are further characterized by the pattern of the level of gene expression on days 0, 4, 8 and 12 following the end of the DSS treatment. Many of the isolated clones were further characterized in Tables 2 and 3. Their nucleotide sequences are provided as SEQ ID NO:l- 62 in the Sequence Listing below.

The data shown in Figure 1 were generated with a 5'-PCR primer (C-G-A-C-G-G-T- A-T-C-G-G-C-G-C-G, SEQ ID NO: 68) paired with the "universal" 3' primer (SEQ ID NO: 66) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus. PCR reaction products were resolved by gel electrophoresis on 4.5% acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer).

The results of TOGA analysis using a 5' PCR primer with parsing bases CGCG (SEQ ED NO: 68) are shown in Figure 1, which presents the results of TOGA analysis using a 5' PCR primer with parsing bases CGCG, showing PCR products produced from mRNA extracted from (top to bottom panels) colons isolated from mice 0 (Figure 1A), 4 (Figure IB) , 8 (Figure IC) or 12 days (Figure ID) after a seven day course of treatment with DSS. The vertical index line indicates a PCR product of about 458 b.p. that is present on day 0, reduced on day 4, much increased on day 8 and whose expression relatively decreases but is still elevated on day 12.

Some products, which were also differentially represented, appeared to migrate in positions that suggest that the products were novel based on comparison to data extracted from GenBank. In these cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands. Examples are found in Table 4, below. In order to verify that the clones isolated are from the same peak, PCR primers were designed based on the determined sequence and PCR was performed using the cDNA produced in the first PCR reaction as substrate. For example, for the 458 b.p. product disclosed above, oligonucleotides were synthesized using the universal 3' PCR primer and a 5' PCR primer corresponding to the 5' PCR primer in the second PCR step extended at the 3' end with additional nucleotides from the clone sequence 3' to the parsing bases (in this case, CGCG). This extended 5' PCR primer had the sequence: GATCGAATCC GGCGCGCACG GGGACCAGAC (SEQ ID NO: 78). The products were separated by electrophoresis and the length of the clone was compared to the length of the original PCR product as shown in Figure 2. Figure 2A shows the length (as peak position) of the PCR product derived as described above. Figure 2B shows the PCR products produced using the original PCR primers, SEQ ID NO: 68 and SEQ ID NO: 66 (compare to Figure 1 A). In Figure 2C, the traces from the top and middle panels are overlaid, demonstrating that the PCR product using the sequence of the isolated clone is the same length as the original PCR product.

The same procedure was used to verify candidate matches to database entries. The results are shown in Table 4, below. In each case, oligonucleotides were synthesized using the universal 3' PCR primer and a 5' PCR primer corresponding to the 5' PCR primer in the second PCR step extended at the 3' end with additional nucleotides from the sequences adjacent to the terminal Mspl sites in the identified corresponding GenBank sequences. In three cases (IMX2_33, SEQ ID NO:21, IMX2_34, SEQ ID NO:61 and IMX2_70, SEQ ID NO:62) the DST sequence listed was obtained from the verified database match sequence in GenBank.

Figure 3 is a graphical representation of the results of Northern Blot analysis of clone EMX 2_46, SEQ ID NO: 10, where an agarose gel containing poly A enriched mRNA from the four experimental samples (0, 4, 8 or 12 days post-treatment) as well as size standards was blotted after electrophoresis and probed with radiolabelled IMX 2_46, SEQ ED NO: 10, imaged using a phosphorimager and quantified. Quantitative results showing the relative expression levels of the 1.6 kb transcript were: 0 day, 64; 4 days, 53; 8 days, 223; and 12 days, 269. The amount of RNA loaded on the gel was determined by probing for cyclophilin ("eye").

EST = Expressed Sequence Tag, NA = Not Applicable, R = Reverse Transcπptase-Polymerase Chain Reaction, N = Northern Blot Analysis *Relatιve DST Amount = Relative DST Amount for Northern blots have been corrected for background and normalized to the hybridization signal of cyclophihn The values were obtained by exposing the Northern blot to a phosphoπmaging screen and quantitated using the Phosphorimager SI

EST = Expressed Sequence Tag, N/A = Not Applicable

EST = Expressed Sequence Tag

EST = Expressed Sequence Tag

EXAMPLE 2 RT-PCR Validation

RT-PCR validation of cloned DSTs was performed, and results are presented in Table 2. The starting amount of template was chosen based on a control curve that was generated to accurately define the linear range of amplication for the given cloned DST. Based upon the intensity of the peak cloned from the TOGA™ panel, the following amounts of template were chosen:

TOGA peak intensity Low TcDNAl High TcDNAl

0 - 400 2000 pg 5000 pg

300-1000 400 pg 2000 pg

> 1000 50 pg 250 pg

The PCR primers used for validation of cloned DSTs are listed in Table 5. Duplicate reaction mixtures were assembled using the appropriate low and high concentration of cDNA template chosen from the time point sample showing the strongest TOGA™ signal. The reaction mixtures were cycled for 23, 26, 29, 32, 35, and 38 cycles. The resulting amplification products from the duplicate reactions were quantitated and plotted against the cycle number to generate a standard curve. From these data, the cycle number and cDNA concentration combination which yielded acceptable levels of PCR product within the linear range of amplification were chosen for RT-PCR validation across the various time-points.

The RT-PCR validation consisted of assembling triplicate reactions using the chosen concentration of cDNA cycled to the defined cycle number. For example, the data in Table 2 for IMX2_55 (SEQ ID NO: 13) were generated using 50 pg cDNA template and 29 cycles. An internal control primer pair amplified under the same conditions was also performed to provide the basis for normalizing any differences between the cDNA templates.

EXAMPLE 3

RT-PCR Analysis Using Fluorimetry Two DSTs were validated using an alternative protocol. The primers used for RT- PCR are listed in Table 5. For each DST examined, the optimal annealing temperature and reagent conditions were determined for the corresponding set of primers (see Table 5) based on results from a preliminary experiment. In eight separate reactions, each set of primers was assayed to find the optimal conditions by adjusting the following four parameters: primer concentration, dNTP concentration, MgCl? concentration, and Taq polymerase. Once optimal conditions were determined, each DST was run in duplicate multiple simultaneous reactions which usually included at least four dilutions of template, plus control reactions lacking template, and six sequential data points for numbers of cycles.

Reactions were performed using "Hot Start" PCR with the Clontech TaqStart antibody system (Cat. #5400-1). Each reaction contained lμl of the cDNA library dilution as template, determined amounts of AmpliTaq DNA polymerase (cat. #N808-0156), MgCl₂, dNTPs (GibcoBRL cat. #10297-018), primer, and Clontech TaqStart Antibody in a 20μl reaction volume using lOx Taq buffer II (without MgCl₂). Typically, a master mix containing all components except the template was prepared and aliquoted. Various templates were then added to these master mix samples and 20 μl volumes were subsequently dispensed into individual reaction tubes. During the PCR run, tubes were removed sequentially on a predetermined schedule in order to quantitate expression of the target DST over a "window" of cycles. After amplification, the samples were quantified via fluorimetry.

PCR was performed at annealing temperatures about 5 degrees above the lowest melting temperature of each primer pair using the following program: 1) 95 degrees Celsius, 3 minutes; 2) 95 degrees Celsius, 30 seconds; 3) TM+5 degrees Celsius, 30 seconds; 4) 72 degrees Celsius, for a time dependent on target length at 16 bp/second; 5) repeat steps 2-4 33 more cycles; 6) 72 degrees Celsius, 3 minutes; 7) 14 degrees Celsius, forever.

Following temperature cycling, 2μl of the PCR reaction was added to 140μl of a 1 :280 dilution of PicoGreen (Molecular Probes cat. #P-11495 (10xl00μl)) in TE pH 7.5 in a 96-well Costar UV microtiter plate (Fisher cat. #07-200623). The samples were mixed gently for 1.5 minutes and allowed to equilibrate at room temperature in the dark for 15 minutes. The concentration of the PCR products was quantified by fluorimetry using a PerSeptive Biosystems CytoFluor series 4000 multi-well plate reader. Background fluorescence was determined by using duplicate control samples that were cycled with all reaction components except the template. The mean value from these duplicate background control samples was subtracted from the corresponding experimental values prior to analyzing results. The sensitivity of the PicoGreen dsDNA assay is reported to be 250pg/ml (50pg dsDNA in a 200μl assay volume) using a fluorescence microplate reader such as was used in these measurements.

The results of the quantified RT-PCR of MX 2_48 for a 1 :2000 dilution of the library are shown in Figure 4 (in arbitrary fluorescence units) and in Table 6 (normalized to the control value at each time point). The results of the quantified RT-PCR of IMX 2_74 for a 1 :500 dilution of the library are shown in Figure 5 (in arbitrary fluorescence units) and in Table 6 (normalized to the control value at each time point).

EXAMPLE 4 Extended Sequence Clone for IMX 2 4

The clone was isolated from a D4 D8 DSS colon library using the Clonecapture procedure from Edge Biosystems. The library was constructed by Edge Biosystems for Immunex from RNA isolated from D4 and D8 inflamed colons from DSS treated C57BL/6 mice. The clone is in an Edge vector pEAK12.

IMX2_04 DST (SEQ ID NO:l) matches extended sequence for IMX2_04 from base 688 to 957 (SEQ ID NO:2) (See Table 7, above). There is an open reading frame starting at base 1 that may encode a partial gene product. The amino acid sequence of the encoded partial protein is given in SEQ ID NO: 129.

When the extended sequence was compared to NCBI nr database w/BLAST, linear segments of the nucleotide sequence for bases 1-936 show about 70% identity with segments of the nucleotide sequence of a cosmid clone from human chromosome 19, AC007565.1 (See Table 7, above). This indicates that the human homologue of IMX2_4 is, at least in part, found on the cosmid. However, several exons predicted on the cosmid (by GRAIL program or by some homology to mouse ESTs) are skipped by the linear sequence of IMX2-4- ElO.seq. It appears that one predicted, yet skipped exon, is real in that a perfect match is found in the DERWENT database. The Derwent entry is for a "secreted" molecule with the protein fragment in the Derwent protein entry being a signal peptide containing amino acid sequence not found on the cosmid (or the nucleotide Derwent entry). However, the Derwent nucleotide entry also has a match to another more 5' segment of the cosmid which does show a match with the 5' end of IMX2-4-E10.seq at the 75% identity level. These forms may represent different splice variants of a secreted protein.

EXAMPLE 5 Extended Sequence Clone for IMX 2 36

The IMX2_36.EXT sequence information was derived from the clone IMX2_36pT7T3-2.seq which is an EST clone derived from FVB/N mouse proximal colon obtained from IMAGE consortium. The accession number for the EST is AA529850, and the clone id is MAGE:921608. The EST is in the pT7T3 vector. The IMX2_36 DST (SEQ ID NO:6) matches the extended sequence for IMX2_36.EXT (SEQ ID NO 7) from base 293 to 427 (See Table 7, above). Blast of the EST to GenBank gives hits to 'mouse KAR (killer activating receptor)' and 'DAP12 protein' (See Table 7, above).

EXAMPLE 6 Extended Sequence Clone for IMX 2 43

The clone was isolated from a D4/D8 DSS colon library using the Clonecapture procedure from Edge Biosystems. The library was constructed by Edge Biosystems for Immunex from RNA isolated from pooled D4 and D8 inflamed colons from DSS treated C57BL/6 mice The clone is m the Edge vector pEAK12 The IMX2_43 DST (SEQ ID NO:8) matches the IMX2_43 extended sequence (SEQ ID NO:9) from base 1079 to 1298 (See Table 7, above).

EXAMPLE 7

Extended Sequence Clone for IMX 2 46 The IMX2_46.EXT sequence information was deπved from the two clones IMX2_46pT7T3-6.seq and IMX2_46pT7T3-7.seq, which are EST clones in the pT7T3 vector that were obtained from IMAGE consortium. IMX2_46pT7T3-6.seq (accession number AA290194, clone id IMAGE:750847) was deπved from C57BL/6 mouse lymph node. IMX2_46pT7T3-7.seq (accession number AA174968, clone id IMAGE: 617717) was deπved from C57BL/6 mouse spleen. The IMX2_46 DST (SEQ ID NO: 10) aligns with the IMX2_46.EXT extended sequence (SEQ ID NO:l 1) from base 157 to 561 (See Table 7, above).

Blast of ESTs against GenBank gives hits to human TOSO: regulator of fas-mduced apoptosis (See Table 7, above).

EXAMPLE 8

Extended Sequence Clone for IMX 2 55 The IMX2_55.EXT extended sequence information was denved from the two clones IMX2_55pT7T3-8.seq and IMX2_55pT7T3-24.seq, which are EST clones that were obtained from IMAGE consortium. IMX2_55pT7T3-8.seq (accession number AA823573, clone id IMAGE: 1079189) was derived from FVB irradiated mouse colon. IMX2_55pT7T3-24.seq (accession number AA690372, clone id IMAGE: 1164692) was derived from from FVB mouse proximal colon.

The accession number for the EST for IMX2_55pT7T3-8.seq is AA823573, and the clone id is AGE:1079189. The accession number for the EST for IMX2_55pT7T3-24.seq is AA690372, and the clone id is IMAGE: 1164692. The both ESTs are in the pT7T3 vector and were obtained from IMAGE consortium. IMX2_55pT7T3-8.seq clone was derived from FVB irradiated mouse colon and IMX2_55pT7T3-24.seq clone was derived from FVB mouse proximal colon. The IMX2_55 DST (SEQ ID NO: 13) aligns bases of 322 to 744 with the extended sequence IMX2_55.EXT (SEQ ID NO: 14) (See Table 7, above).

Blast of the extended IMX2_55 sequence to GenBank disclosess ESTs with homology to C reactive protein (See Table 7, above).

EXAMPLE 9 Extended Sequence Clone for IMX 2 57 PCR primers were designed for IMX2_57 based on sequence information obtained from the sequences of two EST clones that were commercially unavailable. The EST clones used for deriving sequence information were (1) an EST with accession number AA240177 and clone id IMAGE:679264 derived from mouse liver and (2) an EST with accession number AV005227 and clone id 0910001G08 derived from C57BL/6 spleen.

The primers used were forward primer IMX2_57-FP and reverse primer IMX2_57-RP which prime off the sequence obtained from electronically assembling the DST and the EST AV005227. The PCR reaction was performed on cDNA derived from RNA prepared from C57BL/6 mouse colon. The PCR product was cloned into the pGEM vector resulting in the clone IMX2_57PCR1. The IMX2_57 DST, (SEQ ID NO: 15) aligns with bases 283 to 408 of the extended sequence IMX2_57.EXT (SEQ ID NO: 16) (See Table 7, above). Blast of assembled ESTs to Genbank gives hits to 'human chymotrypsin-like (CTRL) mRNA' and 'human proteosome-like subunit (MECL-1), chymotrypsin-like protease (CTRL- 1) and protein-serine kinase (PSK-H1) last exon (See Table 7, above).

EXAMPLE 10 Extended Sequence Clone for IMX 2 61 The IMX2_61 extended sequence information was derived from the EST clone IMX2_61pBS-47.seq, an EST clone that was obtained from IMAGE consortium. The accession number for the EST for IMX2_61pBS-47.seq is AA981092, and the clone ID is IMAGE: 1279287. The IMX2_61pBS-47.seq clone was derived from WEHI3 mouse macrophage cells. The IMX2_61 DST (SEQ ID NO: 17) aligns with bases 204 to 425 of the IMX2_61 extended sequence (SEQ ID NO: 18) (See Table 7, above).

EXAMPLE 11

Extended Sequence Clone for IMX 2 17: Crvpt-ductin alpha scavenger receptor (CRP-ductin)

TOGA analysis indicated that IMX2_17 (SEQ ID NO:3) coπesponds to Mus musculus CRP-ductin-alpha mRNA, accession number U37438 (Table 3). CRP-ductin localizes to the apical portion of crypt cells in the small intestine. In the colon, it is seen predominantly in surface epithelial cells (EC). It is also seen in the apical portion of the EC lining pancreatic and larger hepatic ducts. The CRP-ductin-alpha sequence predicts a mosaic protein with a short cytoplasmic region, a transmembrane domain and a large extracellular region composed of many repeats. The extracellular region contains 21 potential N- glycosylation sites in the C-terminal half of the protein. There are also two potential phosphorylation sites in the cytoplasmic domain.

Forward primer mCRP.5179-FP and reverse primer mCRP.6106-RP were used to PCR a 936 bp product from a cDNA template derived from RNA isolated from C57BL/6 colon. The PCR product was subcloned into the pGEM vector and the sequence was verified before being used as a probe for Northern blot analysis. Figure 6 presents the results of Northern blot analysis of clone IMX 2_17, SEQ ID NO: 3, where an agarose gel containing poly A enriched mRNA from the experimental samples from and size standards was blotted after electrophoresis, probed with a labeled probe coπesponding to U37438 bases 5179-6106, imaged using a phosphorimager and quantified. Figure 6A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with CD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knock-out mice without colitis and mdr knock-out mice with colitis. Figure 6B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8% DSS, and Balb/c mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment. The predicted transcript size for IMX2_17 CRP-ductin is 6.6 Kb; the actual transcript size found in this study was approximately 6.5 Kb.

Northern blots were performed on mRNA samples from several models of IBD. Analysis of samples from C57BL/6 mice with DSS colitis showed high levels of constitutive expression in the large intestine, maximal at day 8 of DSS colitis (Figure 6 A), consistent with the results of the initial TOGA analysis (Table 1). Analysis of samples from Balb/c mice with DSS colitis showed similar results (Figure 6B).

Analysis of samples from C57BL/6 mice with αCD3 ileitis showed low levels of constitutive expression in the small intestine that increases early in inflammation (Figure 6A) reaching a maximum at six hours and declining thereafter. Analysis of samples from Balb/c mice with αCD3 ileitis showed similar, though less intense, results (Figure 6B).

Constitutive expression was seen in FVB large intestine samples (Figure 6A).

Increased expression in healthy mdr knock-out mice with no signs of colitis, with little further increase in expression in mdr knock-out mice with active colitis (Figure 6A).

The expression shown on the Northern blot of Figure 6 was quantified and normalized to the amount of G3PDH in each lane. The normalized results are shown in Table 8, below.

EXAMPLE 12

Extended Sequence Clone for IMX 2 22

IMX2 22 Hematopoietic Progenitor Kinase HPKl

TOGA analysis indicated that IMX2_22 coπesponds to Mus musculus mRNA for serine/threonine kinase, accession number Y09010 (Table 3). HPKl is a hematopoietic protein kinase activating the SAPK/JNK pathway.

Forward primer mHPKl .1640-FP and reverse primer mHPKl .2420-RP were used to

PCR a 780 bp product from a cDNA template was derived from RNA isolated from C57BL/6 colon. The PCR product was subcloned into the pGEM vector and was sequence verified before being used as a probe for Northern blot analysis. The predicted transcript size for IMX2_22 HPKl was 2.7 Kb and published transcript sizes are 2.8 Kb and 3.6 Kb. The actual transcript size found in this study was 2.8Kb.

Figure 7 presents the results of Northern blot analysis of clone IMX 2_22, SEQ ED NO: 4, where an agarose gel containing poly A enriched mRNA from the experimental samples and size standards was blotted after electrophoresis, probed with a labeled probe coπesponding to bases 1640-2420 of Y09010, imaged using a phosphorimager and quantified. Figure 7A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knock-out mice without colitis, mdr knock-out mice with colitis and C57BL/6 spleen. Figure 7B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment and C57BL/6 normal lymphoid tissue samples (MLM, PP, spleen and thymus).

Northern blots were performed on mRNA samples from several models of IBD. Analysis of samples from C57BL/6 mice with DSS colitis showed low levels of constitutive expression in the large intestine, maximal at day 12 of DSS colitis (Figure 7A), not very consistent with the results of the initial TOGA analysis (Table 1). Analysis of samples from Balb/c mice with DSS colitis showed similar results with a maximum at day 8 (Figure 7B).

Analysis of samples from C57BL/6 mice with αCD3 ileitis showed constitutive expression in the small intestine that decreases at six hours (Figure 7 A). Analysis of samples from Balb/c mice with αCD3 ileitis showed similar results (Figure 7B).

Increased expression in mdr knock-out mice with active colitis (Figure 7A), in contrast to little change in corresponding TOGA analysis (data not shown).

Strong expression was found in lymphoid tissues in MLN, PP, thymus, especially the spleen (Figure 7 A & B).

The expression shown on the Northern blot of Figure 7 was quantified and normalized to the amount of G3PDH in each lane. The normalized results are shown in Table 9, below.

EXAMPLE 13

Extended Sequence Clone for IMX 2 28

Down-Regulated in Adenoma protein (DRA)

TOGA analysis indicated that IMX2_28 corresponds to Mus musculus DRA down- regulated in adenoma protein, accession number AF136751 (Table 3).

Forward primer Dra.1551 -FP and reverse primer Dra.2390-RP were used to PCR an

840 bp product from a cDNA template derived from RNA isolated from C57BL/6 colon. The PCR product was subcloned into the pGEM vector and was sequence verified before being used as a probe for Northern blot analysis. The predicted transcript size for IMX2_28 DRA is 2.6 Kb; the actual transcript size found in this study was approximately 3 Kb.

Figure 8 presents the results of Northern blot analysis of clone IMX 2_28, SEQ ID NO: 5, where an agarose gel containing poly A enriched mRNA from the experimental samples and size standards was blotted after electrophoresis, probed with a labeled probe corresponding to bases 1551-2390 of AF136751, imaged using a phosphorimager and quantified. Figure 8A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knock-out mice without colitis and mdr knock-out mice with colitis. Figure 8B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8% DSS, and Balb/c mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment. Northern blots were performed on mRNA samples from several models of IBD Analysis of samples from C57BL/6 mice with DSS colitis showed high levels of constitutive expression in the large intestine that decrease with inflammation, minimal at day 8 of DSS colitis (Figure 8 A), consistent with the results of the initial TOGA analysis (Table 1) Analysis of samples from Balb/c mice wfth DSS colitis showed the same pattern (Figure 8B)

Analysis of samples from C57BL/6 mice with αCD3 ileitis showed levels of constitutive expression in the small intestine lower than that seen in the large intestine with DSS colitis Expression decreases with inflammation rapidly by 6 hours and remains low (Figure 8 A) Analysis of samples from Balb/c mice with αCD3 ileitis showed the same pattern (Figure 8B) TOGA analysis in this model (data not shown) did not show a corresponding peak

Constitutive expression was seen in FVB large intestine samples (Figure 8A) Constitutive expression was seen in healthy mdr knock-out mice with no signs of colitis, with a dramatic decrease in expression seen m mdr knock-out mice with active colitis (Figure 8A) TOGA analysis in this model (data not shown) did not show a corresponding peak

The expression shown on the Northern blot of Figure 8 was quantified and normalized to the amount of G3PDH in each lane The normalized results are shown in Table 10, below

EXAMPLE 14 Extended Sequence Clone for IMX2 33

Secretory Leukocyte Protease Inhibitor (SLPI)

TOGA analysis indicated that IMX2_33 corresponds to Mus musculus secretory leukocyte protease inhibitor, accession number U73004 (Table 3). Secretory leukocyte protease inhibitor is an epithelial cell and macrophage derived inhibitor of leukocyte serine proteases. SLPI expression is suppressed by gamma-IFN. SLPI is an LPS induced gamma- IFN suppressible phagocyte product that serves to inhibit LPS responses.

Forward primer mSLPI.447-FP and reverse primer mSLPI.800-RP were used to PCR a 350 bp product from a cDNA template was derived from RNA isolated from C57BL/6 colon. The PCR product was subcloned into the pGEM vector and the sequence was verified before being used as a probe for Northern blot analysis. The probe used corresponded to bases 447-800 of U73004. The predicted transcript size for IMX2_33 SLPI is 1.1 Kb; the actual transcript size found in this study was 1.1 Kb. Figure 9 presents the results of Northern blot analysis of clone IMX 2_33, SEQ ID NO:21, where an agarose gel containing poly A enriched mRNA from the experimental samples and size standards was blotted after electrophoresis, probed, imaged using a phosphorimager and quantified. Figure 9A shows the results from C57BL/6 mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with αCD3 ileitis 0, 6, 30 or 72 hours after treatment, as well as samples from large intestines from FVB, mdr knock-out mice without colitis and mdr knock-out mice with colitis. Figure 9B shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8% DSS. and Balb/c mice with αCD3 ileitis (0, 6, 30, 72 hours).

Northern blots were performed on mRNA samples from several models of IBD. Analysis of samples from C57BL/6 mice with DSS colitis showed minimal constitutive expression in the large intestine that increases significantly with inflammation, maximal at day 8 of DSS colitis and still high at day 12 (Figure 9A), consistent with the results of the initial TOGA analysis (Table 1). Analysis of samples from Balb/c mice with DSS colitis showed the same pattern (Figure 9B).

Analysis of samples from C57BL/6 mice with αCD3 ileitis showed low to moderate levels of constitutive expression in the small intestine with less regulation than seen in the large intestine with DSS colitis. There was a significant increase with inflammation maximal at 30 hours, then decreasing (Figure 9A). Analysis of samples from Balb/c mice with αCD3 ileitis showed the same pattern (Figure 9B). The pattern was consistent with that seen in TOGA analysis in this model (data not shown).

Minimal constitutive expression was seen in healthy mdr knock-out mice with no signs of colitis, with an increase in expression seen in mdr knock-out mice with active colitis (Figure 9A). The pattern was consistent with that seen in TOGA analysis in this model (data not shown).

The expression shown on the Northern blot of Figure 9 was quantified and normalized to the amount of G3PDH in each lane. The normalized results are shown in Table 11, below.

EXAMPLE 15

Extended Sequence Clone for IMX 2 48

IMX2 48 macrophage inflammatory protein 2 (MIP2)

TOGA analysis indicated that IMX2_48 coπesponds to Mus musculus MIP-2, macrophage inflammatory protein 2, accession number X53798 (Table 3).

Forward primer MIP2.61-FP and reverse primer MIP2.345-RP were used to PCR a

284 bp product from a cDNA template was derived from RNA isolated from C57BL/6 colon. The PCR product was subcloned into the pGEM vector and the sequence was verified. The predicted transcript size for IMX2_48 MIP2 is 1.1 Kb.

Claims

We claim:

1. An isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47. SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62.

2. An isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ DD NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ED NO:17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ED NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ED NO:33, SEQ ED NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ED NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ED NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ED NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ED NO:61 and SEQ ID NO:62.

3. An isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to the isolated nucleic acid molecule of claim 1.

4. An isolated nucleic acid molecule at least ten bases in length that is hybridizable to the isolated nucleic acid molecule of claim 1 under stringent conditions.

5. An isolated nucleic acid molecule encoding the polypeptide of claim 2.

6. An isolated nucleic acid molecule encoding a fragment of the polypeptide of claim 2.

7. An isolated nucleic acid molecule encoding a polypeptide epitope of the polypeptide of claim 2.

8. The polypeptide of claim 2 wherein the polypeptide has biological activity.

9. An isolated nucleic acid encoding a species homologue of the polypeptide of claim 2.

10. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N- terminus.

11. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.

12. A recombinant host cell comprising the isolated nucleic acid molecule of claim 1.

13. A method of making the recombinant host cell of claim 12.

14. The recombinant host cell of claim 12 comprising vector sequences.

15. The isolated polypeptide of claim 2, wherein the isolated polypeptide comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

16. An isolated antibody that binds specifically to the isolated polypeptide of claim 2.

17. The isolated antibody of claim 16 wherein the antibody is a monoclonal antibody.

18. The isolated antibody of claim 16 wherein the antibody is a polyclonal antibody.

19. A recombinant host cell that expresses the isolated polypeptide of claim 2.

20. An isolated polypeptide produced by the steps of:

(a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and

(b) isolating the polypeptide.

21. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 2 or the polynucleotide of claim 1.

22. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:

(a) determining the presence or absence of a mutation in the polynucleotide of claim 1 ; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

23. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:

(a) determining the presence or amount of expression of the polypeptide of claim 2 in a biological sample; and

(b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

24. A method for identifying a binding partner to the polypeptide of claim 2 comprising: (a) contacting the polypeptide of claim 2 with a binding partner; and

(b) determining whether the binding partner effects an activity of the polypeptide.

25. The gene corresponding to the cDNA sequence of the isolated nuclei acid of claim 1.

26. A method of identifying an activity of an expressed polypeptide in a biological assay, wherein the method comprises:

(a) expressing the polypeptide of claim 2 in a cell;

(b) isolating the expressed polypeptide;

(c) testing the expressed polypeptide for an activity in a biological assay; and (d) identifying the activity of the expressed polypeptide based on the test results.

27. A substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in gastrointestinal inflammation, chosen from the group consisting of the DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in gastrointestinal inflammation.

28. A kit suitable for detecting the presence of the polypeptide of the claim 2 in a mammalian tissue sample comprising a first antibody which immunoreacts with a mammalian protein encoded by a gene coπesponding to the polynucleotide of claim 1 or with a polypeptide of claim 2 in an amount sufficient for at least one assay, instructions for use and suitable packaging material.

29. A kit of claim 28 further comprising a second antibody that binds to the first antibody.

30. The kit of claim 29 wherein the second antibody is labeled.

31. The kit of claim 30 wherein the label comprises enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.

32. A kit for suitable for detecting the presence of a gene regulated in gastrointestinal inflammation, comprising: at least one polynucleotide of claims 1 or 4, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay; label means; instructions for use; and suitable packaging material.

33. An isolated polypeptide comprising SEQ ID NO: 129.