WO2002031114A2 - Expression genique module dans l'ileite - Google Patents

Expression genique module dans l'ileite Download PDF

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Publication number
WO2002031114A2
WO2002031114A2 PCT/US2001/032091 US0132091W WO0231114A2 WO 2002031114 A2 WO2002031114 A2 WO 2002031114A2 US 0132091 W US0132091 W US 0132091W WO 0231114 A2 WO0231114 A2 WO 0231114A2
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WIPO (PCT)
Prior art keywords
seq
polypeptide
ofthe
ileitis
polynucleotide
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PCT/US2001/032091
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English (en)
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WO2002031114A3 (fr
Inventor
Joanne L. Viney
John E. Sims
Robert F. Dubose
Peter R. Baum
Karl W. Hasel
Brian S. Hilbush
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Digital Gene Technologies, Inc.
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Priority to AU2002213207A priority Critical patent/AU2002213207A1/en
Publication of WO2002031114A2 publication Critical patent/WO2002031114A2/fr
Publication of WO2002031114A3 publication Critical patent/WO2002031114A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • IBD Inflammatory bowel diseases
  • CD Crohn's disease
  • UC ulcerative colitis
  • Ileitis is a general term for inflammation ofthe small intestine, and encompasses chronic IBD, such as Crohn's, and also acute enteropathies.
  • the pathological features of IBD include massive gut damage, especially in the mucosal epithelium, and intestinal inflammation.
  • the histological features suggest a dysregulation ofthe lymphoid tissue.
  • the immunological dysregulation is a primary cause of IBD or whether the inflammatory response is secondary to another mucosal insult is unclear.
  • T cell activation Injection of mice with anti-CD3 mAb causes T cell activation. This activation displays the normal characteristics of T cell activation, including increased ' IL-2R expression, colony stimulating factor secretion, and extra-medullary hematopoiesis in the spleen (R. Hirsch et al, J. Immunol. 142:737-743, 1989).
  • Anti-CD3 antibody also induces T cell activation in explants of fetal human small intestine (T.T. MacDonald and J. Spencer, J. Exp. Med. 167:1341-1349, 1988).
  • the CD3 antigen is intimately associated with the T cell receptor (TCR) on the surface of T cells.
  • binding of CD3 by anti-CD3 mAb may cause T cell activation through the TCR.
  • T cell activation along with release of proinfla matory cytokines, has been implicated in the pathogenesis of IBD. Intestinal damage in human IBD is associated with evidence of enhanced T cell activation in the mucosal tissues.
  • activation of T cells in explants of human small intestine in vitro induces crypt hyperplasia and villous atrophy, two hallmarks of IBD (T.T. MacDonald and J. Spencer, J. Exp. Med. 167:1341- 1349, 1988).
  • the anti-CD3 mAb mouse model of EBD mimics several ofthe characteristics of human IBD.
  • Anti-CD3 mAb-induced intestinal damage is characterized by villus atrophy, crypt destruction, loss of goblet cells and enhanced apoptosis in the epithelium and lamina propria — pathology similar to that seen in human IBD.
  • the anti-CD3 mAb-induced IBD model system can be used to examine (i) the mechanism by which activated T cells initiate intestinal damage and (ii) the mechanisms that direct recovery and trigger the repair of damaged tissues.
  • This model system can serve to identify genes and proteins that modulate or prevent intestinal damage or stimulate recovery ofthe mucosa. Given the diversity of factors that may contribute to these processes, a clear need is evident for the identification of new proteins involved in IBD and the elucidation of their roles.
  • TOGATM Total Gene Expression Analysis
  • the TOGATM 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 ofthe 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.
  • the PCR-based Total Gene Expression Analysis (TOGA TM ) differential display system has been used in studies to examine how IBD is regulated in the anti-CD3 mAb- induced IBD model system. Such studies have examined the mechanism of disease in response to anti-CD3 mAb and have examined protems and genes that may prevent IBD.
  • Molecules have been identified that correspond to genes that are regulated by the administration of anti-CD3 mAb to mice. Such molecules are useful in therapeutic and diagnostic applications in the treatment of IBD.
  • the present invention provides identified molecules that correspond to genes that are regulated by the anti-CD3 mAb treatment. Such molecules are useful in therapeutic and diagnostic applications in the treatment of IBD and other gut pathologies.
  • the present invention provides novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides. Also provided are diagnostic methods for detecting disorders related to the polypeptides and the polynucleotides encodmg them, and therapeutic methods for treating such disorders. The invention further relates to screemng methods for identifying binding partners ofthe polypeptides.
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 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
  • 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:l-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • 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:l-76, SEQ ID NO: 134, and SEQ ID NOs:147-159, under stringent conditions.
  • the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:
  • the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in gastrointestinal inflammation, chosen from the group consisting ofthe DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in gastrointestinal inflammation.
  • the present invention also provides a system and method for detecting the presence of a gene regulated in gastrointestinal inflammation.
  • the present invention provides a kit for suitable for detecting the presence of a gene regulated in gastrointestinal inflammation, comprising at least one polynucleotide ofthe present invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay; label means; instructions for use; and suitable packaging material.
  • the polynucleotide is chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, 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 ED NO:34, SEQ ID NO:31
  • Another embodiment comprises a polynucleotide at least 95% identical to an isolated nucleic acid molecule of selected from the group consisting of SEQ ID NOs:l-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • 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:l-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 under stringent conditions.
  • a polynucleotide is chosen from the group consisting of DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in gastrointestinal inflammation.
  • Another embodiment ofthe invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as ileitis comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide ofthe invention or a polynucleotide ofthe invention.
  • a further embodiment of the invention provides an isolated antibody that binds specifically to the isolated polypeptide ofthe invention.
  • a preferred embodiment ofthe invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as ileitis, comprising administering to a mammalian subject a therapeutically effective amount ofthe antibody.
  • An additional embodiment ofthe 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 ileitis is diagnosed based on the presence or absence ofthe mutation.
  • Even another embodiment ofthe invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition, such as ileitis in a subject.
  • the method comprises detectmg an alteration in expression of a polypeptide encoded by the polynucleotide ofthe invention, wherein the presence of an alteration in expression ofthe polypeptide is indicative ofthe 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.
  • a first biological sample is obtained from a patient suspected of having ileitis and a second sample from a suitable comparable control source is obtained.
  • the amount of at least one polypeptide encoded by a polynucleotide ofthe invention is determined in the first and second sample.
  • a patient is diagnosed as having ileitis if the amount ofthe polypeptide in the first sample is greater than or less than the amount ofthe polypeptide in the second sample.
  • a polynucleotide ofthe invention is down-regulated and exacerbates a pathological condition, such as IBD
  • the expression ofthe polynucleotide can be increased or the level ofthe 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 ofthe invention to the mammalian subject.
  • TOGA detected decreased expression ofthe polynucleotide with SEQ ID NO: 23 (IMX 3_65).
  • a polynucleotide ofthe invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as IBD
  • the expression ofthe polynucleotide can be blocked or reduced or the level ofthe 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, triple helix base pairing methodology or ribozymes.
  • drugs or antibodies that bind to and inactivate the polypeptide product can be used.
  • TOGA detected increased expression ofthe polynucleotide with SEQ ID NO: 7 (IMX 3_16).
  • Figure 1 is a graphical representation ofthe results of TOGA TM runs using a 5' PCR primer with parsing bases AGTG (SEQ ID NO: 132) and the universal 3' primer (SEQ ID NO: 145), showing PCR products produced from mRNA extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours (control) ( Figure IA), 6 hours (Figure IB), 30 hours (Figure 1C), and 72 hours ( Figure ID).
  • the horizontal axis represents the number of base pairs ofthe molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA analysis (which corresponds to the relative expression ofthe molecule of that address).
  • Figure 2 shows aNorthern Blot analysis of clone IMX3_67 (SEQ ID NO: 25; AGTG
  • Figure 3 is a graphical representation ofthe results of reverse transcriptase polymerase chain reaction (RT-PCR) of clone IMX3_25 (SEQ ID NO:31) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • Figure 4 is a graphical representation ofthe results of RT-PCR of clone IMX3_29 (SEQ ID NO:32) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 5 is a graphical representation ofthe results of RT-PCR of clone IMX3_30 (SEQ ID NO: 14) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 6 is a graphical representation ofthe results of RT-PCR of clone IMX3_32 (SEQ ED NO : 15) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 7 is a graphical representation ofthe results of RT-PCR of clone IMX3_37 (SEQ ID NO: 16) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 8 is a graphical representation ofthe results of RT-PCR of clone IMX3_39 (SEQ ID NO: 18) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 9 is a graphical representation ofthe results of RT-PCR of clone IMX3_41 (SEQ ID NO:33) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 10 is a graphical representation ofthe results of RT-PCR of clone IMX3_42 (SEQ ID NO:34) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 11 is a graphical representation ofthe results of RT-PCR of clone IMX3_44 (SEQ ID NO: 35) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 12 is a graphical representation ofthe results of RT-PCR of clone IMX3_45 (SEQ ID NO: 52) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 13 is a graphical representation ofthe results of RT-PCR of clone IMX3_48 (SEQ ID NO: 55) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 14 is a graphical representation ofthe results of RT-PCR of clone IMX3 51 (SEQ ID NO:56) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 15 is a graphical representation ofthe results of RT-PCR of clone IMX3_53 (SEQ ID NO-.19) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 16 is a graphical representation ofthe results of RT-PCR of clone IMX3_59 (SEQ ID NO:21) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 17 is a graphical representation ofthe results of RT-PCR of clone IMX3_60 (SEQ ID NO:22) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 18 is a graphical representation ofthe results of RT-PCR of clone IMX3_64 (SEQ ID NO:37) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 19 is a graphical representation ofthe results of RT-PCR of clone IMX3_65 (SEQ ID NO:23) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 20 is a graphical representation ofthe results of RT-PCR of clone EMX3_69 (SEQ ID NO: 26) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 21 is a graphical representation ofthe results of RT-PCR of clone IMX3_71 (SEQ ID NO:27) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 22 is a graphical representation ofthe results of RT-PCR of clone IMX3 73 (SEQ ID NO:28) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 23 is a graphical representation ofthe results of RT-PCR of clone IMX3 83 (SEQ ID NO: 66) where poly A enriched mRNA was exfracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 24 is a graphical representation ofthe results of RT-PCR of clone IMX3_85 (SEQ ID NO:38) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 25 is a graphical representation ofthe results of RT-PCR of clone IMX3 94 (SEQ ID NO:29) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 26 is a graphical representation ofthe results of RT-PCR of clone IMX3_99 (SEQ ID NO:39) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 27 is a graphical representation ofthe results of RT-PCR of clone IMX3 102 (SEQ ID NO:73) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 28 is a graphical representation ofthe results of RT-PCR of clone IMX3_103 (SEQ ID NO:74) where poly A enriched mRNA was extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50 ug) and sacrificed after 0 hours, 6 hours, 30 hours, and 72 hours.
  • Figure 29 A-D is a graphical representation similar to Figure 1 ofthe results of
  • Figure 30 A-C presents a graphical example ofthe results obtained when a DST is verified by the Extended TOGA TM Method using a primer generated from a cloned product (as described below).
  • the length ofthe PCR product corresponding to SEQ ID NO:69 (IMX3_95) was cloned and a 5 ' PCR primer was built from the cloned DST (SEQ ID NO:141).
  • 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 TOGATM as IMX3_95 (SEQ ID NO:69). Panel C thus illustrates that IMX3_95 (SEQ ID NO:69).
  • 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 ofthe coding sequences that flank that part ofthe molecule in the genome ofthe organism in which it naturally occurs; (b) a nucleic acid inco ⁇ orated 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
  • 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.
  • 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 occu ⁇ ing), 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-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • the polynucleotide can contain all or part ofthe nucleotide sequence ofthe 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 ofthe present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • 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.
  • 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” ofthe present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NOs:l-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 or the complement thereof, or the cDNA.
  • 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.
  • BLOTTO 5% w/v non-fat dried milk in phosphate buffered saline
  • heparin 5% w/v non-fat dried milk in phosphate buffered saline
  • denatured salmon sperm DNA and other commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems with compatibility.
  • 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 ofthe 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 translation of these alternative open reading frames are specifically contemplated by the present invention.
  • the polypeptide ofthe 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.
  • 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
  • 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 ofthe biological activity is measured by its dose-response characteristics; in the case of polypeptides with activity similar to the polypeptide ofthe present invention, the dose-response dependency will be substantially similar in a given activity as compared to the polypeptide ofthe present invention.
  • Polypeptides may derive their "biological activity" through binding to specific cellular receptors, which mediate secondary signals to the target cell or tissue.
  • 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 co ⁇ esponding 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.
  • Standard 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 ofthe 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.
  • Constant amino acid substitution refers to a substitution between similar amino acids that preserves an essential chemical characteristic ofthe 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.
  • Multiple 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.
  • mAb Monoclonal antibody
  • mAb 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 ofthe 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.
  • protem 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 ofthe 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 ofthe present invention.
  • 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 prefe ⁇ ed embodiment ofthe 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.”
  • an "immunogenic epitope” is defined as apart 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 co ⁇ esponding 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 ofthe following characteristics with respect to the polypeptides ofthe 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 inhibitors.
  • 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 ofthe present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition ofthe 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.
  • 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.
  • a 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 ofthe present invention.
  • 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 ofthe 3' end of an mRNA.
  • IBD irritable bowel disease
  • mice were injected intraperitoneally with 50 ug of anti-CD3 monoclonal antibody.
  • the small intestines were removed from the mice at the following time points: 0 hours (control), 6 hours, 30 hours, and 72 hours.
  • the dissected small intestines were immediately placed in guanidinium thiocyanate buffer and homogenized. Homogenized lysates were centrifuged briefly to remove large debris. The lysates were layered onto a CsCl gradient and centrifuged to isolate the RNA. The RNA was isolated from the gradient using conventional methods.
  • TOGA TM TOtal Gene expression Analysis
  • the isolated RNA was enriched to form a starting polyA-containing mRNA population by methods known in the art.
  • 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 1 PCR primers in separate reactions produced PCR products that were cDNA fragments that co ⁇ esponded to the 3'-region ofthe starting mRNA population.
  • the produced PCR products were then identified by: a) the imtial 5' sequence comprising the sequence remainder ofthe recognition site ofthe restriction endonuclease used to cut and isolate the 3' region plus the sequence ofthe preferably four parsing bases immediately 3' to the remainder ofthe recognition site, preferably the sequence ofthe entire fragment, and b) the length ofthe 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 ofthe insert, the sequence ofthe insert provided in the sequence listing is shorter than the fragment length that forms part ofthe digital address.
  • the method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags ofthe 3' end of mRNAs. DSTs that showed changes in relative levels during anti-CD3 -induced ileitis were selected for further study. The intensities of the laser-induced fluorescence ofthe labeled PCR products were compared across samples isolated from small intestine of mice injected with anti-CD3 antibody after 0, 6, 30, and 72 hours. The results are presented in Tables 1 and 2 below.
  • 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-V-N-N (SEQ ID NO: 142), 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' endpomt 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 co ⁇ esponding to the 3' region ofthe 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 prefe ⁇ ed streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Lake Success, NY).
  • 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 ofthe cDNAs.
  • Notl which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion ofthe cDNAs.
  • the 3' Mspl-Notl cDNA fragments which are of uniform length for each mRNA species, were directionally ligated into Clal- Notl-cleaved plasmid pBC SK + (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli SURE cells (Stratagene).
  • the ligation regenerates the Notl site, but not the Mspl site, leaving CGG as the first 3 bases of the 5' end of all PCR products obtained.
  • Each library contained in excess of 5 x 10 5 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.
  • 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.
  • T3 RNA polymerase MEGAscript kit, Ambion
  • each ofthe cRNA preparations was processed in a three-step fashion.
  • 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:143).
  • step two 400 pg of cDNA product was used as PCR template in four separate reactions with each ofthe 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:144), each paired with a "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-G-T (SEQ ID NO: 145).
  • step three the product of each subpool was further divided into 64 subsubpools (2 ng in 20 ul) for the second PCR reaction, with 100 ng each ofthe fluoresceinated "universal" 3' PCR primer, the oligonucleotide (SEQ ID NO:145) conjugated to 6-FAM and the appropriate 5' PCR primer ofthe form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 146), using a program that included an annealing step at a temperature X slightly above the T ra of each 5' PCR primer to minimize artifactual mispriming and promote high fidelity copying.
  • Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).
  • N4 reaction products The products (“N4 reaction products”) from the final polymerase chain reaction step for each ofthe 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 ofthe four pools established by the 5' PCR primers ofthe first PCR reaction, for a total of 256 product subpools for the entire 5' PCR primer set ofthe second PCR reaction. The mRNA samples from each timepoint after induction of ileitis by anti-CD3 antibody as described above were analyzed. Table 1 is a summary ofthe expression levels of 372 mRNAs determined from cDNA.
  • cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length ofthe molecule, as well as the relative amount ofthe 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 ofthe parsing bases ofthe 5' PCR primer used in the final PCR step.
  • the digital address length ofthe 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.
  • the entry in Table 1 that describes a DNA molecule identified by the digital address Mspl AGTG 267 is further characterized as having a 5' terminus partial nucleotide sequence of CGGAGTG and a digital address length of 267 b.p.
  • the DNA molecule identified as Mspl AGTG 267 is further described as being expressed at decreased levels during early induction of ileitis (6 hours and 30 hours after anti-CD3 injection) and near control levels after 72 hours.
  • the DNA molecule identified as Mspl AGTG 267 is described by its nucleotide sequence, which co ⁇ esponds with SEQ ID NO:25 (DST IMX3_67).
  • the other DNA molecules identified in Table 1 by their Mspl digital addresses are further characterized by the pattern of gene expression found in small intestine after exposure to anti-CD3 antibody for various time intervals, namely after 0 hours, 6 hours, 30 hours and 72 hours of anti-CD3 antibody treatment. Additionally, several ofthe DSTs were further characterized in Tables 2-32.
  • Figure 1 is a graphical representation ofthe results of TOGA runs using a 5' PCR primer with parsing bases AGTG (SEQ ID NO: 132) and the universal 3 ' primer (SEQ ID NO: 145), showing PCR products produced from mRNA extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50ug) and sacrificed after 0 hours (control) (Figure IA), 6 hours (Figure IB), 30 hours (Figure 1C), and 72 hours ( Figure ID).
  • the horizontal axis represents the number of base pairs ofthe molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA analysis (which co ⁇ esponds to the relative expression ofthe molecule of that address).
  • the results ofthe TOGA runs have been normalized using the methods described n pending U.S . Patent Application Serial No. 09/318,699/U.S., and pending PCT Application Serial No. PCT/US00/14159, 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 index line drawn through the four panels represents the DST molecule identified as IMX3_67 (SEQ ID NO:25).
  • the vertical index line indicates a PCR product of about 267 b.p. that is present in the control sample and which expression shows a transient decreases during induction of ileitis at early time points (6 and 30 hours) and increases slightly thereafter (
  • the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands. Putative database matches for each cloned DST sequence are listed in Table 2A.
  • PCR primers ("Extended TOGA TM primers) were designed from sequence determined using using one of two methods: (1) in suitable cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands; or (2) in other cases, the sequences listed for the TOGATM PCR products were derived from candidate matches to sequences present in available GenBank, EST, or proprietary databases. PCR was performed using the Extended TOGATM 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 ofthe DST.
  • the 5' PCR primer was G-A-T-C-G-A-A-T-C-C-G-G-G-G-G-A-G-G-C-T-G-C-T-G-C-T-G-C-G-A (SEQ ID NO: 141).
  • This 5' PCR primer was paired with the fluorescence labeled universal 3' PCR primer (SEQ ID NO: 145) in a PCR reaction using the PCR NI reaction product as substrate.
  • the products are separated by electrophoresis.
  • the length ofthe PCR product generated with the Extended TOGA TM primer was compared to the length ofthe original PCR product that was produced in the TOGA TM reaction.
  • the results for SEQ ID NO: 69, for example, are shown in Figure 30.
  • the results ofthe original TOGA TM analysis are shown in Figure 29. Analysis was performed using a 5' PCR primer with parsing bases GGGA (SEQ ID NO: 133) and the universal 3' primer (SEQ ID NO: 145), with results showing PCR products produced from mRNA extracted from the small intestine of mice injected peritoneally with anti-CD3 antibody (50ug) and sacrificed after 0 hours (control, Figure 29 A), 6 hours (Figure 29B), 30 hours (Figure 29C), and 72 hours ( Figure 29D).
  • the vertical index line indicates a PCR product of about 430 b.p. that is not detectable in the control sample but shows a transient increase in expression at 6-30 hours and a return to control level after 72 hours. For the 430 b.p.
  • the 5' PCR primer was C-G-A-C-G-G-T-A-T-C-G-G-G-G- G-A (SEQ ID NO: 133).
  • This 5' PCR primer was paired with the fluorescent labeled 3' PCR primer (SEQ ID NO: 145) in PCR reactions using the cDNA produced in the first PCR reaction as substrate.
  • Figure 30 A-C presents a graphical example ofthe results obtained when a DST is verified by the Extended TOGA Method using a primer generated from a cloned product (as described above).
  • the length ofthe PCR product co ⁇ esponding to SEQ ID NO:69 (IMX3_95) was cloned and a 5' PCR primer was built from the cloned DST (SEQ ID NO: 141).
  • 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 TOGATM as IMX3_95 (SEQ ID NO:69).
  • Panel C thus illustrates that IMX3_95 (SEQ ID NO :69) was the DST amplified in Extended TOGATM.
  • IMX3_95 SEQ ID NO :69
  • a similar process was used to determine whether TOGA TM PCR products of interest were derived from sequences of candidate matches from GenBank. Extended primers in these cases were synthesized based on the sequence 3' to the terminal Mspl site ofthe GenBank entries listed in Table 2B.
  • a prefe ⁇ ed method for determining the best overall match between a query sequence (a sequence ofthe present invention) and a subject sequence, also refe ⁇ ed 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 . (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, DJ.
  • sequence includes nucleotide and amino acid sequences.
  • 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 ofthe 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 inte ⁇ reted by the experienced investigator to determine real relationships versus random similarities.
  • the BLAST program supports four different search mechanisms:
  • Extended sequences for DSTs IMX3_3 (SEQ ID NO:3), IMX3_53 (SEQ ID NO: 19), IMX3_65 (SEQ ID NO:23), IMX3_69 (SEQ ID NO:26), and IMX3_101 (SEQ ID NO:30) were generated by doing 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 IMX3_20 (SEQ ID NO:9), IMX3_24 (SEQ ID NO: 12), IMX3_26 (SEQ ID NO: 13), IMX3_32 (SEQ ID NO:15), IMX3_60 (SEQ ED NO:22), IMX3_67 (SEQ ID NO:25), IMX3_71 (SEQ ID NO:27), and IMX3_94 (SEQ ID NO:29) were obtained by performing library PCR on a cDNA lambda Zap II library (Stratagene) constructed from cDNA from small intestines of control mice or mice teated with anti-CD3, followed by sequencing ofthe PCR product.
  • a cDNA lambda Zap II library (Stratagene) constructed from cDNA from small intestines of control mice or mice teated with anti-CD3, followed by sequencing ofthe PCR product.
  • 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 (50ng) was labeled with 32 [P]-dCTP and 32 [P]-dATP using asymmetric PCR labeling.
  • the membrane was probed with radiolabeled DNA (2-5 x 10 6 cpm/ ml) overnight at 42°C in hybridization buffer.
  • the northern blots were probed with radiolabelled cyclophilin DNA to normalize the amount of mRNA in each sample.
  • the relative band intensities of 18 other DST clones are reported in Table 4 below.
  • Several clones showed an expression pattern with an increase at the earlier timepoints following anti-CD3 induced ileitis (at 6 hours and 30 hours) and a decrease by 72 hours to within about 100% to 200% of control levels (IMX3_3 (SEQ ID NO: 3), IMX3_12 (SEQ ID NO: 5), IMX3_16 (SEQ ID NO: 7), IMX3_38 (SEQ ID NO: 17)) or about 50-100% control levels (IMX3_1 (SEQ ID NO: 1), IMX3_15 (SEQ ID NO: 6), IMX3_26 (SEQ ID NO: 13), IMX3_75 (SEQ ID NO: 62), IMX3_101 (SEQ ID NO: 30)).
  • clone IMX3_11 is slightly increased after 6 hours of anti-CD3 induced ileitis and subsequently decreased after longer periods of anti-CD3 induced ileitis (30 and 72 hours).
  • Table 4 also shows that the expression of clone IMX3_9 peaks after 30 hours of anti-CD 3 induced ileitis and begins to decrease by 72 hours.
  • Table 4 shows that the gene expression of several clones decreases at early time points following anti-CD3 induced ileitis and subsequently increases after sustained induced ileitis.
  • clones IMX3_20 (SEQ ID NO: 9), IMX3_21 (SEQ ID NO: 10), and IMX3_56 (SEQ ID NO: 20) demonstrate decreased expression after 6 hours of induced ileitis, while expression increases 30-72 hours later.
  • the expression of clones X3_2 (SEQ ID NO: 2), IMX3_24 (SEQ ID NO: 12), IMX3_66 (SEQ ID NO: 24), and X3_88 (SEQ ID NO: 134) decreases after 6-30 hours of induced ileitis and begins to increase after 72 hours.
  • RT-PCR Twenty-six ofthe 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.
  • Table 6 For each DST examined, the optimal annealing temperature and reagent conditions were determined for the co ⁇ esponding 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 2 concentration, and Taq polymerase. Once optimal conditions were determined RT-PCR was performed for each DST in duplicate reactions which usually included at least four dilutions of template (TOGA TM cDNA library), 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 double-stranded cDNA prepared from mRNA (lug) for each ofthe time points, determined amounts of AmpliTaq DNA polymerase (cat. #N808-0156), MgCl 2 , dNTPs (GibcoBRL cat. #10297-018), primer, and Clontech TaqStart Antibody in a 20ul final reaction volume using lOx Taq buffer II (without MgCl 2 ). Typically, a master mix containing all components except the template was prepared and aliquoted.
  • 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.
  • IMX3_29 was lowest 6 hours after induction of ileitis and continued to increase between 30 and 72 hours after ileitis induction.
  • the results ofthe quantified RT-PCR of DST IMX3_30 (SEQ ID NO: 14) for a 1 :2500 dilution of cDNA are shown in Figure 5 (in arbitrary fluorescence units) and in Table 9 (normalized to the control value at each time point).
  • the initial TOGATM analysis showed that the expression of IMX3_30 was decreased during anti-CD3 induced ileitis. (See Table 1).
  • the expression was lowest 6 hours after induction of ileitis (about 50-fold lower than control) and increased at 30-72 hours after ileitis induction until it was slightly below control level.
  • the RT-PCR study verified that expression of IMX3_30 was decreased immediately after ileitis induction (at 6 hours) and increased thereafter.
  • the RT-PCR study verified that expression of IMX3_44 was increased about 2 to 8-fold 6 hours after ileitis induction and decreased thereafter.
  • the results ofthe quantified RT-PCR of DST IMX3_45 (SEQ ID NO: 52) for a 1 :500 dilution ofthe cDNA are shown in Figure 12 (in arbitrary fluorescence units) and in Table 16 (normalized to the control value at each time point).
  • the initial TOGA TM analysis showed that the expression of IMX3_45 was decreased immediately after anti-CD3 induced ileitis and returned to near control level by 30 hours. (See Table 1).
  • the initial TOGA TM analysis showed that the expression of IMX3_48 was decreased at earlier time points after anti-CD3 induced ileitis and began to increase at 72 hours after induction. (See Table 1). The expression was lowest 6 -30 hours after induction of ileitis (about 3 to 5-fold lower than control) and increased to slightly below control level by 72 hours. The RT-PCR study verified that expression of IMX3_48 was decreased about 1.5 to 3 times 6-30 hours after ileitis induction and increased to slightly below control level thereafter.
  • the RT-PCR study verified that expression of IMX3_59 was increased about 1.5 to 2-fold 6 hours after ileitis induction and returned to control level by 30 hours.
  • the results ofthe quantified RT-PCR of DST IMX3 60 (SEQ ID NO: 22) for a 1 :2500 dilution ofthe cDNA are shown in Figure 17 (in arbitrary fluorescence units) and in Table 21 (normalized to the control value at each time point).
  • the initial TOGATM analysis showed that the expression of IMX3_60 was decreased during anti-CD3 induced ileitis. (See Table 1).
  • the expression decreased 6-30 hours after induction of ileitis (about 3-fold lower than control) and then slightly increased at 72 hours after ileitis induction.
  • the RT-PCR study verified that expression of IMX3_60 was decreased about 1.5 to 2-fold 6-30 hours after ileitis induction.
  • EST L08187.Gb_Pr was identified as an EST encoding a protein belonging to the hematopoietin receptor family. Devergne et al, J. Virol. 70:1143-1153 (1996). The predicted structure is a secreted protein with the features of a complete, approximately 200 bp hematopoietin receptor domain. The closest identified homologues are the p40 subunit of IL- 12, which encodes an lg domain on the N-terminal end ofthe hematopoietin domain, and CNTF receptor, which is structurally similar to IL12p40, but contains a GPI linkage to the cell membrane.
  • Full length cDNA clones were isolated from a lambda phage library utilizing oligonucleotides designed from the original EST sequence.
  • the insert was cloned into Bluescript SK-.
  • Clone L081-19a comprised approximately 60 base pairs of 5' untranslated region and 450 base pairs of 3' untranslated region including a poly A tail. See SEQ ID NOs: 138, 139, and 140.
  • the insert was subcloned into the transient mammalian expression vector pDC409.
  • An Fc fusion protein was generated by engineering a Bgl ⁇ site in frame with the BgKI site in mutein Fc.
  • L081-Fc protein was produced by transient transfection, purified, and used to generate antibodies in rats.
  • the L081-Fc protein failed to find a hit in Biacore screening.
  • the positive control of IL-12 receptor-Fc also failed to bind IL-12 indicating a possible inactivation of receptor function in this family by Fc tags.
  • C-terminal tags were exchanged on L081 by using a Sa l to BgKI fragment from L081-Fc ligated into existing expression vectors engineered to express tags downstream of an in-frame BgKI site.
  • L081 was further modified by PCR to contain an Spel site in the proper reading frame just upstream ofthe predicted mature N-terminus.
  • the Spel to Notl fragment of this version was ligated into an expression vector comprising Igkl, sFlag, and pHis elements just upstream of the Spel site.
  • the resulting construct was named IgKL-sFlag-pHis-L081. See SEQ ID ⁇ Os: 135, 136, and 137.
  • IgKL-sFlag-pHis-L081 was co-transfected with the ⁇ 35 subunit of IL-12 ( ⁇ 35/L081).
  • L081 expression levels were low and the resulting product was about 50% pure.
  • the biological activities identified using this protein preparation of L081 include: inhibition of CD3 induced EF ⁇ -gamma production PHA blasts, inhibition of CD3, but not IL-15 induced proliferation of PHA blasts, inhibition of IL-12 and IL-15 induced IF ⁇ -gamma production by ⁇ K cells.
  • quantitative PCR of L081 mR ⁇ A indicated that L081 expression is up- regulated by CD40L stimulation of Dendritic cells. Expression is also up-regulated by LPS stimulation in monocytes, but down regulated by IF ⁇ -gamma stimulation.
  • p35/L081 expressed in CHO cell supernatants were used for FACS analysis to find a source for a membrane bound receptor.
  • Binding was detected on MP-1 cells and to a lesser degree on CB23 cells. Negative cell lines included U937, Jurkat, THP-1, NK, and HSB-2. Analysis by FACS and western blot of tagged versions of L081 show that both N-terminal and C-terminal tagged versions of L081 are inactivated by the tags. FACS binding was reduced by about 20 fold. A fully functional binding molecule was isolated by capturing CHO expressed p35/L081 with alpha-L081 antibody bound to a Protein A-Sepharose and released from the beads complexed with the antibody. Further screening of an MP-1 library can be accomplished with this functional p35/L081 to isolate a membrane bound receptor for L081.
  • the present invention comprises isolated nucleic acid molecules comprising a polynucleotide chosen from the group consisting of SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • a prefe ⁇ ed embodiment ofthe present invention comprises isolated nucleic acid molecules comprising a polynucleotide chosen from the group consisting of SEQ ID NOs.T-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 and associated with ileitis.
  • the polynucleotide can contain all or part ofthe nucleotide sequence ofthe 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 ofthe nucleic acid sequence.
  • a "polypeptide" refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • a polynucleotide ofthe present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NOs:l-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159, or the complement thereof, or the cDNA.
  • 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.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).
  • blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems with compatibility.
  • a polynucleotide which hybridizes only to polyA+ sequences such as any
  • a polynucleotide ofthe present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • 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.
  • 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.
  • polynucleotide embraces chemically, enzymatically, or metabolically modified forms.
  • Another prefe ⁇ ed embodiment ofthe present invention is an isolated nucleic acid molecule encoding a polypeptide ofthe present invention.
  • a prefe ⁇ ed embodiment ofthe present invention is a polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NOs:l-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • the polypeptide ofthe 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
  • a prefe ⁇ ed embodiment ofthe 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:l-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 and the translations of SEQ ID NOs:l-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 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 hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods ofthe invention.
  • polypeptides identified from the translations of SEQ ID NOs:l-76, SEQ ID NO: 134, and SEQ ID NOs: 147- 159 may be used to generate antibodies which bind specifically to the secreted proteins encoded by the cDNA clones identified.
  • DNA sequences generated by sequencing reactions can contain sequencing e ⁇ ors.
  • the e ⁇ ors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
  • the e ⁇ oneously inserted or deleted nucleotides cause frame shifts in the reading frames ofthe predicted amino acid sequence.
  • 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 co ⁇ esponding to SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 and translations of SEQ ID NOs:l-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • the co ⁇ esponding 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 co ⁇ esponding gene from appropriate sources of genomic material.
  • 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.
  • polypeptides ofthe 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.
  • polypeptides may be in the form ofthe 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.
  • polypeptides ofthe 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 ofthe invention also can be purified from natural or recombinant sources using antibodies ofthe invention raised against the secreted protein in methods which are well known in the art.
  • the deduced amino acid sequence ofthe 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.
  • Signal P Neelsen et al, Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • McGeoch and von Heinje are inco ⁇ orated.
  • the present invention provides secreted polypeptides having a sequence co ⁇ esponding to the translations of SEQ. ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 which have an N-terminus beginning within 5 residues (i.e., plus or minus 5 residues) ofthe predicted cleavage point.
  • SEQ. ID NOs: 1-76 amino acid sequence sequence
  • SEQ ID NO: 134 amino acid sequence
  • SEQ ID NOs:147-159 which have an N-terminus beginning within 5 residues (i.e., plus or minus 5 residues) ofthe predicted cleavage point.
  • cleavage ofthe signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species.
  • the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence.
  • the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
  • the predicted signal sequence will be capable of directing the secreted protein to the ER.
  • 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.
  • the above polypeptides should exhibit at least one biological activity ofthe protein.
  • polypeptides ofthe 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • 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 AppMaih., 2:482-489 (1981)).
  • the parameters are set such that the percentage of identity is calculated over the full length ofthe reference polynucleotide and that gaps in identity of up to 5% ofthe total number of nucleotides in the reference polynucleotide are allowed.
  • a prefe ⁇ ed method for determining the best overall match between a query sequence (a sequence ofthe present invention) and a subject sequence, also refe ⁇ ed 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)).
  • sequence includes nucleotide and amino acid sequences.
  • 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.
  • a polynucleotide having a nucleotide sequence of at least 95% "identity" to a sequence contained in SEQ ID Nos: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 means that the polynucleotide is identical to a sequence contained in SEQ ID Nos: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 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).
  • a polynucleotide having a nucleotide sequence at least 95% identical to SEQ ID Nos: 1-76, SEQ ID NO: 134, and SEQ ED NOs:147-159 up to 5% ofthe nucleotides in the sequence contained in SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.
  • 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 ofthe 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 ofthe total length ofthe reference polypeptide.
  • the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids ofthe total length ofthe reference polypeptide.
  • up to 5% ofthe 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% ofthe total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations ofthe reference sequence may occur at the amino or carboxy terminal positions ofthe 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.
  • polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities ofthe encoded polypeptide are prefe ⁇ ed.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also prefe ⁇ ed.
  • Polynucleotide variants can be produced for a variety of reasons.
  • 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 prefe ⁇ ed 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)).
  • allelic variants can vary at either the polynucleotide and/or polypeptide level.
  • non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. See, e.g.., Curr. Prot. Mol. Bio., Chapter 8.
  • variants may be generated to improve or alter the characteristics ofthe polypeptides ofthe present invention.
  • 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.
  • 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.,l0:307-377 (1993)).
  • 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)).
  • ample evidence demonstrates that variants often retain a biological activity similar to that ofthe naturally occurring protein.
  • 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 ofthe molecule. Multiple mutations were examined at every possible amino acid position.
  • 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 ofthe residues ofthe 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.
  • the invention further includes polypeptide variants which show substantial biological activity.
  • variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • 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 su ⁇ risingly 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.
  • the most buried or interior (within the tertiary structure ofthe protein) amino acid residues require nonpolar side chains, whereas few features of surface or exterior side chains are generally conserved.
  • 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 ofthe acidic residues Asp and Glu, replacement ofthe amide residues Asn and Gin, replacement ofthe basic residues Lys, Arg, and His, replacement ofthe aromatic residues Phe, Tyr, and T ⁇ , and replacement ofthe small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants ofthe present invention include: (i) substitutions with one or more ofthe 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 ofthe mature polypeptide with another compound, such as a compound to increase the stability and/or solubility ofthe polypeptide (e.g., polyethylene glycol); (iv) fusion ofthe polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification.
  • substitutions with one or more ofthe non-conserved amino acid residues where the substituted amino acid residues may or may not be one encoded by the genetic code
  • substitution with one or more of amino acid residues having a substituent group such as a compound to increase the stability and/or solubility ofthe polypeptide (e.g., poly
  • a "polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence contained in that shown in SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159. 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 prefe ⁇ ed.
  • polynucleotide fragments ofthe 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, 401-760, to the end of SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • “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.
  • these fragments encode a polypeptide which has biological activity.
  • polypeptide fragment refers to a short amino acid sequence contained in the translations of SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159. 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 ofthe invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end ofthe coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, or 60 amino acids in length.
  • Prefe ⁇ ed polypeptide fragments include the secreted protein as well as the mature form. Further prefe ⁇ ed 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 ofthe secreted protein or mature form.
  • any combination ofthe above amino and carboxy terminus deletions are prefe ⁇ ed.
  • polynucleotide fragments encoding these polypeptide fragments are also prefe ⁇ ed.
  • 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 ofthe translations of SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotide fragments encoding these domains are also contemplated. Other prefe ⁇ ed fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide ofthe present invention. The biological activity ofthe fragments may include an improved desired activity, or a decreased undesirable activity.
  • 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).
  • 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)).
  • 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 prefe ⁇ ed 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).
  • the immunogenic epitope may be prescribed without a carrier, if the sequence is of sufficient length (at least about 25 amino acids).
  • 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.)
  • antibody 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 prefe ⁇ ed, as well as the products of a Fab or other immunoglobulin expression library.
  • antibodies ofthe present invention include chimeric, single chain, and 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 immunogenicity when the antibodies are administered to humans.
  • 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.
  • 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.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 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.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147- 159 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 inco ⁇ orate 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. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, which are inco ⁇ orated by reference herein.
  • Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule. The spleen cells may be fused with myeloma cells to produce hybridomas, by conventional procedures.
  • a method for producing a hybridoma cell line comprises immunizing such a fransgemc 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- 76, SEQ ID NO: 134, and SEQ ID NOs:147-159; 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- 76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • Disorders, such as IBD which may be caused or exacerbated (directly or indirectly) by the interaction of such polypeptides ofthe present invention with cell surface receptors thus may be treated.
  • inflammation associated with IBD is the result of locally produced cytokines and chemokines that bind to cell surface receptors on various cells ofthe immune system, thus triggering changes in immune cell physiology that lead to disease.
  • An antibody that binds a polypeptide ofthe 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: 1-76, SEQ ED NO: 134, and SEQ ID NOs:147-159.
  • a prefe ⁇ ed embodiment ofthe present invention is a therapeutic method comprismg administering to a mammalian subject a therapeutically effective amount of an antibody induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1- 76, SEQ ID NO: 134, and SEQ ID NOs:147-159, to prevent, treat, ameliorate, or modulate a disease or condition, such as IBD.
  • conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ED NOs:147-159.
  • detectable or therapeutic agent attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ED NOs:147-159.
  • agents are well known, and include but are not limited to diagnostic radionuchdes, therapeutic radionuchdes, 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.
  • any polypeptide ofthe present invention can be used to generate fusion proteins.
  • the polypeptide ofthe present invention when fused to a second protein, can be used as an antigenic tag.
  • Antibodies raised against the polypeptide ofthe present invention can be used to indirectly detect the second protein by binding to the polypeptide.
  • secreted proteins target cellular locations based on trafficking signals, the polypeptides ofthe present invention can be used as targeting molecules once fused to other proteins.
  • domains that can be fused to polypeptides ofthe 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.
  • fusion proteins may also be engineered to improve characteristics ofthe polypeptide ofthe present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus ofthe 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 ofthe polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
  • polypeptides ofthe present invention, including fragments and, specifically, epitopes can be combined with parts ofthe constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • Fusion proteins facilitate purification and show an increased half-life in vivo.
  • One reported example describes chimeric proteins consisting ofthe first two domains ofthe human CD4- ⁇ olypeptide 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)).
  • EP A 0 464 533 (Canadian counte ⁇ art 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • 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).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the pu ⁇ ose 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)).
  • the polypeptides ofthe present invention can be fused to marker sequences, such as a peptide which facilitates purification ofthe fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in apQE 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 ofthe fusion protein (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)).
  • HA hemagglutinin protein
  • Other fusion proteins may use the ability ofthe polypeptides of die 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.
  • any of these above fusions can be engineered using the polynucleotides or the polypeptides ofthe present invention. See, e.g., Curr. Prot. Mol. Bio., Chapter 9.6. Fusion proteins can be used to prevent, treat, ameliorate, or modify IBD.
  • the present invention also relates to vectors containing the polynucleotide ofthe present invention, host cells, and the production of polypeptides by recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • 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, frp, 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 ofthe 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 ofthe polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracychne, kanamycin or ampicillin resistance genes for culturing inE. 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.
  • vectors prefe ⁇ ed for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNH16A, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
  • prefe ⁇ ed eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from
  • the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, elecfroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides ofthe 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.
  • HPLC high performance liquid chromatography
  • Polypeptides ofthe present invention 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.
  • a prokaryotic or eukaryotic host including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • the polypeptides ofthe present invention may be glycosylated or may be non-glycosylated.
  • polypeptides ofthe invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N- terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.
  • Polypeptides ofthe present invention 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.
  • a polynucleotide ofthe invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as ileitis
  • the expression ofthe 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.
  • drugs or antibodies that bind to and inactivate the polypeptide product can be used.
  • a polynucleotide ofthe invention is down-regulated and exacerbates a pathological condition, such as ileitis
  • the expression ofthe polynucleotide can be increased or the level ofthe 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 ofthe invention to the mammalian subject.
  • TOGA detected decreased expression ofthe polynucleotide with SEQ ID NO: 23 (DST IMX3_65).
  • a polynucleotide ofthe 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 or a polynucleotide which is at least 98%> identical to a nucleic acid sequence shown in SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs.T 47-159.
  • the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%, or 95% identical to a polynucleotide comprising SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • the administration of a polynucleotide or recombinant expression vector ofthe invention to a mammalian subject can be used to express a polynucleotide in said subject for the freatment of, for example, ileitis. Expression of a polynucleotide in target cells, including but not limited to colon cells, would effect greater production ofthe encoded polypeptide.
  • the regulation of other genes may be secondarily up- or down-regulated.
  • High expression ofthe polynucleotide would be advantageous since decreased expression of some polynucleotides (e.g. SEQ ID NO: 23; DST IMX3_65), as detected by TOGA, was associated with the development of EBD.
  • a naked polynucleotide can be administered to target cells.
  • Polynucleotides and recombinant expression vectors ofthe 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, adso ⁇ tion, or penetration ofthe 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
  • 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 ofthe IBD, the age, weight, sex, medical condition ofthe 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 ofthe 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 ofthe desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement ofthe 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.
  • U.S. Patent No. 5,672,344 describes an in vivo viral- mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector.
  • 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.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
  • nucleic acids and/or vectors ofthe invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors ofthe invention or other agents.
  • 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 ofthe 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, CaPO 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).
  • a polynucleotide ofthe invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as IBD
  • the expression ofthe 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.
  • drugs or antibodies that bind to and inactivate the polypeptide product can be used.
  • TOGA detected increased expression ofthe polynucleotide with SEQ ID NO: 7 (DST IMX3_16).
  • 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 translation. 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 ofthe 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 internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • triple helix pairing is useful because it causes inhibition ofthe ability ofthe 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.Y., 1994).
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes .
  • 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.
  • 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 ofthe invention. These modifications can be internal or at one or both ends ofthe antisense molecule.
  • 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.
  • 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).
  • ribozyme action involves sequence-specific hybridization ofthe 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 ofthe 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
  • 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.
  • RNA sequences of between 15 and 20 ribonucleo tides co ⁇ esponding to the region ofthe 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:l-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 and their complements provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity ofthe hybridization sequence for the target.
  • the hybridizing and cleavage regions ofthe ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region ofthe 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 ofthe 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 terminator signal, for controlling transcription of ribozymes in the cells.
  • 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.
  • Pathological conditions or susceptibility to pathological conditions can be diagnosed using methods ofthe invention.
  • Testing for expression of a polynucleotide of the invention or for the presence ofthe polynucleotide product can co ⁇ elate with the severity of a condition such as IBD and can also indicate appropriate treatment for the condition.
  • the presence or absence of a mutation in a polynucleotide ofthe invention can be determined and a pathological condition or a susceptibility to a pathological condition (for example, IBD) is diagnosed based on the presence or absence ofthe mutation.
  • an alteration in expression of a polypeptide encoded by a polynucleotide ofthe invention can be detected, where the presence of an alteration in expression ofthe 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.
  • a first biological sample from a patient suspected of having a pathological condition 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.
  • the average concentrations and distribution of a polynucleotide or polypeptide ofthe 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 ofthe invention is determined in the first and second sample.
  • the amounts ofthe polypeptide in the first and second samples are compared.
  • a patient is diagnosed as having a pathological condition if the amount ofthe polypeptide in the first sample is greater than or less than the amount ofthe polypeptide in the second sample.
  • 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 ofthe polypeptide ofthe 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.
  • a diagnostic system preferably in kit form, for assaying for the presence ofthe polypeptide ofthe 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.
  • a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide ofthe present invention, a gene co ⁇ esponding to a polynucleotide ofthe 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.
  • PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence ofthe gene to be detected.
  • the nucleotide sequence is a portion ofthe nucleotide sequence of a polynucleotide ofthe present invention.
  • Particularly prefe ⁇ ed 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.
  • PCR primer pairs useful for detecting the genes co ⁇ esponding to the polynucleotides ofthe present invention and expression of these genes are described in the Examples, including the co ⁇ esponding Tables. Nucleotide primers from the co ⁇ esponding region ofthe polypeptides ofthe present invention described herein are readily prepared and used as PCR primers for detection ofthe presence or expression ofthe co ⁇ esponding gene in any of a variety of tissues.
  • a diagnostic system for diagnosing a disease or condition, such as IBD.
  • the diagnostic system includes, in an amount sufficient to perform at least one assay, a subj ect polypeptide of the present invention, a subj ect antibody or monoclonal antibody, and/or a subject nucleic acid molecule probe ofthe present invention, as a separately packaged reagent.
  • a diagnostic system preferably in kit form, is contemplated for assaying for the presence ofthe polypeptide ofthe 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 ofthe 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 ofthe present invention and/or a subject antibody as a separately packaged immunochemical reagent.
  • a diagnostic system ofthe present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule ofthe present invention.
  • Any label or indicating means can be linked to or inco ⁇ orated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition ofthe 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 protems 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 fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethyhhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
  • fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethyhhodamine iso
  • the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like.
  • HRP horseradish peroxidase
  • glucose oxidase or the like.
  • additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed.
  • 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 124 1, 125 1, 128 I, ] I and 51 Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly prefe ⁇ ed is 125 I.
  • Another group of useful labeling means are those elements such as ⁇ C, 18 F, 15 O and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body.
  • a beta emitter such 1 1 mdium or ⁇ H.
  • antibody molecules produced by a hybridoma can be labeled by metabolic inco ⁇ oration 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., Aura eas, 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.
  • the specific binding agent binds the reagent species when that species is present as part of a complex.
  • the specific binding agent is labeled.
  • the agent is typically used as an amplifying means or reagent.
  • 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 ofthe present invention can be used in an "ELISA" format to detect the quantity ofthe polypeptide ofthe present invention in a sample.
  • ELISA ELISA
  • 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 inco ⁇ orated herein by reference.
  • a polypeptide ofthe present invention an antibody or a monoclonal antibody ofthe present invention can be affixed to a sohd 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 adso ⁇ tion 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 adso ⁇ tion 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 ( ⁇ m) 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.
  • SEPHADEX Pharmacia Fine Chemicals
  • agarose agarose
  • polystyrene beads of about 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, IL)
  • polyvinyl chloride polystyrene
  • 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.
  • 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.
  • the polynucleotides ofthe 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 polymo ⁇ hisms) are presently available. Each polynucleotide ofthe present invention can be used as a chromosome marker. Currently no diagnostic markers exist to detect and diagnose IBD. Each polynucleotide ofthe present invention can be used as a chromosome marker.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159. 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 co ⁇ esponding to the SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ED NOs: 147-159 will yield an amplified fragment.
  • 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 ofthe 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.
  • FISH fluorescence in situ hybridization
  • 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). Prefe ⁇ ed polynucleotides co ⁇ espond to the noncoding regions ofthe cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross-hybridization during chromosomal mapping.
  • 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.
  • polynucleotide andthe co ⁇ esponding gene between affected and unaffected individuals can be examined.
  • the polynucleotides of SEQ ID NOs: 1-76, SEQ ID NO: 134 and SEQ ID NOs: 147-159 can be used for this analysis of individuals.
  • a genetic etiology is suggested by the prevalence of EBD in certain populations among 1 st degree relatives and by familial clustering ofthe disease.
  • 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, prefe ⁇ ed polynucleotides are usually 20 to 40 bases in length and complementary to either the region ofthe gene involved in transcription (see, Lee et al., Nuc.
  • Polynucleotides ofthe 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 co ⁇ ect 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 ofthe present invention.
  • a polypeptide ofthe present invention can be used to assay protein levels in a biological sample using antibody-based techniques.
  • 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).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; and radioisotopes, such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc); fluorescent labels, such as fluorescein and rhodamine; and biotin.
  • enzyme labels such as glucose oxidase
  • radioisotopes such as iodine ( 125 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc)
  • fluorescent labels such as fluorescein and rhodamine
  • 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 inco ⁇ orated 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., 13l I, 112 In, 99m Tc), a radio-opaque substance, or a material detectable by NMR, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the mammal.
  • a radioisotope e.g., 13l I, 112 In, 99m Tc
  • a radio-opaque substance e.g., a radio-opaque substance, or a material detectable by NMR
  • 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., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Eds., Masson Publishing Inc. (1982)).
  • the invention provides a diagnostic method of a disorder, such as IBD, which involves (a) assaying the expression of a polypeptide ofthe 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.
  • IBD 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.
  • polypeptides ofthe present invention can be used to treat disease, for example IBD.
  • patients can be administered a polypeptide ofthe present invention in an effort to replace absent or decreased levels ofthe 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).
  • a polypeptide ofthe present invention to replace decreased levels of a polypeptide that makes a person susceptible to IBD or that causes some ofthe symptoms of IBD
  • antibodies directed to a polypeptide ofthe present invention can also be used to treat disease.
  • administration of an antibody directed to a polypeptide of the present invention can bind and reduce ove ⁇ roduction ofthe polypeptide.
  • administration 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 ove ⁇ roduced polypeptide can be used to modulate the T cell response. Treatment of patients with IBD with a polypeptide or polynucleotide ofthe 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 ofthe host cell. Moreover, the polypeptides ofthe present invention can be used to test the following biological activities. Biological Activities
  • polynucleotides and polypeptides ofthe 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.
  • a polypeptide or polynucleotide ofthe present invention may be useful in treating deficiencies or disorders ofthe 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.
  • a polynucleotide or polypeptide ofthe 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 ofthe 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.
  • 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.
  • a polypeptide or polynucleotide ofthe present invention could also be used to modulate hemostatic (bleeding cessation) or thrombolytic activity (clot formation).
  • a polynucleotide or polypeptide ofthe 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.
  • a polynucleotide or polypeptide ofthe 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.
  • 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 ofthe host tissue. Therefore, the admimsfration of a polypeptide or polynucleotide ofthe 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.
  • autoimmune disorders examples 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, Pu ⁇ ura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Ba ⁇ e Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
  • IBD has several aspects that suggest an autoimmune 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 ftirther 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 ofthe present invention. Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • a polynucleotide or polypeptide ofthe 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 ofthe transplanted tissue through an immune response.
  • an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
  • the administration of a polypeptide or polynucleotide ofthe 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.
  • a polypeptide or polynucleotide ofthe present invention may also be used to modulate inflammation.
  • the polypeptide or polynucleotide may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
  • These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, IBD, Crohn's disease, or resulting from over production of cytokines (e.g., TNF orIL-l).
  • infection e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury e.g., endotoxin lethality, arthritis, complement-mediated hypera
  • a polynucleotide or polypeptide ofthe 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.
  • organs e.g., pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • nervous hematopoietic
  • skeletal bone, cartilage, tendon, ligament
  • skeletal bone, cartilage, tendon, ligament
  • a polynucleotide or polypeptide ofthe present invention could also be used prophylactically in an effort to avoid damage.
  • Specific diseases that could be treated include of tendinitis, ca ⁇ al tunnel syndrome, and other tendon or ligament defects.
  • tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
  • nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide ofthe 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).
  • diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases e.g., Alzheimer's disease,
  • Parkinson's disease Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome
  • Shy-Drager syndrome could all be treated using the polynucleotide or polypeptide ofthe present invention.
  • a polynucleotide or polypeptide ofthe 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 hype ⁇ roliferation.
  • the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
  • a polynucleotide or polypeptide ofthe present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hype ⁇ roliferative 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 ofthe present invention can also atfract fibroblasts, which can be used to treat wounds.
  • a polynucleotide or polypeptide ofthe present invention may inhibit chemotactic activity. Such molecules could also be used to treat a variety of disorders. Thus, a polynucleotide or polypeptide ofthe 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 ofthe 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.
  • a polypeptide ofthe present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds.
  • the binding ofthe polypeptide and the molecule may activate (i.e., agonist), increase, inhibit (i.e., an antagonist), or decrease activity ofthe polypeptide or the molecule bound.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to the natural ligand ofthe polypeptide, e.g., a fragment ofthe 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)).
  • the molecule can be closely related to the natural receptor to which the polypeptide binds or, at least, related to a fragment ofthe receptor capable of being bound by the polypeptide (e.g., an active site).
  • the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane.
  • Prefe ⁇ ed 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
  • 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.
  • the assays can discover agents which may inhibit or enhance the production ofthe polypeptide from suitably manipulated cells or tissues.
  • IBD diagnosis depends on a number of relatively invasive and expensive clinical tests.
  • Assays for the presence of markers, in easily obtained specimens may provide an important diagnostic tool.
  • the invention includes a method of identifying compounds which bind to a polypeptide ofthe invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide ofthe invention; and (b) determining if binding has occuned.
  • the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide ofthe invention, (b) assaying a biological activity, and (c) determining if a biological activity ofthe polypeptide has been altered.
  • a polypeptide or polynucleotide ofthe 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 EBD 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.
  • nucleic acid molecule comprising a nucleotide sequence which is at least 80%, preferably at least
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide ofthe clone sequence and ending with the nucleotide at about the position of the 3' nucleotide ofthe clone sequence.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ED NOs:147-159 in the range of positions beginning with the nucleotide at about the position ofthe 5' nucleotide ofthe start codon and ending with the nucleotide at about the position ofthe 3' nucleotide ofthe clone sequence as defined for SEQ ID NOs: 1-76, SEQJD NO: 134, and SEQ ED NOs:147-159.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence ofSEQ ED NOs: 1-76, SEQ D NO: 134, and SEQ ID NOs:147-159 in the range of positions beginning with the nucleotide at about the position ofthe 5' nucleotide ofthe first amino acid ofthe signal peptide and ending with the nucleotide at about the position ofthe 3' nucleotide ofthe clone sequence as defined for SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159. Further prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • a further prefe ⁇ ed embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159 beginning with the nucleotide at about the position ofthe 5' nucleotide ofthe first amino acid ofthe signal peptide and ending with the nucleotide at about the position ofthe 3' nucleotide ofthe clone sequence as defined for SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • a further prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • 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 prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ED NOs:147-159, 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.
  • 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.
  • 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 prefe ⁇ ed 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 ED NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • Also prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147- 159.
  • the nucleic acid molecules can comprise DNA molecules or RNA molecules.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159. Also preferred 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 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159, in the range of positions beginning with the residue at about the position ofthe first amino acid ofthe secreted portion and ending with the residue at about the last amino acid ofthe open reading frame.
  • 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 ED NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159. Further prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ED NOs: 147- 159.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159, 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.
  • 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • Also prefe ⁇ ed 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 prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • Also prefe ⁇ ed 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 prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159.
  • the step of detecting said polypeptide molecules includes using an antibody.
  • nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-76, SEQ ED NO: 134, and SEQ ID NOs:147- 159.
  • prefe ⁇ ed is an isolated nucleic acid molecule, wherem said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host. Also prefe ⁇ ed 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: 1-76, SEQ ID NO: 134, and SEQ ID NOs: 147-159. Further prefe ⁇ ed is a method of making a recombinant vector comprising inserting any ofthe above isolated nucleic acid molecule into a vector.
  • prefe ⁇ ed is the recombinant vector produced by this method. Also prefe ⁇ ed 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 prefe ⁇ ed 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 prefe ⁇ ed is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-76, SEQ ID NO: 134, and SEQ ID NOs:147-159.
  • the isolated polypeptide produced by this method is also prefe ⁇ ed.
  • Also prefe ⁇ ed 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 ofthe claimed invention effective to increase the level of said protein activity in said individual.
  • EST Expressed Sequence Tag
  • RT-PCR Reverse Transcriptase Polymerase Chain Reaction
  • EST Expressed Sequence Tag
  • RT-PCR Reverse Transcriptase Polymerase Chain Reaction

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Abstract

L'invention concerne des polynucléotides, des polypeptides, des kits et des méthodes en relation avec des caractéristiques géniques de l'iléite.
PCT/US2001/032091 2000-10-11 2001-10-11 Expression genique module dans l'ileite WO2002031114A2 (fr)

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EP2695947A1 (fr) * 2011-04-01 2014-02-12 Chugai Seiyaku Kabushiki Kaisha Procédé de production de polypeptide recombiné
WO2023250459A3 (fr) * 2022-06-24 2024-03-21 The University Of Chicago Procédés et compositions pour le traitement de conditions inflammatoires et auto-immunes

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WO2000077166A2 (fr) * 1999-06-10 2000-12-21 Digital Gene Technologies, Inc. Expression genique modulee dans des inflammations gastro-intestinales
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US6365352B1 (en) * 1997-08-22 2002-04-02 Yale University Process to study changes in gene expression in granulocytic cells

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US6228585B1 (en) * 1998-09-04 2001-05-08 Washington University Gene markers for chronic mucosal injury
WO2000077166A2 (fr) * 1999-06-10 2000-12-21 Digital Gene Technologies, Inc. Expression genique modulee dans des inflammations gastro-intestinales

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2695947A1 (fr) * 2011-04-01 2014-02-12 Chugai Seiyaku Kabushiki Kaisha Procédé de production de polypeptide recombiné
EP2695947A4 (fr) * 2011-04-01 2014-10-29 Chugai Pharmaceutical Co Ltd Procédé de production de polypeptide recombiné
US11028149B2 (en) 2011-04-01 2021-06-08 Chugai Seiyaku Kabushiki Kaisha Recombinant polypeptide production method
US11905325B2 (en) 2011-04-01 2024-02-20 Chugai Seiyaku Kabushiki Kaisha Recombinant polypeptide production method
WO2023250459A3 (fr) * 2022-06-24 2024-03-21 The University Of Chicago Procédés et compositions pour le traitement de conditions inflammatoires et auto-immunes

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