EP1181385A2 - Modulation d'expression genique dans une inflammation gastro-intestinale - Google Patents

Modulation d'expression genique dans une inflammation gastro-intestinale

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Publication number
EP1181385A2
EP1181385A2 EP00941187A EP00941187A EP1181385A2 EP 1181385 A2 EP1181385 A2 EP 1181385A2 EP 00941187 A EP00941187 A EP 00941187A EP 00941187 A EP00941187 A EP 00941187A EP 1181385 A2 EP1181385 A2 EP 1181385A2
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EP
European Patent Office
Prior art keywords
seq
polypeptide
polynucleotide
sequence
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00941187A
Other languages
German (de)
English (en)
Other versions
EP1181385A4 (fr
Inventor
Adel Youakim
Robert Finley Dubose
John E. Sims
Thomas M. Pribyl
Brian S. Hilbush
Karl W. Hasel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NEUROME Inc
Original Assignee
NEUROME Inc
Digital Gene Technologies Inc
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Publication of EP1181385A2 publication Critical patent/EP1181385A2/fr
Publication of EP1181385A4 publication Critical patent/EP1181385A4/fr
Withdrawn legal-status Critical Current

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Definitions

  • Oxidative Damage to the intestinal epithelial barrier is a hallmark of inflammatory bowel diseases (IBD).
  • IBD inflammatory bowel diseases
  • Studying the factors that influence the integrity of the intestinal epithelial barrier in vivo is a difficult task for a number of reasons, including the complexity of the tissue itself (there are numerous cell types in the gut including epithelial, stromal, endocrine, neuronal and hematopoietic) and the technical problems associated with tissue manipulation in animals or in isolated organs.
  • IBD inflammatory bowel diseases
  • T84 intestinal epithelial barrier system (Dharmsathaphorn, K., J.A. McRoberts, K.G. Mandel, L.D. Tisdale, and H. Masui. 1984. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am J. Physiol 246- G204-0208; Madara, J.L., and K. Dharmsathaphorn. 1985. Occluding junction structure-function relationships in a cultured epithelial monolayer. J. Cell Biol. 101. 2124-2133). Although T84 cells were derived from a human colonic adenocarcinoma, they have retained many of the properties associated with normal colonic crypt cells.
  • T84 cells form polarized monolayers that exhibit high electrical resistance as well as vectorial fluid and chloride secretion reminiscent of colonic crypts in vivo. These properties are directly dependent on a complex of proteins referred to as tight junctions and T84 cells express many of the known members of this complex. These cells also respond to proinflammatory cytokines, such as interferon-gamma (IFN- ⁇ ), by decreasing barrier function (Madara, J.L., and J. Stafford. 1989. Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers. J. Clin. Invest. 83: 724-727; Adams, R.B., S.M. Planchon, and J.K. Roche. 1993.
  • IFN- ⁇ interferon-gamma
  • IFN-gamma modulation of epithelial barrier function Time course, reversibility, and site of cytokine binding. /. Immunol. 150: 2356-2363.
  • MHC Class II molecules and antigen presenting activity Hershberg, R.M., D.H. Cho, A. Youakim, M.B. Bradley, J.S. Lee, P.E. Framson and G.T. Nepom. 1998. Highly polarized HLA class II antigen processing and presentation by human intestinal epithelial cells. J. Clin. Invest. 102: 792-803).
  • the T84 cell model system has been used in studies to examine how the epithelial barrier is regulated by various agents such as interferon-gamma and cells of the immune system (i.e., neutrophils). Such studies have examined the mechanism of barrier breakdown and recovery in response to such agents and to identify proteins and genes that may prevent barrier breakdown or stimulate barrier recovery.
  • Molecules have been identified that correspond to genes that are regulated by the treatment of T84 cells with proinflammatory cytokines, such as interferon-gamma. 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 encoding them, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying binding partners of the polypeptides.
  • the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOJ, 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 NOJ 1, SEQ ID NO.12, SEQ ID NO:
  • SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NOJ 6, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO.21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO.31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43.
  • Another embodiment comprises an isolated nucleic acid molecule at least 95% identical to the isolated nucleic acid molecule of SEQ ID NO: 1-43.
  • a further embodiment comprises an isolated nucleic acid molecule at least ten bases in length that is hybridizable to the isolated nucleic acid molecule of SEQ ID NO: 1-43 under stringent conditions.
  • the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ 3D NOJ, 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,
  • the invention provides an isolated nucleic acid molecule encoding the amino acid sequence of the invention.
  • SEQ ID NO:10 SEQ ID NOJ 1, SEQ ID NOJ2, SEQ ID NOJ3, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO.21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43.
  • the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in gastrointestinal inflammation, chosen from the group consisting of the DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in T84 cells treated with interferon-gamma.
  • 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 of the present invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay; label means; instructions for use; and suitable packaging material.
  • the polynucleotide is chosen from the group consisting of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOJ, 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 NOJ 1, SEQ ID NO: 12, SEQ ID NOJ3, SEQ ID NOJ4, SEQ ID NOJ5, SEQ ID NOJ6, SEQ ID NOJ7, SEQ ID NOJ 8, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36
  • Another embodiment comprises a polynucleotide at least 95% identical to the isolated nucleic acid molecule of SEQ ID NO: 1-43.
  • a further embodiment comprises a polynucleotide at least ten bases in length that is hybridizable to the isolated nucleic acid molecule of SEQ ID NO: 1-43 under stringent conditions.
  • the polynucleotide is chosen from the group consisting of the DNA molecules identified in Table 1, having a 5' partial nucleotide sequence and length as described by their digital address, and having a characteristic regulation pattern in T84 cells treated with interferon-gamma.
  • Figure 1 is a graphical representation of the results of TOGA analysis (TOtal Gene expression Analysis) using a 5' PCR primer with parsing bases GGCC, showing PCR products produced from mRNA extracted from untreated T84 cells (Figure 1 A), T84 cells 4 hours after treatment with interferon-gamma (Figure IB), 24 hours after treatment with interferon-gamma (Figure 1C), and T84 cells 44 hours after treatment with interferon- gamma ( Figure ID), where the vertical index line indicates a PCR product of about 374 b.p.
  • T84 cells that is enriched in treated T84 cells, where the ordinate is in arbitrary units of fluorescence intensity and the abscissa is length of PCR product in nucleotides;
  • Figure 2 is a graphical representation of more detailed analysis of the 374 b.p.
  • Figure 1 shows the PCR product obtained using an extended 5' primer as described in the text
  • Figure 2B shows the PCR products obtained using the original PCR primers
  • Figure 2C the traces from Figure 2A and 2B are overlaid, demonstrating that the PCR product of the isolated and sequenced clone is the same length as the original PCR product, where the ordinate is in arbitrary units of fluorescence intensity and the abscissa is length of PCR product in nucleotides; and
  • Figure 3 is a graphical representation of the results of Northern Blot analysis of clone IMX 43, SEQ ID NO: 6, where an agarose gel containing poly A enriched mRNA from the four T84 experimental samples (control, 4 hour, 24 hour and 44 hour) as well as size standards was blotted after electrophoresis, the Northern blot probed with radiolabelled IMX 43, imaged using a phosphorimager and quantified.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
  • a "polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NOJ-43.
  • the polynucleotide can contain all or part of the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • a “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO: 1 -43, or the complement thereof, or the cDNA.
  • Stringent hybridization conditions refers to an overnight incubation at 42° C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 microgram/milliliter denatured, sheared salmon sperm DNA, followed by washing the filters in OJx SSC at about 65°C.
  • 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).
  • salt concentrations e.g. 5X SSC.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • 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).
  • polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • 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.
  • polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • 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.
  • a polypeptide having biological activity refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about ten-fold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.).
  • the translated amino acid sequence, beginning with the methionine is identified although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by the translation of these alternative open reading frames are specifically contemplated by the present invention.
  • SEQ ID NO: 1-43 and the translations of SEQ ID NO: 1-43 are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from the translations of SEQ ID NOJ -43 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 errors.
  • the errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
  • the erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than
  • the present invention also relates to the genes corresponding to SEQ ID NO: 1-43, and translations of SEQ ID NOJ -43.
  • the corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
  • species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.
  • the polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified by the one- step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods which are well known in the art.
  • Variant refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention. "Identity" per se has an art-recognized meaning and can be calculated using published techniques.
  • identity is well known to skilled artisans. (Carillo, H., and Lipton, D., SIAM J Applied Math 48:1073 (1988).) Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers,” Martin J. Bishop, ed., Academic Press, San Diego, (1994), and Carillo, H., and Lipton, D., SIAM J Applied Math 48:1073 (1988).
  • the parameters are set so that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence 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 in percent identity.
  • a polynucleotide having a nucleotide sequence of at least 95% "identity" to a sequence contained in SEQ ID NO: 1-43 means that the polynucleotide is identical to a sequence contained in SEQ ID NO: 1-43 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).
  • nucleotide having a nucleotide sequence at least 95% identical to SEQ ID NOJ-43 up to 5% of the nucleotides in the sequence contained in SEQ ID NOJ-43 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.
  • polynucleotides having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity will encode a polypeptide identical to an amino acid sequence contained in the translations of SEQ ID NOJ-43.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the above polypeptides should exhibit at least one biological activity of the protein.
  • polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of SEQ ID NOJ-43.
  • 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 of the encoded polypeptide are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. col ⁇ ).
  • 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.
  • 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.
  • variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N- terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem.
  • 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, J. U. et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Tip, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification.
  • substitutions with one or more of the non-conserved amino acid residues where the substituted amino acid residues may or may not be one encoded by the genetic code
  • substitution with one or more of amino acid residues having a substituent group or fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity.
  • a "polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence contained in that shown in SEQ ID NOJ- 43.
  • the short nucleotide fragments are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length.
  • a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in SEQ ID NOJ-43.
  • These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, and more nucleotides) are preferred.
  • polynucleotide fragments of the invention include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, to the end of SEQ ID NOJ-43.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • 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 NOJ-43. Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50 or 60, amino acids in length. In this context "about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.
  • polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha- helix forming regions, beta-sheet and beta-sheet- forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Polypeptide fragments of the translations of SEQ ID NOJ-43 falling within conserved domains are specifically contemplated by the present invention.
  • polynucleotide fragments encoding these domains are also contemplated.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • epitopes refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human.
  • a preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • an "immunogenic epitope” is defined as a part of a protein that elicits an antibody response. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81 :3998-4002 (1983).)
  • Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,211.)
  • antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids.
  • Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, for instance, Wilson et al., Cell 37:767- 778 (1984); Sutcliffe, J. G. et al., Science 219:660-666 (1983).)
  • immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).)
  • a preferred immunogenic epitope includes the secreted protein.
  • the immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
  • 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 As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
  • 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 an antibody comprises immunizing a non-human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOJ-43, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ ID NOJ-43 are generated in said animal.
  • Procedures have been developed for generating human antibodies in non-human animals.
  • the antibodies may be partially human, or preferably completely human.
  • Non-human animals (such as transgenic mice) into which genetic material encoding one or more human immunoglobulin chains has been introduced may be employed. Such transgenic mice may be genetically altered in a variety of ways.
  • the genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization.
  • Antibodies produced by immunizing transgenic animals with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOJ-43 are provided herein.
  • mice in which one or more endogenous immunoglobulin genes are inactivated by various means have been prepared.
  • Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes.
  • Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for production and use of such transgenic animals are described in U.S. Patents 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.
  • Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule.
  • the spleen cells may be fused with myeloma cells to produce hybridomas, by conventional procedures.
  • a method for producing a hybridoma cell line comprises immunizing such a transgenic animal with a immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOJ-43; 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 NOJ-43.
  • Such hybridoma cell lines, and monoclonal antibodies produced therefrom, are encompassed by the present invention.
  • Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques.
  • Antibodies may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOJ-43. Disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated.
  • a therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOJ-43.
  • 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 NOJ-43.
  • detectable or therapeutic agent e.g., an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOJ-43.
  • agents include but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drugs.
  • the conjugates find use in in vitro or in vivo procedures.
  • Fusion Proteins Any polypeptide of the present invention can be used to generate fusion proteins.
  • the polypeptide of the present invention when fused to a second protein, can be used as an antigenic tag.
  • Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
  • secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
  • domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
  • polypeptides of the present invention can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • fusion proteins facilitate purification and show an increased half-life in vivo.
  • chimeric proteins consisting of the first two domains of the human CD4- polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins.
  • Fusion proteins having disulf ⁇ de-linked dimeric structures can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP-A 0 232 262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • HA hemagglutinin protein
  • the present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, Bowes melanoma cells and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNHl ⁇ a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
  • a polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the 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 of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the 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 of the amino acid to which the N-terminal methionine is covalently linked.
  • the polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NOJ-43. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NOJ-43 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 of the polynucleotides can be achieved with panels of specific chromosome fragments.
  • Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • 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).
  • Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.
  • Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease.
  • Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).) Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
  • polynucleotide and the co ⁇ esponding gene between affected and unaffected individuals can be examined.
  • the polynucleotides of SEQ ID NOJ-43 can be used for this analysis of individual humans.
  • a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 3:173 (1979); Cooney et al., Science 241 :456 (1988); and Dervan et al., Science 251 :1360 (1991) ) or to the mRNA itself (antisense - Okano, j. Neurochem.
  • Polynucleotides of the present invention are also useful in gene therapy.
  • One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect.
  • the polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
  • Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.
  • the polynucleotides are also useful for identifying individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the polynucleotides of the present invention can be used as additional DNA markers for RFLP.
  • polynucleotides of the present invention can also be used as an alternative to
  • RFLP RFLP
  • DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc.
  • DNA sequences amplified from polymorphic loci such as DQa class II HLA gene
  • PCR PCR Technology
  • DQa class II HLA gene a polymorphic loci
  • polymorphic loci such as DQa class II HLA gene
  • reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin.
  • Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
  • the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
  • a polypeptide of the 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.
  • 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 include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( I, I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine ( I, 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.
  • proteins can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 131 1, 1 12 In, 99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal.
  • a radioisotope for example, 131 1, 1 12 In, 99m Tc
  • a radio-opaque substance for example, parenterally, subcutaneously, or intraperitoneally
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)
  • the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
  • polypeptides of the present invention can be used to treat disease.
  • patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B), to inhibit the activity of a polypeptide (e.g., an oncogene), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth).
  • a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B), to inhibit the activity of a polypeptide (
  • antibodies directed to a polypeptide of the 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 overproduction of the polypeptide.
  • administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
  • polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.
  • polynucleotides and polypeptides of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides could be used to treat the associated disease.
  • a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
  • Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
  • the etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious.
  • a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
  • a polynucleotide or polypeptide of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells.
  • a polypeptide or polynucleotide of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells.
  • immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g.
  • agammaglobulinemia agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge 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.
  • SIDs severe combined immunodeficiency
  • a polypeptide or polynucleotide of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation).
  • a polynucleotide or polypeptide of the present invention could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
  • a polynucleotide or polypeptide of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment of heart attacks (infarction), strokes, or scarring.
  • a polynucleotide or polypeptide of the present invention may also be useful in treating or detecting autoimmune disorders.
  • Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoinunune 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, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
  • allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polypeptide or polynucleotide of the present invention.
  • these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • a polynucleotide or polypeptide of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD).
  • Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
  • an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
  • the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
  • a polypeptide or polynucleotide of the 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, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)
  • SIRS systemic inflammatory response syndrome
  • a polypeptide or polynucleotide can be used to treat or detect hyperproliferative disorders, including neoplasms.
  • a polypeptide or polynucleotide of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions.
  • a polypeptide or polynucleotide of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
  • hyperproliferative disorders can be treated.
  • This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
  • hyperproliferative disorders that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
  • neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
  • hyperproliferative disorders can also be treated or detected by a polynucleotide or polypeptide of the present invention.
  • hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated.
  • the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • the polypeptide or polynucleotide of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention.
  • viruses include, but are not limited to the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbilli virus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccini
  • Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
  • bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellu
  • parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
  • These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombicuhasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
  • treatment using a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy).
  • the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
  • a polynucleotide or polypeptide of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues.
  • the regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
  • Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue.
  • organs e.g., pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • nervous hematopoietic
  • skeletal bone, cartilage, tendon, and ligament
  • skeletal bone, cartilage, tendon, and ligament
  • regeneration occurs without or decreased scarring.
  • Regeneration also may include angiogenesis.
  • a polynucleotide or polypeptide of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage.
  • 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 of the present invention to proliferate and differentiate nerve cells.
  • Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stroke).
  • diseases associated with peripheral nerve injuries e.g., resulting from chemotherapy or other medical therapies
  • peripheral neuropathy e.g., resulting from chemotherapy or other medical therapies
  • localized neuropathies e.g., central nervous system diseases
  • central nervous system diseases e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome
  • a polynucleotide or polypeptide of the present invention may have chemotaxis activity.
  • a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproHferation.
  • the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
  • a polynucleotide or polypeptide of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
  • a polynucleotide or polypeptide of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, a polynucleotide or polypeptide of the present invention could be used as an inhibitor of chemotaxis.
  • a polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds.
  • the binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic.
  • the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
  • the assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
  • 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.
  • 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 of the polypeptide from suitably manipulated cells or tissues.
  • the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occurred.
  • the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.
  • a polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
  • a polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).
  • a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
  • a polypeptide or polynucleotide of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
  • a polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
  • nucleic acid molecule comprising a nucleotide sequence which is at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NOJ-43.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOJ-43 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the clone sequence and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO: 1 -43 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the start codon and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NOJ-43.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NOJ-43 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NOJ-43.
  • 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 NOJ-43.
  • 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 NOJ-43.
  • a further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NOJ-43 beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for SEQ ID NOJ-43.
  • a further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NOJ-43.
  • nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
  • a further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NOJ-43, 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.
  • said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group.
  • said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group.
  • the nucleic acid molecules can comprise DNA molecules or RNA molecules.
  • a further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NOJ-43.
  • Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of nucleotide sequences of SEQ ID NOJ-43.
  • 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.
  • 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 NOJ-43.
  • 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 NOJ-43.
  • polypeptide wherein said sequence of contiguous amino acids is included in an amino acid sequence translated from SEQ ID NOJ-43, in the range of positions beginning with the residue at about the position of the first amino acid of the secreted portion and ending with the residue at about the last amino acid of the open reading frame.
  • an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in an amino acid sequence translated from SEQ ID NOJ-43.
  • 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 NOJ-43.
  • an isolated polypeptide comprising an amino acid sequence at least 95% identical to acids in an amino acid sequence translated from SEQ ID NOJ-43.
  • 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 NOJ-43, 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.
  • said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOJ-43.
  • step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
  • Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOJ-43.
  • the above method for identifying the species, tissue or cell type of a biological sample comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.
  • Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOJ-43.
  • 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 NOJ-43.
  • nucleic acid molecule wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
  • 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 NOJ-43.
  • a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
  • Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOJ- 43. The isolated polypeptide produced by this method is also preferred.
  • 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 of the claimed invention effective to increase the level of said protein activity in said individual.
  • the present invention also includes a diagnostic system, preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, such brain tissue, cell suspensions or tissue sections; or a body fluid sample, such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.
  • a diagnostic system preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, such brain tissue, cell suspensions or tissue sections; or a body fluid sample, such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.
  • a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide of the present invention, a gene corresponding to a polynucleotide of the present invention, or a mRNA in a cell that is diagnostic for the presence or expression of a polypeptide of the present invention in the cell.
  • the nucleic acid molecule probes can be of a variety of lengths from at least about 10, suitably about 10 to about 5000 nucleotides long, although they will typically be about 20 to 500 nucleotides in length. Hybridization methods are extremely well known in the art and will not be described further here.
  • PCR primer extension reactions such as the polym erase chain reaction (PCR).
  • PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence of the gene to be detected.
  • the nucleotide sequence is a portion of the nucleotide sequence of a polynucleotide of the 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 of the present invention and expression of these genes are described in the Examples, including the co ⁇ esponding Tables. Nucleotide primers from the co ⁇ esponding region of the polypeptides of the present invention described herein are readily prepared and used as PCR primers for detection of the presence or expression of the co ⁇ esponding gene in any of a variety of tissues.
  • the diagnostic system includes, in an amount sufficient to perform at least one assay, a subject polypeptide of the present invention, a subject antibody or monoclonal antibody, and/or a subject nucleic acid molecule probe of the present invention, as a separately packaged reagent.
  • a diagnostic system preferably in kit form, is contemplated for assaying for the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample.
  • Such diagnostic kit would be useful for monitoring the fate of a therapeutically administered polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention.
  • the system includes, in an amount sufficient for at least one assay, a polypeptide of the present invention and/or a subject antibody as a separately packaged immunochemical reagent.
  • a diagnostic system preferably in kit form, is contemplated for assaying for the presence of a nucleic acid in a body fluid or tissue sample that is characteristic of inflammatory bowel disease, using a polynucleotide of the present invention.
  • the system includes, in an amount sufficient for at least one assay, at least one polynucleotide of the present invention or fragments thereof as well as other reagents.
  • 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 of the 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.
  • a diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.
  • complex refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction.
  • exemplary complexes are immunoreaction products.
  • label means 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.
  • Any label or indicating means can be linked to or incorporated in a polynucleotide, an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents.
  • Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
  • Suitable label means comprise enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.
  • the labeling means can be a fluorescent labeling agent that chemically binds to polynucleotides, antibodies or antigens, without preventing interaction with the binding partner, to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
  • Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), 6-
  • FAM fluorescein isothiocyante
  • DANSC 5-dimethylamine-l-naphthalenesulfonyl chloride
  • TRITC tetramethylrhodamine isothiocyanate
  • lissamine rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
  • FITC fluorescein isothiocyante
  • DANSC 5-dimethylamine-l-naphthalenesulfonyl chloride
  • TRITC tetramethylrhodamine isothiocyanate
  • lissamine rhodamine 8200 sulphonyl chloride
  • 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 radiolabehng agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124 I, 125 1, 128 1, 132 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 U 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. Also useful is a beta emitter, such indium or H.
  • antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium (see, e.g., Galfre et al., Meth. Enzymol, 73:3-46 (1981)).
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable (see, for example, Aurameas, et al., Scand. J. Immunol, Vol. 8 Suppl. 7:7-23 (1978); Rodwell et al., Biotech., 3:889-894 (1984); and U.S. Pat. No. 4,493,795).
  • the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
  • a "specific binding agent” is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention.
  • 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 of the present invention can be used in an "ELISA" format to detect the quantity of the polypeptide of the present invention in a sample.
  • 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 description of the ELISA technique is found in Sites et al., Basic and Clinical Immunology, 4 th Ed., Chap. 22, Lange Medical Publications, Los Altos, CA (1982) and in U.S. Patent No. 3,654,090; Patent No. 3,850,752; and Patent No. 4,016,043, which are all incorporated herein by reference.
  • a polypeptide of the present invention an antibody or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.
  • a reagent is typically affixed to a solid matrix by adsorption from an aqueous medium, although other modes of affixation applicable to polynucleotides, proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adsorption methods are described herein.
  • Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ), agarose; polystyrene beads of ut 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 ut 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers (e.g., Abbott Laboratories, Chicago, IL),
  • 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.
  • packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
  • T84 cells were plated on 75 mm polycarbonate transwell filter inserts (Costar) and grown in DME/F12 (1 :1) containing 10% heat-inactivated bovine calf serum. The cells were maintained at confluence for 2-3 days and integrity of the epithelial barrier was determined by measuring transepithelial electrical resistance (TER) using an EVOM epithelial voltohmmeter (World Precision Instruments). When the TER values were greater than 1000 ohms/cm ⁇ and were stable, cells were treated with interferon- ⁇ (30 ng/ml, Genzyme) added to the basolateral side of the membrane.
  • TER transepithelial electrical resistance
  • TERs were measured to monitor the interferon-induced disruption of the barrier and RNA was harvested from the cells at those time points using TRIzol reagent (Life Technologies). The isolated RNA was subsequently used for TOGA analysis.
  • RNA was analyzed using a method of simultaneous sequence-specific identification of mRNAs known as TOGA (TOtal Gene expression Analysis) described in Sutcliffe, J.G., et al Proc Natl Acad Sci USA 2000 Feb 29; 97(5):1976-1981, International published application PCT/US99/23655, U.S. Patent No. 5,459,037, U.S. Patent No. 5,807,680, and U.S. Patent No. 6,030,784, hereby incorporated herein by reference.
  • the isolated RNA was enriched to form a starting polyA-containing mRNA population by methods known in the art.
  • the TOGA method further comprises an additional Polymerase Chain Reaction ("PCR") step performed using one of four 5' PCR primers and cDNA templates prepared from a population of antisense complimentary RNA ("cRNAs").
  • PCR Polymerase Chain Reaction
  • a final PCR step using one of a possible 256 5' PCR primers and a universal 3' PCR primer produced as PCR products, cDNA fragments that co ⁇ esponded to a 3 '-region of the starting mRNA population.
  • the produced PCR products were then identified by a) the sequence of at least the 5' seven base pairs, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences.
  • the method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags (ESTs) of the 3' end of mRNAs. DSTs that showed changes in relative levels during barrier breakdown were selected for further study. The intensities of the laser-induced fluorescence of the labeled PCR products were compared across samples isolated at different time intervals after treatment.
  • DSTs Digital Sequence Tags
  • ESTs expressed sequence tags
  • double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture of all 48 5'- biotinylated anchor primers of a set to initiate reverse transcription.
  • One such suitable set is G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 44), where V is A, C or G and N is A, C, G or T.
  • One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double stranded cDNA.
  • Each biotinylated double stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG.
  • the 3' fragments of cDNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate.
  • Suitable streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass particles.
  • a 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 of the cDNAs.
  • Notl which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • the 3' Mspl-Notl fragments which are of uniform length for each mRNA species, were directionally ligated into Clal-, Notl-cleaved plasmid pBC SK + (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli SURE cells (Stratagene).
  • the ligation regenerates the Notl site, but not the Mspl site.
  • Each library contained in excess of 5 x 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 of the 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: 45).
  • step two 400 pg of cDNA product was used as PCR template in four separate reactions with each of the four 5' PCR primers of the form G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 46), 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: 47).
  • step three the product of each subpool was further divided into 64 subsubpools (2ng in 20 ⁇ l) for the second PCR reaction, with 100 ng each of the fluoresceinated "universal" 3' PCR primer, the oligonucleotide (SEQ ID NO: 47) conjugated to 6-FAM and the appropriate 5' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 48), using a program that included an annealing step at a temperature X slightly above the T m of each 5' PCR primer to minimize artifactual misprinting and promote high fidelity copying.
  • Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).
  • Table 1 is a summary of the expression levels of 79 mRNAs determined from cDNA. These cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule, as well as the relative amount of the molecule produced at different time intervals after treatment. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. The digital address length of the fragment was determined by interpolation on a standard curve and, as such, may vary ⁇ 1-2 b.p. from the actual length as determined by sequencing.
  • the entry in Table 1 that describes a DNA molecule identified by the digital address Mspl GTGC 257 is further characterized as having a 5' terminus partial nucleotide sequence of CGGTGC and a digital address length of 257 b.p.
  • the DNA molecule identified as Mspl GTGC 257 is further described as being expressed at increasing levels after the induction of barrier breakdown.
  • the treatment results in a marked decrease of the expression of Mspl ACTG 191, producing a 13-fold decrease between the untreated control cells and the induced cells 44 hours after induction.
  • the other 77 DNA molecules identified in Table 1 by their Mspl digital addresses are further characterized by 1) level of gene expression in untreated T84 cells, 2) level of gene expression 4 hours after barrier breakdown induction, 3) level of gene expression 24 hours after barrier breakdown induction and 4) level of gene expression 44 hours after barrier breakdown induction.
  • Many of the isolated clones were further characterized in Table 2 and their nucleotide sequences are provided as SEQ ID NO: 1-43 in the Sequence Listing below.
  • FIG. 1 presents the results of TOGA analysis using a 5' PCR primer with parsing bases GGCC, showing PCR products produced from mRNA extracted from untreated T84 cells (Figure 1 A), T84 cells 4 hours after treatment with interferon-gamma (Figure IB), 24 hours after treatment with interferon-gamma (Figure 1C), and T84 cells 44 hours after treatment with interferon-gamma ( Figure ID).
  • the vertical index line indicates a PCR product of about 374 b.p. that is present in T84 cells and whose expression increases after induction of barrier breakdown in T84 cells.
  • PCR primers were designed based on the determined sequence and PCR was performed using the cDNA produced in the first PCR reaction as substrate. For example, for the 374 b.p. product disclosed above, oligonucleotides were synthesized eo ⁇ esponding to the 5' PCR primer in the second PCR step extended at the 3' end with additional nucleotides from the sequences adjacent to the terminal Mspl sites in the GenBank sequences. This primer had the sequence: GAT CGA ATC CGG GGC CCA GGC CTA TGC CTC (SEQ ID NO: 50).
  • Figure 2 shows the upper panel shows the length (as peak position) of the PCR product derived from the isolated clone as described above.
  • Figure 2A shows the PCR product obtained using an extended 5' PCR primer.
  • Figure 2B shows the PCR products obtained using the original PCR primers SEQ ID NO: 49 and SEQ ID NO: 47 (compare to the top panel in Figure 1).
  • Figure 2C the traces from Figure 2A and 2B are overlaid, demonstrating that the PCR product of the isolated and sequenced clone is the same length as the original PCR product.
  • Table 4 shows the results of the verification of candidate matches to database entries.
  • IMX 2 and 53 were cloned and assayed as described above for GGCC 374 to verify these clones.
  • oligonucleotides were synthesized co ⁇ esponding to the 5' PCR primer in the second PCR step extended at the 3' end with additional nucleotides from the sequences adjacent to the terminal Mspl sites in the identified co ⁇ esponding GenBank sequences.
  • IMX 2 (SEQ ID NOJ) and IMX 53 (SEQ ID NO:37) were cloned and assayed as described above for GGCC 374 to verify that the clones isolated are from the TOGA peaks (GAAT 140 and TGCC 265, respectively).
  • the 5' oligonucleotides used in the above mentioned assay were synthesized which contain a portion of the Mspl site (CGG) and the parsing bases (GAAT or TGCC, respectively) with clone sequence 3' to the parsing bases. The sequences of these oligonucleotides are listed in Table 4.
  • oligonucleotides were synthesized that extend at the 3' end with additional nucleotides from the sequences adjacent to the terminal Mspl sites in the identified corresponding GenBank sequences.
  • Other PCR products notably IMX 22 (SEQ ID NO: 1) and IMX 38 (SEQ ID NO: 2), were also differentially represented, and appeared to migrate in positions that indicate that the PCR products were novel based on comparison to data extracted from GenBank. In these cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands.
  • Figure 3 is a graphical representation of the results of Northern Blot analysis of clone IMX 43, SEQ ID NO: 6, where an agarose gel containing poly A enriched mRNA from the four T84 experimental samples (control, 4 hour, 24 hour and 44 hour) as well as size standards was blotted after electrophoresis and probed with radiolabelled IMX 43, SEQ ID NO: 6, imaged using a phosphorimager and quantified.
  • GSP Genome Sequencing Project
  • EST Expressed Sequence Tag
  • NA Not Applicable
  • R Reverse Transc ⁇ ptase- Polymerase Cham Reaction
  • NBA Northern Blot
  • Relat ⁇ ve DST Amount Relative DST Amount for Northern blots have been corrected for background and normalized to the hybridization signal of cyclophihn The values were obtained by exposing the Northern blot to a phospho ⁇ maging screen and quantitated using the Phospho ⁇ mager SI
  • GSP Genome Sequencing Pro j ect
  • EST Expressed Sequence Tag
  • NA Not Applicable
  • R Reverse Transc ⁇ ptase-Polymerase Chain Reaction
  • NBA Northern Blot Analysis
  • GSP Genome Sequencing Project
  • EST Expressed Sequence Tag
  • NA Not Applicable
  • GSP Genome Sequencing Project
  • EST Expressed Sequence Tag
  • NA Not Applicable
  • RT-PCR validation of cloned DSTs was performed, and results are presented in Table 2.
  • RT-PCR validation of RACE clones was also performed, with results presented in Table 10.
  • the starting amount of template was chosen based on a control curve that was generated to accurately define the linear range of amplication for the given cloned DST. Based upon the intensity of the peak cloned from the TOGATM panel, the following amounts of template were chosen:
  • PCR primers used for validation of cloned DSTs are listed in Table 7.
  • Duplicate reaction mixtures were assembled using the appropriate low and high concentration of cDNA template chosen from the time point sample showing the strongest TOGATM signal.
  • the reaction mixtures were cycled for 23, 26, 29, 32, 35, and 38 cycles.
  • the resulting amplification products from the duplicate reactions were quantitated and plotted against the cycle number to generate a standard curve, shown in tabular form below for one RACE clone, IMX 9R1. From these data, the cycle number and cDNA concentration combination which yielded acceptable levels of PCR product within the linear range of amplification were chosen for RT-PCR validation across the various time- points.
  • RT-PCR validation consisted of " assembling triplicate reactions using the chosen concentration of cDNA cycled to the defined cycle number. An internal control primer pair amplified under the same conditions was also performed to provide the basis for normalizing any differences between the cDNA templates. The results obtained were consistent with the initial TOGA findings, and are shown in Table 10. An example of the results from RT-PCR validation for an extended sequence obtained by the RACE method is shown in Table 6, below.
  • RACE Clone Amount Relative RACE Clone Amount for Northerns have been corrected for background and normalized to the hybridization signal of cyclophilin The values were obtained by exposing the Northern blot to a phosphorimaging screen and quantitated using the Phosphorimager SI
  • the sequences of several DSTs were extended using Gibco BRL Life Technologies 5' RACE (Rapid Amplification of cDNA Ends) system using primers described in Table 8. Briefly, for each DST selected, two DST-specific primers were designed. The first, a gene-specific primer, was located close to the poly- A tail. The second, overlapping the first, but more toward the 5' end of the DST was a nested RACE primer. First-strand cDNA synthesis was performed on the mRNA corresponding to the time point that displayed the highest level of expression of that individual DST, based on intensity of the fluorescently labeled PCR product. The first-strand cDNA reactions were initiated using the 3 '-most DST-specific primer (closest to the poly- A tail) and Superscript II reverse transcriptase, in the buffer and under the conditions described by the manufacturer.
  • the first-strand cDNA was purified using Amicon
  • PCR products from both of the serial PCR reactions were analyzed on acrylamide gels. An aliquot of each of the products was then ligated into Invitrogen 's TOPO II vector and transformed into TOP 10 cells. Bacterial colonies were picked and grown overnight for mini-preparation of the plasmid DNA. The plasmid DNAs were then digested with a restriction enzyme to determine the presence and size of a PCR insert. The insert in the plasmid DNA was then sequenced and analyzed. The RACE derived sequences for a specific DST were aligned both with each other and with the original DST sequence. Due to the design of the DST-specific PCR primers, RACE products generated from the second serial PCR reaction only contained a portion of the original DST sequence.
  • Validation of the RACE-PCR generated clones was performed by RT-PCR (see Table 10) as described above in Example 2.
  • the database matches and alignments to DSTs are presented in Table 9, above.
  • the results of validation by Northern blot analysis (IMX 6R1) or RT-PCR (IMX 9R1, IMX 12R1 and IMX 41R1) are presented in Table 10, above.
  • IMX 1 was found to have a region of high homology to human K12 mRNA. Further studies are described below, and in Lyman, S.D., et al., Identification of CD7 as a cognate of the human K12 (SECTM1) protein, J. Biol. Chem. 275(2):3431-3437 (2000), which is incorporated by reference herein.
  • the murine K12-Fc fusion protein was tested in an LPS/D-gal septic shock model.
  • K12-Fc administration 50 ⁇ g/mouse did not appear to enhance the toxicity of 5 ng LPS in combination with D- gal, producing about 30% mortality in both groups.
  • K12 protein was tested in a variety of T cell proliferation assays, including anti-CD3 induced proliferation of naive OVA TcR transgenic lymph node cells in vitro; antigen induced proliferation of naive OVA TcR transgenic lymph node cells in vitro; antigen - induced proliferation of memory OVA TcR transgenic lymph node cells in vitro.
  • K12 inhibited the concanavalin-A induced, but not the anti-T cell receptor induced, proliferation of mouse lymph node T cells. Numerous other molecules also have this effect, and its significance is unclear.
  • K12 was found to have no effect on human NK cell and mouse thymocyte proliferation. K12 induced CD25, CD54 and CD69 on human NK cells. Human K12 binds to the human T cell molecule CD7. Identification of a Cognate for the K12 Protein
  • Mouse K12 also binds to mouse CD7. In both the human and mouse genomes, the K12 gene maps very near to the CD7 gene. See Lyman, et al. (2000) for further details and discussion.

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Abstract

Cette invention a trait à des polynucléotides, des polypeptides ainsi qu'à des nécessaires et des méthodes en rapport avec des gènes régulés caractéristiques d'une inflammation gastro-intestinale.
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See also references of WO0073324A2 *

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WO2000073324A9 (fr) 2001-08-09
WO2000073324A3 (fr) 2001-07-19
AU5593300A (en) 2000-12-18
WO2000073324A2 (fr) 2000-12-07
JP2003501017A (ja) 2003-01-14
HK1044779A1 (zh) 2002-11-01
EP1181385A4 (fr) 2003-03-19
MXPA01012381A (es) 2002-07-22
CA2375855A1 (fr) 2000-12-07

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