WO2002031116A2

WO2002031116A2 - Gene expression modulated in spontaneous inflammatory bowel disease

Info

Publication number: WO2002031116A2
Application number: PCT/US2001/032176
Authority: WO
Inventors: Joanne L. Viney; John E. Sims; Robert F. Dubose; Peter R. Baum; Karl W. Hasel; Brian S. Hilbush
Original assignee: Digital Gene Technologies, Inc.
Priority date: 2000-10-11
Filing date: 2001-10-11
Publication date: 2002-04-18
Also published as: WO2002031116A9; AU2002211753A1; AU2002211753A8

Abstract

Polynucleotides, polypeptides, kits and methods are provided related to regulated genes characteristic of inflammatory bowel disease.

Description

GENE EXPRESSION MODULATED IN SPONTANEOUS INFLAMMATORY BOWEL DISEASE

BACKGROUND OF THE INVENTION The human inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, are syndromes characterized by a chronic, relapsing inflammation of the gastrointestinal tract. Although the etiology of IBD remains unknown, the induction and pathogenesis of this disease is widely thought to be multifactorial, involving interactions between genetic, immune, and environmental factors. Recent research has provided support for the hypothesis that IBD is caused by an overly aggressive immune response to normal bacterial constituents in the gut, as mediated by Thl lymphocytes and macrophages (Sartor, Aliment. Pharmacol. Ther. 11:17-23 (1997)).

While several models of IBD have been developed in rodents, none of these models has entirely recapitulated the histopathology and clinical features of human IBD. Valuable information concerning the potential mechanisms of IBD, however, has been collected through an examination of currently available IBD models. A number of these models were generated through the disruption or modification of the immune system, thus suggesting that the mucosal immune system plays a central role in the regulation of intestinal inflammation. Such IBD rodent models have been developed through the targeted deletion of genes encoding IL-2, IL-10, TGFβ, and TcRα, or the introduction of transgenes encoding human HLA-B27 or CD3ε (Podolsky, Acta Gastroenterol. Belg. 60(2):163-65 (1997)).

Several models of IBD have emerged as a result of the disruption of epithelial- associated genes. These models include the targeted deletion in mice of genes encoding Gα2_]5 or keratin-8, and the introduction of a dominant negative construct which functionally blocks N-cadherin (Podolsky, Acta Gastroenterol. Belg. 60(2): 163-65 (1997)). The large number and diverse nature of the IBD models underscores the belief that multiple genetic loci modulate the development of IBD. The discovery that many animals with altered T cell populations or cytokine deficiencies spontaneously develop IBD, suggests that immunological dysregulation is a critical factor in the manifestation of this disease (Powrie and Leach, Ther. Immunol. 2(2): 115-23 (1995)). However, it has also been suggested that the immunological changes associated with IBD may not be the primary cause of the disease process, but rather the result of secondary nonspecific inflammation (Panwala et al., J Immuno. 161(10):5733-44 (1998)). Recent research has implicated intestinal microflora as an important cofactor in the initiation and perpetuation of IBD, suggesting that the disease may develop as a result of a dysregulated inflammatory response to components of the normal flora (Duchmann et al., Clin. Exp. Immunol. 102(3):448-55 (1995)). For example, the development of IBD in many mouse models can be readily prevented by generating and maintaining mice in a germ-free environment or through treatment of animals with oral antibiotics. Furthermore, there is some evidence that human patients with IBD suffer from adverse and enhanced reactivity to their autologous resident intestinal flora (Duchmann et al, Clin. Exp. Immunol. 102(3):448-55 (1995)).

Intestinal epithelial cells, which act as a barrier between the mucosal immune system and the large antigenic load of the gut lumen, appear to play a significant role in regulating the contact between immune cells and bacteria in the gut lumen. For example, it has been shown that mouse cells with the multiple disease resistance (mdr) gene knocked-out develop IBD (Panwala et al., J Immuno. 161(10):5733-44 (1998)). In patients with IBD, the disruption of the epithelial barrier may lead to the entrance of intestinal flora into the mucosa and subsequent antigen-specific local response. The mechanisms by which the intestinal microflora and intestinal immune system interact are not well understood. It is particularly unclear how the functioning intestinal immune system maintains its ability to mount a rapid and potent response to pathogenic bacteria, viruses and parasites while remaining tolerant to the resident intestinal microflora (Duchmann et al., Clin. Exp. Immunol. 102(3):448-55 (1995)). Intestinal epithelial cells and some lymphocyte subsets have been shown to express genes conferring the multiple drug resistance (mdr) phenotype (Croop et al., Mol. Cell. Biol. 9(3): 1346-50 (1989); Drach et al., Blood 80(ll):2729-34 (1992)). These genes belong to a family of transmembrane transporters which are characterized by their ability to pump small aliphatic and hydrophobic molecules across membranes in an ATP-dependent manner, and were first identified by their ability to confer resistance to chemotherapeutic agents in tumors and neoplastic cell lines. Three mdr genes have been identified in rodents, each of which has a restricted pattern of tissue expression and, very likely, a different function. The murine multiple resistance gene, mdr la, is expressed on intestinal epithelial cells, CD8⁺ T cells, a subset of CD4⁺ T cells, hematopoietic cells, and cells at the blood- brain barrier (Panwala et al., J Immuno. 161(10):5733-44 (1998)). Despite the evidence of mdr la tissue distribution and the ability of the protein to actively pump toxic drugs out of cells, the natural in vivo role for the mdr la gene product in the different cell types in which it is expressed has not been fully defined.

In order to determine the natural function of mdr la, mice with a targeted deletion of the gene were generated (Schinkel et al., Cell 77(4):491-502 (1994)). Although it has been demonstrated that the mdrla knockout mice have an increased sensitivity to certain drugs, initial analysis of the mice did not uncover any constitutive abnormalities. It has recently been observed, however, that when mdrla ^~ mice are maintained under specific pathogen-free animal facility conditions, they are susceptible to developing severe, spontaneous colitis characterized by a broad inflammatory response along entire length of the colon (Panwala et al., J. Immunol. 161(10):5733-44 (1998)). The intestinal inflammation observed in mdrla mice has a pathology similar to that of human IBD, particularly ulcerative colitis, and is defined by dysregulated epithelial cell growth, ulcerations, crypt abscesses, and leukocytic infiltration into the lamina propria of the large intestine.

Treatment of mdrl a '^~ mice with oral antibiotics prevents the development of IBD (and can therapeutically reverse colitis in mdrla ^~ mice with active inflammation), suggesting that the presence of resident intestinal flora is necessary for the development of IBD in mdrla '^~ mice. The requirement of bacterial flora for the induction of colitis in mdrla '^~ mice is therefore consistent with other models of IBD. While the development of IBD in this model appears to be due to a defect in the intestinal epithelial barrier rather than the result of an immuno logical defect, the precise mechanistic defect resulting in IBD in mdrla ^1' mice remains to be determined (Panwala et al., I Immunol. 161(10):5733-44 (1998)). The mdrla '^" mouse model of IBD permits dissection of the various constituents of the intestinal epithelium and mucosal immune response that are necessary for maintaining normal homeostasis and which, under specific conditions, contribute to the development of IBD. This model also allows for the exploration of interactions between genetic and environmental factors leading to the disorder. Particularly, the model provides the means by which disease resistance and susceptibility genes can be identified and proteins critical for the induction and regulation of IBD determined. By identifying such genes in mice, this information can then be used to design more effective immunomodulatory therapies for the treatment of IBD in humans.

Moreover, a systematic identification and characterization of molecules that are selectively expressed in either control or mdr ^" knockout mice would greatly illuminate the physiology of the intestinal epithelium and the development of IBD. First, the systematic identification of proteins induced or up-regulated in mM^"7" knockout mice would allow the identification of proteins that contribute to the pathology associated with IBD. Such identification would contribute to the understanding of IBD and the factors associated with the development of the disease. Additionally, the identification of potentially harmful gene products is important to identify molecules that could be useful as a diagnostic marker indicating IBD pathology. Further, identification of potentially harmful gene products is important to identify molecules that could be amenable to pharmaceutical intervention.

A systematic characterization of genes selectively expressed in control mice or down-regulated in mice with IBD would also allow the identification of beneficial molecules that contribute to conditions of protection. Such identification of beneficial products could lead to the development of pharmaceutical agents useful in the treatment of IBD. Furthermore, the identification of harmful and beneficial products may lead to new lines of study towards the amelioration of symptoms associated with IBD pathology.

What is needed therefore, is an understanding and identification of the genes associated with IBD. The Total Gene Expression Analysis (TOGA™) method, described in Sutcliffe et al, Proc. Nαtl. Acαd. Sci. USA 97(5): 1976-81 (2000), International published application WO 00/26406, U.S. Patent No. 5,459,037, U.S. Patent No. 5,807,680, U.S. Patent No. 6,030,784, U.S. Patent No. 6,096,503 and U.S. Patent No. 6,110,680, all of which are incorporated herein by reference, is a tool used to identify and analyze polynucleotide expression associated with IBD. The TOGA™ method is an improved method for the simultaneous sequence-specific identification of mRNAs in an mRNA population which allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA. The method can identify changes in expression of mRNA associated with the administration of drugs or with physiological or pathological conditions such as IBD.

SUMMARY OF THE INVENTION

The PCR-based Total Gene Expression Analysis (TOGA™) differential display system has been used in studies to examine how the gene expression is regulated in spontaneous IBD caused by mdrla deletion. Such studies have examined the mechanism of spontaneous IBD and to identify proteins and genes that are linked to the development of IBD. Molecules have been identified that correspond to genes that are regulated in spontaneous IBD. Such molecules are useful in therapeutic, prognostic and diagnostic applications in the treatment of IBD.

The present invention provides novel polynucleotides and the encoded polypeptides regulated in IBD. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides regulated in IBD. 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 NO: 11, 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: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO:102. Another embodiment comprises an isolated nucleic acid molecule at least 95% identical to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1-44. A further embodiment comprises an isolated nucleic acid molecule at least ten bases in length that is hybridizable to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1-44 under stringent conditions. In another embodiment, the invention provides an isolated polypeptide encoded by 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 NO: 11, 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: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO ):: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO ):: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID

NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102. In another embodiment, the invention provides an isolated nucleic acid molecule encoding the polypeptide of the present invention.

Another embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as IBD, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of the invention or a polynucleotide of the invention.

A further embodiment of the invention provides an isolated antibody that binds specifically to the isolated polypeptide of the invention. A preferred embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as IBD, comprising administering to a mammalian subject a therapeutically effective amount of the antibody.

An additional embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject. The method comprises determining the presence or absence of a mutation in a polynucleotide of the invention. A pathological condition or a susceptibility to a pathological condition, such as IBD is diagnosed based on the presence or absence of the mutation.

Even another embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition, such as IBD in a subject. The method comprises detecting an alteration in expression of a polypeptide encoded by the polynucleotide of the invention, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition. The alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression. In a preferred embodiment a first biological sample is obtained from a patient suspected of having IBD and a second sample from a suitable comparable control source is obtained. The amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample. The amount of the polypeptide in the first and second samples is determined. A patient is diagnosed as having IBD if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.

Where a polynucleotide of the invention is down-regulated and exacerbates a pathological condition, such as IBD, the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, administering a polynucleotide or polypeptide of the invention to the mammalian subject. For example, in mdrla-/- mice with spontaneous IBD, TOGA detected decreased expression of the polynucleotide with SEQ ID NO: 27 (DST IMX 5_49). By enhancing the in vivo levels of this polynucleotide or the polypeptide product, it may be possible to prevent, treat, ameliorate, or modulate IBD. Where a polynucleotide of the invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as IBD, the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition. For example, in mdrla-/- mice with spontaneous IBD, TOGA detected increased expression of the polynucleotide with SEQ ID NO: 33 (DST IMX5_8). By decreasing the in vivo levels of this polynucleotide or the polypeptide product, it may be possible to prevent, treat, ameliorate, or modulate IBD. This can be accomplished by, for example, the use of antisense oligonucleotides, triple helix base pairing methodology or ribozymes. Alternatively, drugs or antibodies that bind to and inactivate the polypeptide product can be used.

The present invention also provides a method for identifying a binding partner to the isolated polypeptide of the invention comprising contacting the polypeptide with a binding partner and determining whether the binding partner effects an activity of the polypeptide.

In addition, the present invention provides a method of identifying an activity of an expressed polypeptide in a biological assay, wherein the method comprises expressing the polypeptide of claim 2 in a cell, isolating the expressed polypeptide, testing the expressed polypeptide for an activity in a biological assay, and identifying the activity of the expressed polypeptide based on the test results.

In a further embodiment, the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in IBD, 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 IBD.

The present invention also provides a system and method for detecting the presence of a gene regulated in IBD and the encoded polypeptide. In one embodiment, the present invention provides a kit suitable for detecting the presence of a polypeptide of the invention comprising a first antibody which immunoreacts with the polypeptide in an amount sufficient for at least one assay, instructions for use and suitable packaging material, h another embodiment, the kit further comprises a second antibody that binds to the first antibody.

Also, the present invention provides a kit suitable for detecting the presence of a gene regulated in IBD, comprising at least one polynucleotide of the present invention, or a 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 for the kit is chosen from the group consisting of SEQ ID NO: 1, SEQ LO 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 11, 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: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92,

SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,

SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID

NO: 102. Another embodiment comprises a polynucleotide at least 95% identical to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NOs:

1-44 and 88-102. A further embodiment comprises a polynucleotide at least ten bases in length that is hybridizable to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1-44 and 88-102 under stringent conditions, h 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 IBD.

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^™ runs using a 5' PCR primer with parsing bases GTTC (SEQ ID NO:80) and the universal 3' PCR primer (SEQ ID NO:85) showing PCR products produced from mRNA extracted from large intestines of FNB control mice (having the wild-type (WT) mdrla gene) (Panel A) and mdrla ^~'^~ knockout mice (having the mdrla gene deleted (KO)) (Panel B). The horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA™ analysis (which corresponds to the relative expression of the molecule of that address). The results of the TOGA^™ runs have been normalized using the methods described in pending U.S. Patent Application Serial No. 09/318,699/U.S., and pending PCT Application Serial No. PCT/USOO/14159, both entitled Methods and System for Amplitude Normalization and Selection of Data Peaks (Dennis Grace, Jayson Durham); and pending U.S. Patent Application Serial No. 09/318,679/U.S. and pending PCT Application Serial No. PCT/USOO/14123, both entitled Methods for

Normalization of Experimental Data (Dennis Grace, Jayson Durham) all of which are incorporated herein by reference. The vertical line drawn through the five panels represents the DST molecule identified as X5_5 (SEQ ID NO:32). The vertical index line indicates a PCR product of about 167 b.p. that is present in FNB control mice (Panel A) and up-regulated in mdrla mice having the mdrla gene deleted (Panel B).

Figure 2 presents a graphical example of the results obtained when a DST is verified by the Extended TOGA™ Method using a primer generated from a cloned product (as described below). The length of the PCR product corresponding to SEQ ID ΝO:32 (IMX5_5) was cloned and a 5' PCR primer was built from the cloned DST (SEQ ID NO:87). The product obtained from PCR with this primer (SEQ ID NO:87) and the universal 3' PCR primer (SEQ ID NO: 85) (as shown in Panel A) was compared to the length of the original PCR product that was produced in the TOGA reaction with large intestines of mdrla^"1' knockout mice using a 5' PCR primer with parsing bases GTTC (SEQ ID NO:80) and the universal 3' PCR primer (SEQ ID NO:85) (as shown in Panel B). Again, for all panels, the number of base pairs is shown on the horizontal axis, and fluorescence intensity (which corresponds to relative expression) is found on the vertical axis. In Panel C, the traces from the top and middle panels are overlaid, demonstrating that the peak found using an extended primer from the cloned DST is the same number of base pairs as the original PCR product obtained through TOGA™ as IMX5_5 (SEQ ID NO:32). Panel C thus illustrates that IMX5_5 (SEQ ID NO:32) was the DST amplified in Extended TOGA™.

Figure 3 is a graphical representation similar to Figure 1 of the results of TOGA™ analysis using a 5' PCR primer with parsing bases CAAG (SEQ ID NO: 79) and the universal 3' primer (SEQ ID NO: 85) showing PCR products produced from mRNA extracted from the large intestines of FNB control mice (having the wild-type (WT) mdrla gene) (as shown in Panel A) and mdrla knockout mice (having the mdrla gene deleted (KO)) (as shown in Panel B) . The vertical line drawn through the two panels represents the DST molecule identified as IMX5_49 (SEQ ID ΝO:27).The vertical index line indicates a PCR product of about 98 b.p. that is present in FNB control mice and down-regulated in mdrla^"1" mice having the mdrla gene deleted.

Figure 4 shows a Northern Blot analysis of clone IMX5_49 (SEQ ID NO: 27; CAAG 98) where an agarose gel containing polyA enriched mRNA from the large intestines of FNB control mice and mdrla^'1" knockout mice, as well as cyclophilin (used to normalize loading variations between lanes) was blotted after elecfrophoresis and probed with radiolabeled IMX5_49.

Figure 5 is a graphical representation similar to Figure 1 of the results of TOGA™ analysis using a 5' PCR primer with parsing bases TTGT (SEQ ID NO: 81) and the universal 3' primer (SEQ ID NO: 85) showing PCR products produced from mRNA extracted from the large intestines of FNB control mice (having the wild-type (WT) mdrla gene) (as shown in Panel A) and mdrla^"1" knockout mice (having the mdrla gene deleted (KO)) (as shown in Panel B). The vertical line drawn through the two panels represents the DST molecule identified as IMX5_8 (SEQ ED NO:33). The vertical index line indicates a PCR product of about 216 b.p. that is present in FNB control mice and up-regulated in mdrla^"1" mice having the mdrla gene deleted.

Figure 6 is a graphical representation of the results of a reverse transcriptase polymerase chain reaction (RT-PCR) of clone IMX5_8 (SEQ ID NO: 33) where polyA enriched mRNA was extracted from the large intestines of FNB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 7 is a graphical representation of the results of RT-PCR of clone

IMX5_11 (SEQ ID NO: 34) where polyA enriched mRNA was extracted from the large intestines of FNB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 8 is a graphical representation of the results of RT-PCR of clone

IMX5_16 (SEQ ID NO: 9) where polyA enriched mRNA was extracted from the large intestines of FNB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 9 is a graphical representation of the results of RT-PCR of clone

IMX5_17 (SEQ ID NO: 37) where polyA enriched mRNA was extracted from the large intestines of FNB control mice (labeled WT) and mdrla^{" ~} knockout mice (labeled KO).

Figure 10 is a graphical representation of the results of RT-PCR of clone

IMX5_19 (SEQ ID NO: 10) where polyA enriched mRNA was extracted from the large intestines of FNB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 11 is a graphical representation of the results of RT-PCR of clone

IMX5_22 (SEQ ID NO: 12) where polyA enriched mRNA was extracted from the large intestines of FNB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO). Figure 12 is a graphical representation of the results of RT-PCR of clone IMX5_23 (SEQ ID NO: 13) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 13 is a graphical representation of the results of RT-PCR of clone X5_25 (SEQ ID NO: 15) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 14 is a graphical representation of the results of RT-PCR of clone IMX5_28 (SEQ ID NO: 17) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 15 is a graphical representation of the results of RT-PCR of clone IMX5_30 (SEQ ID NO: 18) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 16 is a graphical representation of the results of RT-PCR of clone IMX5_32 (SEQ ID NO: 19) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 17 is a graphical representation of the results of RT-PCR of clone IMX5_35 (SEQ ID NO: 21) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 18 is a graphical representation of the results of RT-PCR of clone IMX5_39 (SEQ ID NO: 22) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla ^1" knockout mice (labeled KO). Figure 19 is a graphical representation of the results of RT-PCR of clone 1MX5_41 (SEQ ID NO: 23) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla^"1" knockout mice (labeled KO).

Figure 20 is a graphical representation of the results of RT-PCR of clone IMX5_43 (SEQ ID NO: 24) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla '^" knockout mice (labeled KO).

Figure 21 is a graphical representation of the results of RT-PCR of clone IMX5_51 (SEQ ID NO: 28) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla ^1" knockout mice (labeled KO).

Figure 22 is a graphical representation of the results of RT-PCR of clone IMX5_54 (SEQ ID NO: 30) where polyA enriched mRNA was extracted from the large intestines of FVB control mice (labeled WT) and mdrla ^~ knockout mice (labeled KO).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention and the methods of obtaining and using the present invention will be described in detail after setting forth some preliminary definitions.

Definitions

The following definitions are provided to facilitate understanding of certain terms used in the present invention. Many of the techniques described herein are described in Dracopoli et al., Current Protocols in Human Genetics, John Wiley and Sons, New York (1999), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York (2000), both of which are incorporated herein by reference.

An "isolated nucleic acid" refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein; and (e) a nucleic acid synthesized through chemical means.

An "isolated polypeptide" refers to a polypeptide 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. An "isolated antibody" refers to an antibody 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.

"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.

"Polynucleotide" or "polynucleotide of the invention" or "polynucleotide of the present invention" refers to a molecule having a nucleic acid sequence contained in SEQ ID NOs:l-44 and 88-102. 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. A 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, a 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. A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NOs: 1-44 and 88-102 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 (5X SSC = 750 mM NaCl, 75 mM sodium citrate, 50 mM sodium phosphate pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ug/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at about 65°C. 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 NaCl; 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 IX 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 (5% w/v non-fat dried milk in phosphate buffered saline ("PBS"), heparin, denatured salmon sperm DNA, and other 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).

"Polypeptide" or "polypeptide of the invention" or "polypeptide of the present invention" refers to a molecule having a translated amino acid sequence generated from the polynucleotide as broadly defined. 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 franslation of these alternative open reading frames are specifically contemplated by the present invention. 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. See references below. 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, fransfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, e.g., T. E. Creighton, Ed., Proteins - Structure And Molecular Properties, 2nd Ed., W. H. Freeman and Company, New York (1993); B. C. Johnson, Ed., Posttranslational Covalent Modification Of Proteins, Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol, 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci, 663:48-62 (1992)).

A polypeptide has "biological activity" when the polypeptide has structural, regulatory or biochemical functions of a naturally occurring molecule. Biological activity can be measured by several kinds of biological assays, both in vitro (e.g., cell cultures) or in vivo (e.g., behavioral or metabolic assays). In these cases, the potency of the biological activity is measured by its dose-response characteristics; in the case of polypeptides with activity similar to the polypeptide of the present invention, the dose-response dependency will be substantially similar in a given activity as compared to the polypeptide of the present invention. Polypeptides may derive their "biological activity" through binding to specific cellular receptors, which mediate secondary signals to the target cell or tissue. In other cases, they may have direct effects in the absence of receptor mediated binding or signaling. For example, peptides may interact directly with other proteins or other molecules, and alter their conformation of function, or they may block the binding of a third molecule to the same interaction site, thereby affecting the signal normally mediated between the two molecules.

"DNA" refers to deoxyribonucleic acid.

'RNA" refers to ribonucleic acid.

'mRNA" refers to messenger ribonucleic acid.

'cDNA" refers to a deoxyribonucleic acid that is complementary to an mRNA.

"Gene" refers to a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single protein or RNA. This definition includes the entire functional unit encompassing coding DNA sequence, the regions preceding and following the coding region (leader or trailer), noncoding regulatory DNA sequences, and introns.

"Codon" refers to the three-nucleotide sequence of an mRNA molecule that codes for one specific amino acid.

"Vector" refers to a vehicle for transfer of DNA into a recipient cell.

"Silent mutation" or "silent substitution" refers to a mutation that causes no functional change in the gene product.

"Phenotype" refers to the appearance, behavior, or other characteristics of a cell or individual due to actual expression, or pattern of expression, of a specific gene or set of genes. Differences in phenotype may be due to changes in the expression or pattern of expression of a specific gene or set of genes, or to differences in the biological activity of one or more genes. These differences may be a result of polymorphic or allelic differences in the coding region of the specific genes or in their regulatory sequences, or to other genetic variations (e.g., new mutations).

"Hybridization" refers to the time- and temperature-dependent process by which two complementary single-stranded polynucleotides associate to form a double helix.

"Probe" refers to a polynucleotide, often radiolabelled, used to detect complementary sequences, e.g. an mRNA used to locate its gene by a corresponding nucleic acid blotting method.

"Conservative amino acid substitution" refers to a substitution between similar amino acids that preserves an essential chemical characteristic of the original polypeptide.

"Phage" refers to a virus that infects bacteria. Many phage have proved useful in the study of molecular biology and as vectors for the transfer of genetic information between cells.

"Plasmid" refers to a self-replicating extra-chromosomal element, usually a small segment of duplex DNA that occurs in some bacteria; used as a vector for the introduction of new genes into bacteria.

"Retrovirus" refers to a virus with an RNA genome that may be either an mRNA, (+)-RNA, or its complement, (-)-RNA. Class 1 contains (+)-RNA; class 2, (- )-RNA, which is the template for an RNA-dependent RNA polymerase; class 3, double-stranded RNA, in which (+)-RNA is synthesized by an RNA-dependent RNA polymerase; class 4, retrovirus, in which (+)-RNA is a template for an RNA- dependent DNA polymerase (a reverse transcriptase). A Retrovirus may be used as a vector for the introduction of genes into mammalian cells. "Triple Helix" refers to the tertiary structure of collagen that twists three polypeptide chains around themselves; also a triple-stranded DNA structure that involves Hoogstein base pairing between B-DNA and a third DNA strand that occupies the major groove.

"Antibody" refers to an immunoglobulin molecule that reacts specifically with another (usually foreign) molecule, the antigen.

"Monoclonal antibody (niAb)" refers to an immunoglobulin preparation that is completely homogeneous, due to its formation by daughters of a single progenitor cell that has been programmed for the synthesis and secretion of one specific antibody.

"Polyclonal antibody" refers to a heterogeneous immunoglobulin preparation that contains antibodies directed against one or more determinants on an antigen; the product of daughters of several progenitor cells that have been programmed for immunoglobulin synthesis and secretion.

"Complementary" as used in nucleic acid chemistry, is descriptive of the relationship between two polynucleotides that can combine in an antiparallel double helix; the bases of each polynucleotide are in a hydrogen-bonded inter-strand pair with a complementary base, A to T (or U) and C to G. In protein chemistry, the matching of shape and/or charge of a protein to a ligand.

"C-terminus" refers to, in a polypeptide, the end with a free carboxyl group.

"N-terminus" refers to, in a polypeptide, the end with a free amino group.

A "secreted" protein refers to those proteins capable of being directed to the endoplasmic reticulum, 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.

"Variant" refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. In general, variants have close similarity overall and are identical in many regions 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., Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin and Griffin, Eds., Computer Analysis Of Sequence Data, Parti, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov and Devereux, Eds., Sequence Analysis Primer, 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 et al., 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 et al., (1988), Supra.

"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, e.g., Geysen et al, Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1983)).

"Homologous" means corresponding in structure, position, origin or function. A "homologous polynucleotide" refers to a polynucleotide which encodes a homologous polypeptide.

A "homologous nucleic acid molecule" refers to a nucleic acid molecule which encodes a homologous polypeptide.

A "homologous polypeptide" refers to a polypeptide having any of the following characteristics with respect to the polypeptides of the present invention: similar function, similar amino acid sequence, similar subunit structure and formation of a functional heteropolymer, immunological cross-reaction, similar expression profile, similar subcellular location, similar substrate specificity, or similar response to specific inl ibitors.

"ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme- antibody conjugate to detect and quantify the amount of an antigen present in a sample.

A "specific binding agent" refers to a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention.

The word "complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex.

The term "package" refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene, or polycarbonate), paper, foil and the like capable of holding within fixed limits a polypeptide, polyclonal antibody, or monoclonal antibody of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or antibody or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide or antibody have been operatively affixed (i.e., linked) so as to be capable of being immunologically bound by an antibody or antigen, respectively.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like.

"DST" refers to a Digital Sequence Tag, i.e., a polynucleotide that is an expressed sequence tag of the 3' end of an mRNA.

Other terms used in the fields of biotechnology and molecular and cell biology as used herein will be as generally understood by one of ordinary skill in the applicable arts.

Examples of the Present Invention:

The following examples are experiments that were conducted to identify gene expression associated with spontaneous inflammatory bowel disease and further experiments that used the information gathered from the gene expression data. These experiments are intended to illustrate the invention, and are not to be construed as limiting the scope of the invention.

EXAMPLE 1

Identification and Characterization of Polynucleotides

Regulated in ]frfl--mmatory Bowel Disease Caused by mdrla Deletion

As discussed previously, mdrla^"'^" knockout mice, lacking the multiple drug resistance gene mdrla, are susceptible to developing spontaneous inflammatory bowel disease, characterized by a broad inflammatory response along the entire length of the colon. The intestinal inflammation observed in mdrla^"1" mice has a pathology similar to that observed in human inflammatory bowel disease, particularly ulcerative colitis, which is defined by dysregulated epithelial cell growth, ulcerations, crypt abscesses, and leukocytic infiltration into the lamina propria of the large intestine. Thus, the mdrla^{' '} mouse model of inflammatory bowel disease can be used to study the various constituents of the intestinal epithelium and mucosal immune response that contribute to the development of inflammatory bowel disease. In particular, the model can be used to identify genes and proteins critical for the induction and modulation of inflammatory bowel disease.

For the following studies, mRNA isolated from the large intestines of FVB control mice and mdrla^'1" knockout mice was obtained. FVB control mice and mdrla^"1' knockout mice were initially purchased from Taconic Farms (Germantown, NY) and then bred and maintained according to the methods described in Panwala et al., J Immunol, 161: 5733-5744 (1998)). Control and knockout mice were age matched and monitored on alternate days for signs of colitis as indicated by the presence of diarrhea, sticky or bloody stools, and mucous discharge from the anus. Large intestines were removed from mdrla knockout mice that had visible clinical signs of colitis and FVB control mice. Freshly dissected tissue was immediately placed in GT buffer and homogenized. The homogenized lysates were spun briefly to remove large debris before being layered onto a CsCl gradient. RNA was extracted and polyA enriched mRNA was isolated using conventional methods.

The TOGA Process Isolated RNA was analyzed using a method of simultaneous sequence identification of mRNAs known as TOGA™ (TOtal Gene expression Analysis) described in Sutcliffe et al. Proc. Natl Acad. Sci. USA, 97(5).T 976- 1981 (2000); International published application WO 00/26406; U.S. Patent No. 5,459,037; U.S. Patent No. 5,807,680; U.S. Patent No. 6,030,784; U.S. Patent No. 6,096,503 and U.S. Patent 6,110,680, hereby incorporated herein by reference. Preferably, prior to the application of the TOGA™ technique, the isolated RNA was enriched to form a starting polyA-containing mRNA population by methods known in the art. In a preferred embodiment, the TOGA™ method further comprised an additional PCR step performed using four 5' PCR primers in four separate reactions and cDNA templates prepared from a population of antisense cRNAs. A final PCR step that used 256 5' PCR primers in separate reactions produced PCR products that were cDNA fragments that corresponded to the 3'-region of the starting mRNA population. The produced PCR products were then identified by: a) the initial 5' sequence comprising the sequence remainder of the recognition site of the restriction endonuclease used to cut and isolate the 3' region plus the sequence of the preferably four parsing bases immediately 3' to the remainder of the recognition site, 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. Since the length of the obtained PCR products includes known vector sequences at the 5' and 3' ends of the inserts, the sequence of the insert provided in the sequence listing is shorter than the fragment length that forms part of the digital address.

The method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags of the 3' end of mRNAs. DSTs that showed changes in relative levels during inflammatory bowel disease caused by mdrla deletion were selected for further study. The intensities of the laser-induced fluorescence of the labeled PCR products were compared across sample isolated from the large intestines of mdr la^"'^" knockout mice that had visible clinical signs of colitis and FVB control mice. The results are presented in Tables 1 and 2 below.

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-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: 82), 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 resulting fragments of cDNA corresponding to the 3' region of the starting mRNA 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⁺ (Sfratagene, 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 (Sfratagene). The ligation regenerates the Notl site, but not the Mspl site, leaving CGG as the first 3 bases of the 5' end of all PCR products obtained. 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: 83). 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: 84), 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: 85) to yield four sets of PCR reaction products ("NI reaction products").

In step three, the product of each subpool was further divided into 64 subsubpools (2 ng in 20μl) for the second PCR reaction. This PCR reaction comprised adding 100 ng each of the fluoresceinated "universal" 3' PCR primer (SEQ ID NO: 85) conjugated to 6-FAM and 100 ng of 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: 86) and 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 misprinting and promote high fidelity copying. Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).

The products ("N4 reaction 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 mdrla^"1" knockout mice that had visible clinical signs of colitis and FVB control mice as described above were analyzed. Table 1 is a summary of the expression levels of 272 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 (CCGG) 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 GTTC 167 is further characterized as having a 5' terminus partial nucleotide sequence of CGGGTTC and a digital address length of 167 b.p. The DNA molecule identified as Mspl GTTC 167 is further described as being expressed at increased levels in mdrla^"1" knockout mice that have inflammatory bowel disease. Additionally, the DNA molecule identified as Mspl GTTC 167 is described by its nucleotide sequence, which corresponds with SEQ ID NO: 32 (DST IMX5_5). Similarly, the other DNA molecules identified in Table 1 by their Mspl digital addresses are further characterized by the level of gene expression found in the large intestine of FVB confrol mice and mdrla^'1" knockout mice having inflammatory bowel disease. Additionally, several of the isolated clones are further characterized in Tables 2-6, as well as Tables 7-23.

The data for the DNA molecule identified as Mspl GTTC 167, shown in Figure 1, were generated with a 5'-PCR primer (C-G-A-C-G-G-T-A-T-C-G-G-G-T- T-C, SEQ ID NO: 80) paired with the "universal" 3 ' primer (SEQ ID NO: 85) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus. PCR reaction products were resolved by gel elecfrophoresis on 4.5% acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer). The sequences of the PCR products were determined using standard techniques.

Figure 1 is a graphical representation of the results of TOGA runs using a 5' PCR primer with parsing bases GTTC (SEQ ID NO: 80) and the universal 3' PCR primer (SEQ ID NO:85) showing PCR products produced from mRNA extracted from large intestines of FVB control mice (having the wild-type (WT) mdrla gene) (Panel A) and mdrla knockout mice (having the mdrla gene deleted (KO)) (Panel B). The horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA analysis (which corresponds to the relative expression of the molecule of that address). The results of the TOGA™ runs have been normalized using the methods described in pending U.S. Patent Application Serial No. 09/318,699/U.S., and pending PCT Application Serial No. PCT/USOO/14159, both entitled Methods and System for Amplitude Normalization and Selection of Data Peaks (Dennis Grace, Jayson Durham); and pending U.S. Patent Application Serial No. 09/318,679/U.S. and pending PCT Application Serial No. PCT/US00/14123, both entitled Methods for Normalization of Experimental Data (Dennis Grace, Jayson Durham) all of which are incorporated herein by reference. The vertical line drawn through the five panels represents the DST molecule identified as IMX5_5 (SEQ ID NO:32). The vertical index line indicates a PCR product of about 167 b.p. that is present in FVB control mice (Panel A) and up-regulated in mdrla ^1" mice having the mdrla gene deleted (Panel B).

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. Cloning of TOGA™ Generated PCR Products

In suitable cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands. Putative database matches are shown for cloned DSTs in Table 2.

Verification Using the Extended TOGA™ Method

In order to verify that the TOGA™ peak of interest corresponds to the identified DST, an extended TOGA™ assay was performed for DSTs as described below. PCR primers ("Extended TOGA™ primers") were designed from sequence determined using this method: in suitable cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands.

PCR was performed using the Extended TOGA™ primers and the NI PCR reaction products as a substrate. Oligonucleotides were synthesized with the sequence G-A-T-C-G-A-A-T-C extended at the 3' end with a partial Mspl site (C-G-G), and an additional 18 adjacent nucleotides from the determined sequence of the DST. For example, for the PCR product with the TOGA address GTTC167 (IMX5_5; SEQ ID NO: 32), the 5' PCR primer was G-A-T-C-G-A-A-T-C-C-G-G-G-T-T-C-A-G-C-A-C- T-G-T-C-T-G-C-T-C (SEQ ID NO:87). This 5' PCR primer was paired with the fluorescence labeled universal 3' PCR primer (SEQ ID NO:85) in a PCR reaction using the PCR NI reaction product as substrate.

The products were separated by elecfrophoresis. The length of the PCR product generated with the Extended TOGA™ primer was compared to the length of the original PCR product that was produced in the TOGA™ reaction. The results for SEQ ID NO: 32, for example, are shown in Figure 2. The length of the PCR product corresponding to SEQ ID NO:32 (IMX5_5) was cloned and a 5' PCR primer was built from the cloned DST (SEQ ID NO:87). The product obtained from PCR with this primer (SEQ ID NO:87) and the universal 3' PCR primer (SEQ ID NO:85) (as shown Panel A) was compared to the length of the original PCR product that was produced in the TOGA™ reaction with large intestines of mdrla^"'^" knockout mice using a 5' PCR primer with parsing bases GTTC (SEQ ID NO:80) and the universal 3 ' PCR primer (SEQ ID NO: 85) (as shown in the Panel B). Again, for all panels, the number of base pairs is shown on the horizontal axis, and fluorescence intensity (which corresponds to relative expression) is found on the vertical axis. In Panel C, the traces from Panel A and Panel B are overlaid, demonstrating that the peak found using an extended primer from the cloned DST is the same number of base pairs as the original PCR product obtained through TOGA^™ as IMX5_5 (SEQ ID NO:32). Panel C thus illustrates that IMX5_5 (SEQ ID NO:32) was the DST amplified in Extended TOGA™.

Sequence Identification of DSTs

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 sequence database, can be determined using the BLAST computer program based on the algorithm of Altschul and colleagues (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990), "Basic local alignment search tool." J. Mol. Biol. 215:403-410; Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389-3402.). The term "sequence" includes nucleotide and amino acid sequences. In a sequence alignment, the query sequence can be either protein or nucleic acid or any combination therein. BLAST is a statistically driven search method that finds regions of similarity between a query and database sequences. These are called segment pairs, and consist of gapless alignments of any part of two sequences. Within these aligned regions, the sum of the scoring matrix values of their constituent symbol pairs is higher than a level expected to occur by chance alone. The scores obtained in a BLAST search can be interpreted by the experienced investigator to determine real relationships versus random similarities. The BLAST program supports four different search mechanisms: • Nucleotide Query Searching a Nucleotide Database- Each database sequence is compared to the query in a separate nucleotide-nucleotide pairwise comparison.

• Protein Query Searching a Protein Database- Each database sequence is compared to the query in a separate protein-protein pairwise comparison.

• Nucleotide Query Searching a Protein Database- The query is translated, and each of the six products is compared to each database sequence in a separate protein-protein pairwise comparison. • Protein Query Searching a Nucleotide Database- Each nucleotide database sequence is translated, and each of the six products is compared to the query in a separate protein-protein pairwise comparison. By using the BLAST program to search for matches between a sequence of the present invention and sequences in GenBank and EST databases, identities were assigned whenever possible. A portion of these results are listed in Table 2.

Extended sequences for DSTs IMX5_1 (SEQ ID NO:l), IMX5_3 (SEQ ID NO:2), MX5_4 (SEQ ID NO:3), IMX5_7 (SEQ ID NO:5), IMX5_9 (SEQ ID NO:6), IMX5_20 (SEQ ID NO:l 1), IMX5_22 (SEQ ID NO: 12), IMX5_27 (SEQ ID NO:16), IMX5_41 (SEQ ID NO:23), IMX5_43 (SEQ ID NO:24), IMX5_46 (SEQ ID NO:25), IMX5_49 (SEQ ID NO:27), and IMX5_51 (SEQ ID NO: 28) were generated by performing BLAST searches comparing the original DST sequence to published databases, and those sequences that were nearly 100% sequence matches were selected. The DST and BLAST match sequences were aligned, and the 5' -most sequence was used for additional rounds of BLAST searching. Alignments between successive BLAST match sequences were used to compile a single consensus contiguous sequence ("contig"), which then comprises the extended sequence (as listed in Table 3). Extended sequences for IMX5_23 (SEQ ID NO:13) and IMX5_39 (SEQ ID NO: 22) were obtained by performing library PCR on a cDNA lambda Zap II library (Sfratagene) constructed from cDNA from small intestines of control mice with IBD, followed by sequencing of the PCR product.

EXAMPLE 2 Northern Blot Analysis of Polynucleotides Regulated During Spontaneous Inflammatory Bowel Disease

Several of the isolated DST clones were further validated using Northern Blot analyses (see Table 4). RNA was extracted from the large intestines of FVB control mice and mdrla^'1' knockout mice having inflammatory bowel disease and polyA enriched mRNA was isolated using conventional methods. The mRNA samples from both FVB control mice and mdrla^"1' knockout mice were electrophoresed through an agarose gel, blotted and probed using well-known methods. Briefly, 2 ug of poly A+ mRNA was electrophoresed through a 1.2% agarose gel containing formamide along with the appropriate molecular weight standards. The gel was blotted overnight using nylon membrane to transfer the RNA. The membrane was prehybridized for one hour at 42°C in hybridization buffer (5X SSPE, 5X Denhardt's solution, 50% formamide, 0.2% SDS, 100 ug/ml salmon sperm DNA, and water). The DST clone DNA (50 ng) was labeled with ³²[P]-dCTP and ³²[P]-dATP using asymmetric PCR labeling. The membrane was probed with radiolabeled DNA (2-5 x 10⁶ cpm/ ml) overnight at 42°C in hybridization buffer . In addition, the northern blots were probed with radiolabeled cyclophilin DNA (25 ng) to normalize the amount of mRNA in each sample. Band intensities of the probed mRNA samples were quantitated using a Phosphoimager SI and normalized to the hybridization signal of cyclophilin.

For example, one of the clones characterized by Northern Blot analysis is DST IMX5_49 (SEQ ID NO:27), having the digital address Mspl CAAG 98. Figure 3 shows the TOGA analysis for IMX5_49, the sequence of which was obtained using the above-described TOGA^™ analysis methods. The TOGA™ data were generated with a 5'-PCR primer (C-G-A-C-G-G-T-A-T-C-G-G-C-A-A-G, SEQ ID NO: 79) paired with the "universal" 3' PCR primer (SEQ ID NO: 85) labeled with 6- carboxyfluorescein (6FAM, ABI) at the 5' terminus. PCR reaction products were resolved by gel elecfrophoresis on 4.5% acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer).

The TOGA™ analysis shown in Figure 3 using the above-described 5' PCR primer show PCR products produced from mRNA extracted from the large intestine of FVB control mice (Panel A; labeled WT) and mdrla^"'^" knockout mice having inflammatory bowel disease (Panel B; labeled KO). The vertical index line indicates a PCR product of about 98 b.p. that is present in FVB confrol mice and which expression is down-regulated in mdrla^' nockout mice having inflammatory bowel disease as a result of the mdrla gene deletion.

Figure 4 shows the Northern Blot analysis of clone IMX5_49 (SEQ ID NO: 27; CAAG 98) where the described mRNA samples were blotted after elecfrophoresis and probed with radiolabeled IMX5_49 DNA. Band intensities of the probed mRNA samples were normalized and quantitated by exposing the Northern Blot to a phosphoimaging screen and subsequent analysis as described above. As shown in Figure 4, the IMX5_49 probe detected a 8.7 kb and a 4.2 kb mRNA transcript in FVB control large intestine (labeled WT). Figure 4 further shows that IMX5_49 expression is decreased 4-5 fold in the large intestine of mdrla^'1' knockout mice with spontaneous inflammatory bowel disease (labeled KO). The band intensity values for each mRNA sample probed with DvIX5_49 are reported in Table 4.

The relative band intensities from Northern Blot analyses of 14 additional DST clones are reported in Table 4 below. As shown, the expression of several clones is decreased in the large intestine of mice with spontaneous inflammatory bowel disease caused by deletion of the mdrla gene. For example, IMX5_3 (SEQ ID NO: 2) has four transcripts ranging from 0.46 - 12.8 Kb, all of which are decreased in the large intestine of mice with spontaneous inflammatory bowel disease. Similarly, IMX5_6 (SEQ ID NO: 4), IMX5_7 (SEQ ID NO: 5), IMX5_46 (SEQ ID NO: 25), IMX5_48 (SEQ ID NO: 26), IMX5_49 (SEQ ID NO: 27), and IMX5_52 (SEQ ID NO: 29)show decreased expression in mice with inflammatory bowel disease. Interestingly, IMX5_48, which corresponds to a novel gene, is down-regulated by about 30-fold in mice with spontaneous inflammatory bowel disease. As discussed previously, the identification of genes that have decreased expression in mice with IBD can be used to identify beneficial molecules that contribute to conditions of protection. Such beneficial products could lead to the development of pharmaceutical agents useful in the treatment of IBD.

Table 4 also shows that the expression of several clones is increased in the large intestine of mice with spontaneous inflammatory bowel disease caused by deletion of the mdrla gene. For example, flvIX5_l (SEQ ID NO: 1), IMX5_9 (SEQ ID NO: 6), IMX5_10 (SEQ ID NO: 7), IMX5_20 (SEQ ID NO: 11), and IMX5_24 (SEQ ID NO: 14) show increased expression in mice with inflammatory bowel disease. In addition, clones IMX5_4 (SEQ ID NO: 3), IMX5_13 (SEQ ID NO: 8), IMX5_27 (SEQ ID NO: 16) which are not present in FVB control mice (or are present at near undetectable levels) are induced in the large intestine of mice with spontaneous inflammatory bowel disease. The identification of genes that have increased expression or are induced by IBD, can be useful as markers to identify IBD pathology. In addition, these genes can identify potential targets for pharmaceutical intervention.

EXAMPLE 3 RT-PCR Analysis of Polynucleotides Regulated During Spontaneous Inflammatory Bowel Disease Seventeen of the isolated DST clones were further validated using RT-PCR analyses as summarized in Table 5. The primers used for RT-PCR are listed in Table 6. For each DST examined, the optimal annealing temperature and reagent conditions were determined for the corresponding set of primers (see Table 6) 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 number of Taq polymerase units. Once optimal conditions were determined, each DST was run in duplicate multiple simultaneous reactions which usually included at least four dilutions of template (cDNA), 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 1 ul of cDNA 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 20 ul final 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 ul volumes were subsequently dispensed into individual reaction tubes. At various times 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 cycle times. After amplification, the samples were quantified via fluorimetry.

PCR was performed at annealing temperatures that were five degrees above the average melting temperature of each primer pair. For primers greater than 10 bases (in a 0.05M salt solution), melting temperature was calculated using the following formula: Tm = 59.9 + 41[%GC] - [675 / Primer Length], where %GC is the decimal value. Following determination of the average melting temperature of the primer pair, the annealing temperature was determined by adding five degrees to the average melting temperature. PCR was performed 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. Following temperature cycling, 2 ul of the PCR reaction was added to 140 ul of a 1 :280 dilution of PicoGreen (Molecular Probes cat. #P-11495 (10x100 ul)) 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 confrol samples was subtracted from the corresponding experimental values prior to analyzing results. The sensitivity of the PicoGreen dsDNA assay is reported to be 250 pg/ml (50 pg dsDNA in a 200 ul assay volume) using a fluorescence microplate reader such as was used in these measurements. Figure 5 shows the TOGA™ analysis for IMX5_8 (SEQ ID NO: 33), one of the clones further evaluated by RT-PCR. The clone IMX5_8, identified as Mspl

TTGT 216, was obtained using the above-described TOGA analysis methods. The TOGA™ data were generated with a 5'-PCR primer (C-G-A-C-G-G-T-A-T-C-G-G-T- T-G-T; SEQ ID NO: 81) paired with the "universal" 3' PCR primer (SEQ ID NO: 85) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus. PCR reaction products were resolved by gel elecfrophoresis on 4.5% acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software.

The TOGA™ analysis shown in Figure 5 using the above-described 5' PCR primer show PCR products produced from mRNA exfracted from the large intestine of FVB confrol mice and mdrla^'1' knockout mice having inflammatory bowel disease. The vertical index line indicates a PCR product of about 216 b.p. that is present in FVB control mice (in Panel A labeled WT) and which expression is up-regulated in mdrl -^^"knockout mice (in Panel B labeled KO) having inflammatory bowel disease as a result of the mdrla gene deletion.

The results of the quantified RT-PCR of the DST template for IMX5_8 (SEQ ID NO: 33) for a 1 :200 dilution of cDNA are shown in Figure 6 (in arbitrary fluorescence units) and in Table 7 (normalized to the control value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_8 is about 4-fold greater in mdrla^"'^" knockout mice having inflammatory bowel disease than in FVB control mice. The expression pattern was verified in the RT-PCR study, which showed that the expression of IMX5_8 in mdrla^''^" knockout mice is about 2-fold greater than the expression found in control mice. The results of the quantified RT-PCR of DST X5_11 (SEQ ID NO: 34) for a 1 :25 dilution of cDNA are shown in Figure 7 (in arbitrary fluorescence units) and in Table 8 (normalized to the control value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_11 is about 4 fold greater in mdrla^''^" knockout mice having inflammatory bowel disease than in FVB control mice. The expression pattern was verified in the RT-PCR study, which showed that the expression of IMX5_11 is about 1 to 5-fold greater in mdrla^"'^" knockout mice than in control mice.

The results of the quantified RT-PCR of DST IMX5_16 (SEQ ID NO: 9) for a 1:50 dilution of cDNA are shown in Figure 8 (in arbitrary fluorescence units) and in Table 9 (normalized to the control value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_16 is about 7-fold greater in mdrla^"'^" knockout mice having inflammatory bowel disease than in FVB control mice. The expression pattern was verified in the RT-PCR study, which showed that the expression of IMX5_16 is about 2 to 3-fold greater in mdrla^{' '} knockout mice than in control mice.

The results of the quantified RT-PCR of DST IMX5_17 (SEQ ID NO: 37) for a 1 :25 dilution of cDNA are shown in Figure 9 (in arbifrary fluorescence units) and in Table 10 (normalized to the control value at each time point). The initial TOGA analysis indicated that the expression of IMX5_17 is about 6-fold greater in mdrla^{' '} knockout mice having infl-immatory bowel disease than in FVB control mice. The expression pattern was verified in the RT-PCR study, which showed that the expression of DVIX5_17 is also about 2 to 7-fold greater in mdrla^"1" knockout mice than in control mice.

The results of the quantified RT-PCR of DST J-MX5 9 (SEQ ID NO: 10) for a 1 :200 dilution of cDNA are shown in Figure 10 (in arbitrary fluorescence units) and in Table 11 (normalized to the control value at each time point). The initial TOGA analysis indicated that the expression of IMX5_19 is about 3-fold greater in mdrla^{' '} knockout mice having inflammatory bowel disease than in FVB control mice. The expression pattern was verified in the RT-PCR study, which showed that the expression of IMX5_19 is about 2 to 9-fold greater in mdrla^"'^" knockout mice than in confrol mice.

The results of the quantified RT-PCR of DST IMX5_22 (SEQ ID NO: 12) for a 1 :25 dilution of cDNA are shown in Figure 11 (in arbitrary fluorescence units) and in Table 12 (normalized to the control value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_22 is about 6-fold greater in mdrla^"'^" knockout mice having inflammatory bowel disease than in FVB control mice. The expression pattern was verified in the RT-PCR study, which showed that the expression of IMX5_22 at cycle number 34 is about 9-fold greater in mdrla^'1' knockout mice than in confrol mice.

The results of the quantified RT-PCR of DST IMX5_23 (SEQ ID NO: 13) for a 1:100 dilution of cDNA are shown in Figure 12 (in arbitrary fluorescence units) and in Table 13 (normalized to the control value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_23 is about 5-fold greater in mdrla^'1' knockout mice having inflammatory bowel disease. The expression pattern was verified in the RT-PCR study, which showed that the expression of IMX5_23 is about 2 to 27-fold greater in mdrla^{' '} knockout mice than in confrol mice.

The results of the quantified RT-PCR of DST IMX5_25 (SEQ ID NO: 15) for a 1 :50 dilution of cDNA are shown in Figure 13 (in arbitrary fluorescence units) and in Table 14 (normalized to the confrol value at each time point). The initial TOGA^™ analysis indicated that the expression of IMX5_25 is about 6-fold greater in mdrla^'1' knockout mice having inflammatory bowel disease than in FVB control mice. The expression pattern was verified in the RT-PCR study, which showed that the expression of IMX5_25 is about 2 to 7-fold greater in mdrla^"1' knockout mice than in control mice.

The results of the quantified RT-PCR of DST IMX5_28 (SEQ ID NO: 17) for a 1 :100 dilution of cDNA are shown in Figure 14 (in arbitrary fluorescence units) and in Table 15 (normalized to the control value at each time point). The initial TOGA analysis indicated that the expression of IMX5_28 is about 3-fold greater in mdrla-/- knockout mice having inflammatory bowel disease than in FVB confrol mice. The expression pattern in the RT-PCR study showed that the expression of IMX5_28 is 25 to 61 -fold greater in mdrla-/- knockout mice than in control mice.

The results of the quantified RT-PCR of DST IMX5_30 (SEQ ID NO: 18) for a 1 :200 dilution of cDNA are shown in Figure 15 (in arbitrary fluorescence units) and in Table 16 (normalized to the confrol value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_30 is about 6-fold greater in mdrla^"'^" knockout mice having inflammatory bowel disease than in FVB confrol mice. Similarly, the expression pattern in the RT-PCR study showed that the expression of IMX5_30 is about 2 to 4-fold greater in mdrla^{" '} knockout mice than in confrol mice. The results of the quantified RT-PCR of DST IMX5_32 (SEQ ID NO: 19) for a 1 :200 dilution of cDNA are shown in Figure 16 (in arbitrary fluorescence units) and in Table 17 (normalized to the confrol value at each time point). The initial TOGA^™ analysis indicated that the expression of IMX5_32 is about 30-fold greater in mdrla^'1' knockout mice having inflammatory bowel disease than in FVB confrol mice.

Similarly, the expression pattern in the RT-PCR study showed that the expression of IMX5_32 is 3 to 75-fold greater in mdrla^'1' knockout mice than in confrol mice.The identification of genes, such as IMX5_8, IMX5_11, IMX5_16, IMX5_17, EVIX5_19, IMX5_22, IMX5_23, IMX5_25, IMX5_28, IMX5_30, and IMX5_ 32, that are induced or up-regulated in mice with IBD can be useful as a diagnostic marker to indicate IBD pathology. Also, these genes can be useful to identify potential targets for pharmaceutical intervention.

In addition, several clones having decreased expression in mdrla^{' '} knockout mice relative to confrol mice were identified. The results of the quantified RT-PCR of DST IMX5_35 (SEQ ID NO: 21) for a 1 :800 dilution of cDNA are shown in

Figure 17 (in arbitrary fluorescence units) and in Table 18 (normalized to the control value at each time point). The initial TOGA analysis indicated that the expression of IMX5_35 is decreased 3 -fold in mdrla^{' '} knockout mice having inflammatory bowel disease compared to the expression in FVB confrol mice. Similarly, the expression pattern in the RT-PCR study showed that the expression of IMX5_35 is about 2 to 3-fold less in mdrla^'1' knockout mice than in control mice.

The results of the quantified RT-PCR of DST IMX5_39 (SEQ ID NO: 22) for a 1:100 dilution of cDNA are shown in Figure 18 (in arbitrary fluorescence units) and in Table 19 (normalized to the control value at each time point). The initial TOGA analysis indicated that the expression of IMX5_39 is 5 -fold less in mdrla^'1' knockout mice having inflammatory bowel disease than in FVB control mice. Similarly, the expression pattern in the RT-PCR study showed that the expression of IMX5_39 is about 2 to 5-fold less in mdrla^'1' knockout mice than in confrol mice. The results of the quantified RT-PCR of DST IMX5_41 (SEQ ID NO: 23) for a 1:200 dilution of cDNA are shown in Figure 19 (in arbitrary fluorescence units) and in Table 20 (normalized to the confrol value at each time point). The initial TOGA^™ analysis indicated that the expression of IMX5_41 is about 3 -fold less in mdrla^'1" knockout mice having inflammatory bowel disease than in FVB control mice.

Similarly, the expression pattern in the RT-PCR study showed that the expression of IMX5_41 is about 2-fold less in mdrla^"'^" knockout mice than in confrol mice.

The results of the quantified RT-PCR of DST IMX5_43 (SEQ ID NO: 24) for a 1:100 dilution of cDNA are shown in Figure 20 (in arbitrary fluorescence units) and in Table 21 (normalized to the control value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_43 is about 5 -fold less in mdrla^"'^" knockout mice having inflammatory bowel disease than in FVB confrol mice. Similarly, the expression pattern in the RT-PCR study showed that the expression of IMX5_43 is about 2-fold less in mdrla^{' '} knockout mice than in control mice. The results of the quantified RT-PCR of DST IMX5_51 (SEQ ID NO: 28) for a 1:50 dilution of cDNA are shown in Figure 21 (in arbitrary fluorescence units) and in Table 22 (normalized to the confrol value at each time point). The initial TOGA™ analysis indicated that the expression of IMX5_51 is about 3-fold less in mdrla^{' '} knockout mice having inflammatory bowel disease than in FVB control mice. Similarly, the expression pattern in the RT-PCR study showed that the expression of IMX5_51 is about 2-fold less in mdrla^"'^" knockout mice than in control mice.

The results of the quantified RT-PCR of DST IMX5_54 (SEQ ID NO: 30) for a 1:100 dilution of cDNA are shown in Figure 22 (in arbifrary fluorescence units) and in Table 23 (normalized to the control value at each time point). The initial TOGA analysis indicated that the expression of IMX5_54 is about 10-fold less in mdrla^{" "} knockout mice having inflammatory bowel disease than in FVB control mice.

Similarly, the expression pattern in the RT-PCR study showed that the expression of

IMX5_54 is about 10-fold less in mdrla^'1' knockout mice than in control mice. The identification of genes, such as IMX5_35, IMX5_39, IMX5_41, IMX5_43 , IMX5_51 , and IMX5_54, that are down-regulated in IBD could be useful in the development of pharmaceutical agents useful in the treatment of IBD.

Furthermore, the identification of these beneficial products could lead to new lines of study towards the amelioration of symptoms associated with IBD pathology. Descriptions of the Present Invention

Based on the above examples and the results of the experiments performed, the present invention comprises isolated nucleic acid molecules comprising a polynucleotide chosen from the group consisting of SEQ ID NOs: 1-44 and 88-102. A preferred embodiment of the present invention comprises isolated nucleic acid molecules comprising a polynucleotide chosen from the group consisting of SEQ ID NOs: 1-44 and 88-102 and associated with spontaneous inflammatory bowel disease. 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 NOs: 1-44 and 88-102, or the complement thereof, or the cDNA.

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 NaCl; 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 IX SSPE, 0.1% SDS. In

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).

A 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, a 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. Another prefened embodiment of the present invention is an isolated nucleic acid molecule encoding a polypeptide of the present invention.

A prefened embodiment of the present invention is a polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NOs: 1-44 and 88-102. 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, e.g., T. E. Creighton, Ed., Proteins - Structure And Molecular Properties, 2nd Ed., W. H. Freeman and Company, New York (1993); B. C. Johnson, Ed., Posttranslational Covalent Modification Of Proteins, Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol, 182:626-646 (1990); Rattan et al, Ann. N.Y. Acad. Sci., 663:48-62 (1992)). A prefened embodiment of the present invention includes a polypeptide which is upregulated or downregulated in a disease or condition, such as inflammatory bowel disease, as compared to a normal control.

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 ID NOs: 1-44 and 88-102 and the translations of SEQ ID NOs: 1-44 and 88-102 are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. These nucleic acid molecules 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 NOs: 1-44 and 88-102 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 enors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The enoneously 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 1,000 bases).

The present invention also relates to the genes conesponding to SEQ ID NOs: 1-44 and 88-102, and translations of SEQ ID NOs: 1-44 and 88-102. The conesponding 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 et al., 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 uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein (Virus Res., 3:271-286 (1985)). The method of von Heinje uses the information from the residues sunounding the cleavage site, typically residues -13 to +2, where +1 indicates the amino terminus of the secreted protein (Nucleic Acids Res., 14:4683-4690 (1986)). Therefore, from a deduced amino acid sequence, a signal sequence and mature sequence can be identified.

In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called Signal P (Nielsen et al., Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated.

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty.

Accordingly, the present invention provides secreted polypeptides having a sequence conesponding to the translations of SEQ. ID NOs: 1-44 which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention. Polynucleotide and Polypeptide Variants

Polynucleotide or polypeptide variants differ from the polynucleotides or polypeptides of the present invention, but retain essential properties therof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention.

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 NOs: 1-44 and 88-102. 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% identity will encode a polypeptide identical to an amino acid sequence contained in the translations of SEQ ID NOs: 1-44 and 88-102.

Further embodiments of the present invention include polypeptides having at least 80% identity, more preferably at least 85% 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: 1-44 and 88-102. Preferably, the above polypeptides should exhibit at least one biological activity of the protein. In a prefened 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 NOs: 1-44 and 88-102.

Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al, J. Molec. Biol 215:403 (1990)), and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) which uses the local homology algorithm of Smith and Waterman (Adv. in App.Matk, 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 such 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 prefened method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also refened 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 presented in terms of percent identity. Prefened parameters used in a FASTDB search of a DNA sequence to calculate percent identity are: Matrix=Unitary, k-ruple=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. Prefened parameters employed to calculate percent identity and similarity of an amino acid alignment are:

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 ID NOs: 1-44 and 88-102 means that the polynucleotide is identical to a sequence contained in SEQ ID NOs: 1-44 and 88- 102 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 NOs: 1-44 and 88-102, up to 5% of the nucleotides in the sequence contained in SEQ ID NOs: 1-44 and 88-102 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.

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.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially prefened 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 prefened. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also prefened. Polynucleotide variants can be produced for a variety of reasons. For instance, a polynucleotide variant may be produced to optimize codon expression for a particular host (i.e., codons in the human mRNA may be changed to those prefened by a bacterial host, such as E. coli).

The variants may be allelic variants. 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 (Lewin, Ed., Genes II, 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. See, e.g.., Curr. Prot. Mol Bio., Chapter 8. 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 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 decreased aggregation. As known, aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (see, e.g., Pinckard et al, Clin. Exp. Immunol 2:331-340 (1967); Robbins et al., Diabetes, 36: 838-845 (1987); Cleland et al., Crit. Rev. Therap. Drug Carrier Sys., 10:307-377 (1993)). 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 et al. conducted extensive mutational analysis of human cytokine IL-la (J. Biol. Chem., 268:22105-22111 (1993)). These investigators 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 concluded 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 differed significantly in activity from the wild-type sequence. Another experiment demonstrated that 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. Ron et al. reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues (J. Biol Chem. 268: 2984-2988 (1993)).

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 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, the amino acid positions which have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions which are not critical for protein function. Thus, positions tolerating amino acid substitution may 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 (the introduction of single alanine mutations at every residue in the molecule) can be used (Cunningham et al., Science, 244:1081-1085 (1989)). The resulting mutant molecules can then be tested for biological activity. According to Bowie et al., 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, the most buried or interior (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface or exterior 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 Trp, 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; (ii) substitution with one or more of amino acid residues having a substituent group; (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol); (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a 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.

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 ID NOs: 1-44 and 88-102. The short nucleotide fragments are preferably at least about 15 nucleotides (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 NOs: 1-44 and 88- 102. These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, and greater than 150 nucleotides) are prefened. 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- 440, to the end of SEQ ID NOs: 1-44 and 88-102. In this context "about" includes the particularly recited ranges, larger or smaller by several nucleotides (i.e., 5, 4, 3, 2, or 1 nt) 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 NOs: 1-44 and 88-102. Protein fragments may be "free-standing," 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 amino acids (5, 4, 3, 2, or 1) at either extreme or at both extremes.

Prefened polypeptide fragments include the secreted protein as well as the mature form. Further prefened 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 prefened. Similarly, polynucleotide fragments encoding these polypeptide fragments are also prefened.

Also prefened 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 NOs: 1-44 and 88-102 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotide fragments encoding these domains are also contemplated.

Other prefened 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

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, e.g., Wilson et al., Cell, 37:767-778 (1984); Sutcliffe 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, e.g., Sutcliffe et al., (1983) Supra; Wilson et al, (1984) Supra; Chow et al, Proc. Natl Acad. Sci., USA, 82:910-914; and Bittle et al., J. Gen. Virol, 66:2347-2354 (1985)). A prefened immunogenic epitope includes the secreted protein. The immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse). Alternatively, the immunogenic epitope may be prescribed without a carrier, if the sequence is of sufficient length (at least about 25 amino acids). 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 prefened, as well as the products of a Fab or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, human, and humanized antibodies.

The antibodies may 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 immimogenicity 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 a human antibody comprises immunizing a non- human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-44 and 88-102, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-44 and 88-102 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 NOs: 1-44 and 88- 102 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 fransgenic 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 ID NOs: 1-44 and 88-102; 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 NOs: 1-44 and 88-102. 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 NOs: 1-44 and 88-102. Disorders, such as IBD, which may be caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated. For example, inflammation associated with IBD is the result of locally produced cytokines and chemokines that bind to cell surface receptors on various cells of the immxme system, thus triggering changes in immune cell physiology that lead to disease. An antibody that binds a polypeptide of the present invention could prevent the binding of cytokines or chemokines to cell surface receptors on immune cells, thus preventing disease progression. 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 NOs: 1-44 and 88-102. For example in IBD, focal inflammation is mediated by chemotaxis of immune cells that can locally produce cytokines leading to the inflammation. Administration of antibody specific for the identified polypeptides could be used to modulate these events. A prefened embodiment of the present invention is a therapeutic method comprising administering to a mammalian subject a therapeutically effective amount of an antibody induced by a polypeptide translated from a nucleotide sequence chosen from the SEQ ID NOs: 1-44 and 88-102, to prevent, treat, ameliorate or modulate a disease or condition, such as IBD.

Also provided herein are conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody dirested against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-44 and 88-102. Examples of such agents are well known, and include but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drugs. See, e.g., Thrush et. al (Annu. Rev. Immunol, 14:49-71, 1996, p. 41). 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.

In addition, 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 (see, e.g., 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, Bennett et al., J. Mol. Recognition 8:52-58 (1995); 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 prefened embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. As described in Gentz et al., for instance, hexa-histidine provides for convenient purification of the fusion protein (Proc. Natl Acad. Sci. USA 86:821-824 (1989)). Another peptide tag useful for purification, the "HA" tag, conesponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984)). Other fusion proteins may use the ability of the polypeptides of the present invention to target the delivery of a biologically active peptide. This might include focused delivery of a toxin to tumor cells, or a growth factor to stem cells. Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. See, e.g., Curr. Prot. Mol. Bio., Chapter 9.6. Fusion proteins can be used to prevent, treat, ameliorate, or modify IBD.

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 retro viral 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, trp, 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, and Bowes melanoma cells, and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors prefened for use in bacteria include pQΕ70, pQE60 and pQE- 9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Sfratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among prefened eukaryotic vectors are pWLNEO, ρSV2CAT, pOG44, pXTl and pSG available from Sfratagene; 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, elecfroporation, fransduction, 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 franslation initiation codon generally is removed with high efficiency from any protein after franslation 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.

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 prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.

Diagnosis and Treatment

Where a polynucleotide of the invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as IBD, the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, the use of ribozymes to cleave polynucleotides. Alternatively, drugs or antibodies that bind to and inactivate the polypeptide product can be used. Where a polynucleotide of the invention is down-regulated and exacerbates a pathological condition, such as EBD, the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, administering a polynucleotide or polypeptide of the invention to the mammalian subject. For example, in mdrla-/- mice with spontaneous IBD, TOGA™ detected decreased expression of the polynucleotide with SEQ ID NO: 30 (DST EVIX5_54). By enhancing the in vivo levels of this polynucleotide or the polypeptide product, it may be possible to prevent, treat, ameliorate, or modulate IBD.

A polynucleotide of the invention can be administered to a mammalian subject by a recombinant expression vector comprising the polynucleotide. A mammalian subject can be a human, baboon, chimpanzee, macaque, cow, horse, sheep, pig, horse, dog, cat, rabbit, guinea pig, rat or SEQ ID NOs: 1-44 and 88-102 or a polynucleotide which is at least 98% identical to a nucleic acid sequence shown in SEQ ID NOs: 1- 44 and 88-102. Also, preferably, the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%, or 95% identical to a polynucleotide comprising SEQ ID NOs: 1-44 and 88-102.

The administration of a polynucleotide or recombinant expression vector of the invention to a mammalian subject can be used to express a polynucleotide in said subject for the treatment of, for example, IBD. Expression of a polynucleotide in target cells, including but not limited to colon cells, would effect greater production of the encoded polypeptide. In some cases, where the encoded polypeptide is a nuclear protein, the regulation of other genes may be secondarily up- or down-regulated. High expression of the polynucleotide would be advantageous since decreased expression of some polynucleotides (e.g. SEQ ID NO: 30; DST IMX 5_54), as detected by TOGA™, was associated with the development of IBD. There are available to one skilled in the art multiple viral and non-viral methods suitable for introduction of a nucleic acid molecule into a target cell, as described above. In addition, a naked polynucleotide can be administered to target cells. Polynucleotides and recombinant expression vectors of the invention can be administered as a pharmaceutical composition. Such a composition comprises an effective amount of a polynucleotide or recombinant expression vector, and a pharmaceutically acceptable formulation agent selected for suitability with the mode of administration. Suitable formulation materials preferably are non-toxic to recipients at the concentrations employed and can modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. See Remington 's Pharmaceutical Sciences (18^th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990).

The pharmaceutically active compounds (i.e., a polynucleotide or a vector) can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals. Thus, the pharmaceutical composition comprising a polynucleotide or a recombinant expression vector may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The dosage regimen for treating a disease with a composition comprising a polynucleotide or expression vector is based on a variety of factors, including the type or severity of the IBD, the age, weight, sex, medical condition of the patient, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A typical dosage may range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the factors mentioned above.

The frequency of dosing will depend upon the pharmacokinetic parameters of the polynucleotide or vector in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.

The cells of a mammalian subject may be transfected in vivo, ex vivo, or in vitro. Administration of a polynucleotide or a recombinant vector containing a polynucleotide to a target cell in vivo may be accomplished using any of a variety of techniques well known to those skilled in the art. For example, U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector. The above-described compositions of polynucleotides and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration as well as by inhalation spray. The term "parenteral" as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.

While the nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

Another delivery system for polynucleotides of the invention is a "non- viral" delivery system. Techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄ precipitation, gene gun techniques, elecfroporation, lipofection, and colloidal dispersion (Mulligan, R., (1993) Science, 260 (5110): 926-32). Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection. Several such methodologies have been utilized by those skilled in the art with varying success (Mulligan, R., (1993) Science, 260 (5110):926-32). Where a polynucleotide of the invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as IBD, the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, the use of antisense oligonucleotides or ribozymes. Alternatively, drugs or antibodies that bind to and inactivate the polypeptide product can be used. For example, in mdrla^{" "} mice with spontaneous IBD, TOGA detected increased expression of the polynucleotide with SEQ ID NO: 41 (DST IMX 5_31). By decreasing the in vivo levels of this polynucleotide or the polypeptide product, it may be possible to to prevent, treat, ameliorate, or modulate IBD.

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or franslation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of gene products of the invention in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleoti.de linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphora idates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, (1994) Meth. Mol. Biol, 20:1-8; Sonveaux, (1994) Meth. Mol Biol, 26:1-72; Uhlmann et al., (1990) Chem. Rev., 90:543-583. Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a gene of the invention. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are prefened.

Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Can, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N ., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular polynucleotide sequence. Antisense oligonucleotides can be modified without affecting their ability to hybridize to a polynucleotide of the invention. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5'- substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., (1992) Trends Biotechnol, 10:152-158; Uhlmann et al., (1990) Chem. Rev., 90:543-584; Uhlmann et al., (1987) Tetrahedron. Lett, 215:3539-3542. Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, (1987) Science, 236:1532-1539; Cech, (1990) Ann. Rev. Biochem., 59:543-568; Cech, (1992) Curr. Opin. Struct. Biol, 2:605-609; Couture & Stinchcomb, (1996) Trends Genet., 12:510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a polynucleotide of the invention can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide. Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988) Nature, 334:585-591). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, e.g., Gerlach et al, EP 321,201). Specific ribozyme cleavage sites within a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides conesponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NOs: 1-44 and 88-102 and their complements provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target. Ribozymes can be introduced into cells as part of a DNA construct.

Mechanical methods, such as microinjection, liposome-mediated transfection, elecfroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease polynucleotide expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional tenninator signal, for controlling transcription of ribozymes in the cells. As taught in Haseloff et al., U.S. Patent 5,641 ,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Production of Diagnostic Tests

Pathological conditions or susceptibility to pathological conditions, such as IBD, can be diagnosed using methods of the invention. Testing for expression of a polynucleotide of the invention or for the presence of the polynucleotide product can conelate with the severity of a condition such as IBD and can also indicate appropriate treatment for the condition. For example, the presence or absence of a mutation in a polynucleotide of the invention can be determined and a pathological condition (for example, IBD) or a susceptibility to a pathological condition is diagnosed based on the presence or absence of the mutation. Further, an alteration in expression of a polypeptide encoded by a polynucleotide of the invention can be detected, where the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition. The alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.

As an additional riiethod of diagnosis, a first biological sample from a patient suspected of having a pathological condition, such as IBD, is obtained along with a second sample from a suitable comparable control source. A biological sample can comprise saliva, blood, urine, feces, or tissue, such as gastrointestinal tissue. A suitable control source can be obtained from one or more mammalian subjects that do not have the pathological condition. For example, the average concentrations and distribution of a polynucleotide or polypeptide of the invention can be determined from biological samples taken from a representative population of mammalian subjects, wherein the mammalian subjects are the same species as the subject from which the test sample was obtained. The amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample. The amounts of the polypeptide in the first and second samples are compared. A patient is diagnosed as having a pathological condition if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample. Preferably, the amount of polypeptide in the first sample falls in the range of samples taken from a representative group of patients with the pathological condition. Such a method can be used in diagnosing IBD, for example.

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. The present invention also includes a diagnostic system, preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, such brain tissue, cell suspensions or tissue sections; or a body fluid sample, such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.

In a related embodiment, a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide of the present invention, a gene conesponding to a polynucleotide of the present invention, or a mRNA in a cell that is diagnostic for the presence or expression of a polypeptide of the present invention in the cell. The nucleic acid molecule probes can be of a variety of lengths from at least about 10, suitably about 10 to about 5000 nucleotides long, although they will typically be about 20 to 500 nucleotides in length. Hybridization methods are extremely well known in the art and will not be described further here. In a related embodiment, detection of genes conesponding to the polynucleotides of the present invention can be conducted by primer extension reactions such as the polymerase chain reaction (PCR). To that end, PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence of the gene to be detected. Preferably, the nucleotide sequence is a portion of the nucleotide sequence of a polynucleotide of the present invention. Particularly prefened PCR primers can be derived from any portion of a DNA sequence encoding a polypeptide of the present invention, but are preferentially from regions which are not conserved in other cellular proteins.

Prefened PCR primer pairs useful for detecting the genes conesponding to the polynucleotides of the present invention and expression of these genes are described in the Examples, including the conesponding Tables. Nucleotide primers from the conesponding region of the polypeptides of the present invention described herein are readily prepared and used as PCR primers for detection of the presence or expression of the conesponding gene in any of a variety of tissues. Also prefened is a diagnostic system for diasnosing a disease or condition, such as IBD. The diagnostic system includes, in an amount sufficient to perform at least one assay, a subject polypeptide of the present invention, a subject antibody or monoclonal antibody, and/or a subject nucleic acid molecule probe of the present invention, as a separately packaged reagent. In another embodiment, a diagnostic system, preferably in kit form, is contemplated for assaying for the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample. Such diagnostic kit would be useful for monitoring the fate of a therapeutically administered polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention. The system includes, in an amount sufficient for at least one assay, a polypeptide of the present invention and/or a subject antibody as a separately packaged immunochemical reagent.

Instructions for use of the packaged reagent(s) are also typically included. A diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.

Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems. The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochrom.es such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immimofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., Eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference. Other suitable labeling agents are known to those skilled in the art. In prefened embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3-ethyl-benzthiazoline-G- sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴I,

I, I, l and Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly prefened is I. Another group of useful labeling means are those elements such as ^πC, ¹⁸F, ¹⁵O and ¹³N which themselves emit posifrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such ^{ι π} indium or ³H.

The linking of labels or labeling of polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium (see, e.g., Galfre et al., Meth. Enzymol, 73:3-46 (1981)). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable (see, e.g., Aurameas, et al., Scand. J. Immunol, Vol. 8 Suppl. 7:7-23 (1978); Rodwell et al., Biotech., 3:889-894 (1984); and U.S. Pat. No. 4,493,795).

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that species is present as part of a complex.

In prefened embodiments, the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of the polypeptide of the present invention in a sample. A description of the ELISA technique is found in Sites et al., Basic and Clinical Immunology, 4^th Ed., Chap. 22, Lange Medical Publications, Los Altos, CA (1982) and in U.S. Patent No. 3,654,090; Patent No. 3,850,752; and Patent No. 4,016,043, which are all incorporated herein by reference.

Thus, in some embodiments, a polypeptide of the present invention, an antibody or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium, although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption methods are described herein.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ), agarose; polystyrene beads of about 1 micron (μm) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, IL), polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs (sheets, strips or paddles) or tubes, plates or the wells of a microtiter plate, such as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent, or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.

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. Cunently no diagnostic markers exist to detect and diagnose IBD. 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 NOs: 1-44 and 88-102. 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 conesponding to the SEQ ID NOs: 1-44 and 88-102 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 of 2,000-4,000 bp are prefened. For a review of this technique, see Venna 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). Prefened polynucleotides conespond 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 one 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 conesponding gene between affected and unaffected individuals can be examined. The polynucleotides of SEQ ID NOs: 1-44 and 88-102 can be used for this analysis of individuals. A genetic etiology is suggested by the prevalence of IBD in certain populations among 1^st degree relatives and by familial clustering of the disease.

First, visible structural alterations in the chromosomes, such as deletions or franslocations, 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 nonnal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the conesponding 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 reanangement, or mutation) can be used as a diagnostic or prognostic marker of a disease or condition, such as IBD.

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, prefened polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (see, Lee et al., Nuc. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251:1360 (1991) for discussion of triple helix formation) or to the mRNA itself (see, Okano, J. Neurochem, 56:560 (1991); and Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988) for a discussion of antisense technique). 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, such as IBD.

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 conect 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. Such methods can be used to treat IBD using the polynucleotides of the present invention.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, Ed., PCR Technology, M Stockton Press (1989)). 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 conesponding 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, et al., J. Cell. Biol, 101:976-985 (1985); Jalkanen, 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 (¹²⁵1, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^{J 12}fiι), and technetium (^99mTc); 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, nuclear magnetic resonance (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 (e.g., I, In, ^99mTc), a radio-opaque substance, or a material detectable by NMR, is introduced (e.g., parenterally, subcutaneously, or infraperitoneally) 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, the quantity of radioactivity necessary for a human subject 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 Burchiel et al., "Immunoph-irmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Eds., Masson Publishing Inc. (1982)). Thus, the invention provides a diagnostic method of a disorder, such as IBD, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; and (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. Inflammation associated with IBD is the result of locally produced cytokines, chemokines and changes in immune cell physiology involving up- or down-regulation of polynucleotides and polypeptides. These changes can be diagnosed or monitored by assaying changes in polypeptide levels in tissues such as endoscopic biopsy specimens from gut epithelium, in fluids such as blood, or in fecal samples.

Moreover, polypeptides of the present invention can be used to treat disease, for example, IBD. 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 tumor necrosis factor (TNF) receptors used in reducing inflammation); or to bring about a desired response (e.g., blood vessel growth). Of particular relevance to IBD is the use of a polypeptide of the present invention to replace decreased levels of a polypeptide that makes a person susceptible to IBD or that causes some of the symptoms of IBD.

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease. For example, adminisfration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, adminisfration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor). Polypeptides can also be used as antigens to trigger immune responses. Local production of cytokines modulates many aspects of immune cell function. In IBD, local production of cytokines activates and promotes chemotaxis of T cells that are overly aggressive in their responses to normal gut bacteria. Administration of an antibody to an overproduced polypeptide can be used to modulate the T cell response. Treatment of patients with IBD with a polypeptide or polynucleotide of the present invention might act as a vaccine to trigger a more efficient immune response, thus altering the course of disease.

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 Activity 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, or may be useful as a marker or detector of immune changes associated with IBD.

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, Di George's Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polypeptide or polynucleotide of the present invention could also be used to modulate hemostatic (bleeding cessation) 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 the treatment or detection of 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, or in some way results in the induction of tolerance, 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-Baxre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease. IBD has several aspects that suggest an autoimmiine component. Patients with IBD exhibit immune systems that have overly aggressive responses to normal intestinal gut flora. In addition extraintestinal disorders such as arthritis accompany IBD further suggesting an autoimmune mechanism.

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 im iine 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 rej ection, 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.)

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, ligament) tissue. Preferably, regeneration occurs without scarring or with minimal scarring. Regeneration also may include angiogenesis. In the case of IBD the recurrent infl--mmation triggers chronic degeneration and regeneration of the intestinal wall that never resolves leading to formation of scar tissue as part of the wound healing process. Scar tissue replaces normal cell populations thus preventing return to homeostasis within the intestine. The identified molecules can be used to modify the healing process, to prevent scarring and to promote repopulation of the gut by normal epithelial cells.

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. In the case of IBD, ongoing inflammatory responses in the intestinal wall may reflect defective healing of the barrier provided by intestinal epithelium. Polynucleotides or polypeptides of the present invention might be used to treat IBD by boosting the capacity of the epithelium to heal.

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, amyofrophic 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. Such molecules could also be used to treat a variety of disorders. Thus, a polynucleotide or polypeptide of the present invention could be used as an inhibitor of chemotaxis. For IBD, the most dramatic sign of pathology in the gut is the chemotactic recruitment of inflammatory cells. Polynucleotides or polypeptides of the present invention may be used to either inhibit the recruitment of cells driving the pathology or induce the recruitment of cells able to protect the tissue from damage.

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 (i.e., an agonist), increase, inhibit (i.e., an 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, related to a fragment of the receptor capable of being bound by the polypeptide (e.g., an 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. Prefened 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. At present, IBD diagnosis depends on a number of relatively invasive and expensive clinical tests. Assays for the presence of markers, in easily obtained specimens (blood, urine or stool) may provide an important diagnostic tool.

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 occuned. 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 from a lineage other than the above-described hemopoietic lineage. The ulcerative events of IBD destroy the intestinal epithelium. The identified molecules may be used to promote the differentiation and proliferation of stem cells to repopulate the gut epithelium and promote healing.

Other Preferred Embodiments

Other prefened 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 NOs: 1 -44 and 88- 102.

Also prefened is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOs: 1-44 and 88-102 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 prefened is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOs: 1-44 and 88-102 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 NOs: 1-44 and 88-102. Similarly prefened is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOs: 1-44 and 88-102 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 NOs: 1-44 and 88-102.

Also prefened 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 NOs: 1-44 and 88-102. Further prefened 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 NOs: 1-44 and 88-102.

A further prefened embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 1-44 and 88-102 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 NOs: 1-44 and 88-102. A further prefened embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NOs: 1-44 and 88-102.

Also prefened 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 prefened 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 NOs: 1-44 and 88-102, 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 prefened 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 prefened 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 prefened 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 NOs: 1-44 and 88-102. Also prefened is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene (for example, IBD), 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 NOs: 1-44 and 88-102. Also prefened 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 ID NOs: 1-44 and 88-102. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

Also prefened 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 NOs: 1-44 and 88-102.

Also prefened is a polypeptide, wherein said sequence of contiguous amino acids is included in amino acids in an amino acid sequence translated from SEQ ID NOs: 1-44 and 88-102, 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 prefened 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 NOs: 1-44 and 88-102. Further prefened 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 NOs: 1-44 and 88-102.

Further prefened is an isolated polypeptide comprising an amino acid sequence at least 95% identical to amino acids in an amino acid sequence translated from SEQ ID NOs: 1-44 and 88-102.

Further prefened 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 franslated from SEQ ID NOs: 1-44 and 88-102, 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 prefened 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 NOs: 1-44 and 88-102.

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 prefened 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 NOs: 1-44 and 88-102.

Also prefened 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 prefened 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 NOs: 1-44 and 88-102. In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also prefened 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 franslated from SEQ ID NOs: 1-44 and 88-102.

Also prefened 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.

Also prefened 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 ID NOs: 1-44 and 88- 102.

Further prefened is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also prefened is the recombinant vector produced by this method. Also prefened 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 prefened 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 prefened 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 franslated from SEQ ID NOs: 1-44 and 88-102. The isolated polypeptide produced by this method is also prefened.

Also prefened 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.

The polynucleotides, polypeptides, kits and methods of the present invention may be embodied in other specific forms without departing from the teachings or essential characteristics of the invention. The described embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore to be embraced therein.

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

* The extended sequence was generated from alignment to a rat database sequence in which there were several gaps in the alignment accounting for the lower overall % homology

EST = Expressed Sequence Tag; N = Northern Blot Numbers represent arbitrary units after normalization to the hybridization signal of cyclophilin ore than one transcript was found with the Northern assay, the relative DST band intensity of each transcript is listed along with its size in Kb.

EST = Expressed Sequence Tag, RT-PCR = Reverse Transcriptase Polymerase Chain Reaction

Claims

We claim:

1. 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 NO: 11, 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: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102 associated with inflammatory bowel disease.

2. An isolated polypeptide encoded by 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 NO: 11, 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: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO:102 associated with inflammatory bowel disease.

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 3' end or the 5' end.

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, modulating, 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. The method of claim 21 wherein the medical condition is inflammatory bowel disease.

23. A method for preventing, treating, modulating, or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of the antibody of claim 16.

24. The method of claim 23 wherein the medical condition is infl--mmatory bowel disease.

25. 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.

26. The method of claim 25 wherein the pathological condition is inflammatory bowel disease.

27. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising detecting an alteration in expression of a polypeptide encoded by the polynucleotide of claim 1, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.

28. The method of claim 27 wherein the alteration in expression is an increase in the amount of expression or a decrease in the amount of expression.

29. The method of claim 27 wherein the pathological condition is inflammatory bowel disease.

30. The method of claim 29 wherein the method further comprises the steps of: obtaining a first biological sample from a patient suspected of having inflammatory bowel disease and obtaining a second sample from a suitable comparable control source;

(a) determining the amount of at least one polypeptide encoded by a polynucleotide of claim 1 in the first and second sample; and (b) comparing the amount of the polypeptide in the first and second samples; wherein a patient is diagnosed as having inflammatory bowel disease if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.

31. The use of the polynucleotide of claim 1 or polypeptide of claim 2 for the manufacture of a medicament for the treatment of inflammatory bowel disease.

32. The use of the antibody of claim 16 for the manufacture of a medicament for the treatment of inflammatory bowel disease.

33. 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.

34. The gene corresponding to the cDNA sequence of the isolated nucleic acid of claim 1. .

35. 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.

36. A substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in inflammatory bowel disease, 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 infl-tmmatory bowel disease.

37. A kit suitable for detecting the presence of the polypeptide of claim 2 in a mammalian tissue sample comprising a first antibody which immunoreacts with a mammalian protein encoded by a gene corresponding 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.

38. A kit of claim 37 further comprising a second antibody that binds to the first antibody.

39. The kit of claim 38 wherein the second antibody is labeled.

40. The kit of claim 39 wherein the label comprises enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.

41. A kit suitable for detecting the presence of a gene encoding a protein comprising a polynucleotide of claim 1 or 3, or a fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.

,

42. A method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample, comprising the steps of:

(a) hybridizing a polynucleotide of claim 1 or fragment thereof having at least 10 contiguous bases, with the nucleic acid of the sample; and

(b) detecting the presence of the hybridization product.

43. An isolated nucleic acid molecule comprising an antisense polynucleotide to the nucleic acid molecule of Claim 1 wherein the isolated nucleic acid molecule is associated with inflammatory bowel disease.

44. An antisense polynucleotide of Claim 6 wherein the antisense polynucleotide comprises at least three nucleotides of the complementary nucleic acid molecule of Claim 1 wherein the antisense polynucleotide associated with inflammatory bowel disease.

45. An isolated polypeptide comprising the polypeptide of Claim 2 with conservative amino acid substitutions wherein the isolated polypeptide is associated with inflammatory bowel disease.

46. An isolated polypeptide comprising a variant of the polypeptide of Claim 2 which has the same biological activity as the polypeptide of Claim 2 wherein the isolated polypeptide is associated with inflammatory bowel disease.

47. An isolated polypeptide comprising a homologous polypeptide of the isolated polypeptide of Claim 2 wherein the isolated polypeptide is associated with with inflammatory bowel disease.

48. A method of identifying biomolecules associated with with inflammatory bowel disease., comprising the steps of;

(A) developing a cellular experiment specific for with inflammatory bowel disease;

(B) harvesting the RNA from the cells used in the experiment; (C) obtaining a gene expression profile; and

(D) using the gene expression profile for identifying biomolecules which expression was altered during the experiment.

49. The method of claim 48 wherein the biomolecules identified are polynucleotides.

50. The method of claim 48 wherein the biomolecules identified are polypeptides and the method further comprises the step of identifying polypeptides encoded by the polynucleotides identified in claim 49.