| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 08.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 29.08.00 |
| PATENT TITLE |
Mammalian putative phosphatidylinositol-4-phosphate-5-kinase |
| PATENT ABSTRACT | A novel mammalian phosphatidylinositol-4-phosphate-5-kinase (PIP5K) referred to herein as p235 and novel polynucleotides encoding p235, are provided. p235 is specifically expressed in adipocytes and myocytes and is believed to be involved in insulin-induced membrane trafficking. Therapeutic, diagnostic and research methods utilizing the novel polynucleotides and proteins are also provided. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 20.03.98 |
| PATENT REFERENCES CITED |
Boronenkov, I.V. et al., "The Sequence of Phosphatidylinositol-4-phosphate 5-Kinase Defines a Novel Family of Lipid Kinases," J. Biol. Chem. 270:2881-2884 (1995). Czech, M.P., "Molecular Action Of Insulin On Glucose Transport," Annu. Rev. Nutr. 15:441-471 (1995). Hinchliffe, K. et al., "Inositol lipid pathways turn turtle," Nature 390:123-124 (1997). Israel, D.I., "A PCR-based method for high stringency screening of DNA libraries," Nucleic Acids Research 21(11):2627-2631 (1993). Liscovitch, M. et al., "Signal Transduction and Membrane Traffic: The PITP/Phosphoinostide Connection," Cell 81:659-662 (1995). Loijens, J.C. et al., "The Phosphatidylinositol 4-Phosphate 5-Kinase Family," Advanc. Enzyme Regul. 36:115-140 (1996). Rameh, L.E. et al., "A new pathway for synthesis of phosphatidylinositol-4,5-biphosphate," Nature 390:192-196 (1997). Singh, H. et al., "Molecular Cloning of Sequence-Specific DNA Binding Proteins Using Recognition Site Probe," Bio Techniques 7(3):252-261 (1989). Stenmark, H. et al., "Endosomal Localization of the Autoantigen EEA1 is Mediated by a Zinc-binding FYVE Finger," The Journal Of Biological Chemistry 271(39):24048-24054 (1996). Toker, A. et al., "Activation of Protein Kinase C Family Members by the Novel Phosphosphoinositides Ptdlns-3,4-P.sub.2 and Ptdlns-3,4,5-P.sub.3," The Journal Of Biological Chemistry 269(51)32358-32367 (1994). Yamamoto, A. et al., "Novel Pl(4)P 5-Kinase Homologue, Fab1p, Essential for Normal Vacuole Function and Morphology in Yeast," Molecular Biology of the Cell 6:525-539 (1995). Zhang, X. et al., "Phosphatidylinositol-4-phosphate 5-Kinase Isozymes Catalyze the Synthesis of 3-Phosphate-containing Phosphatidylinositol Signaling Molecules," The Journal Of Biological Chemistry 272(28):17756-17761 (1997). |
| PATENT CLAIMS |
What is claimed is: 1. An isolated nucleic acid molecule encoding p235 comprising SEQ ID NO: 1. 2. A vector comprising the nucleic acid molecule of claim 1. 3. A cell transformed with the nucleic acid molecule of claim 1. 4. The cell of claim 3, wherein the cell is a mammalian cell. 5. The cell of claim 3, wherein the cell is a bacterial cell. 6. A transfected cell producing the protein encoded by the nucleic acid molecule of claim 1. 7. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, b) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, c) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2, having kinase activity, and, d) a polynucleotide which is an allelic variant of the polynucleotide of a). 8. An isolated polynucleotide of claim 6 wherein said polynucleotide is operably linked to an expression control sequence. 9. A host cell transformed with a polynucleotide of claim 8. 10. The host cell transformed with a polynucleotide of claim 9, wherein said cell is a mammalian cell. 11. The host cell of claim 9, wherein the cell is a bacterial cell. 12. A process for producing a protein encoded by a polynucleotide of claim 8, which process comprises: a) growing a culture of the host cell of claim 9 in a suitable culture medium; and b) purifying said protein from the culture. 13. A nucleic acid probe comprising at least a fragment of a nucleic acid molecule encoding a protein fragment having kinase activity comprising a nucleotide sequence capable of hybridizing under salt and temperature conditions of 6.times. SSC at about 45.degree. C., followed by a wash of 0.2.times. SSC at 50.degree. C. to the nucleotide sequence of SEQ ID NO: 1, wherein the probe is of a length sufficient to hybridize with a complementary nucleic acid sequence thereto. 14. A nucleic acid probe comprising at least a fragment of a nucleic acid molecule encoding a protein fragment having kinase activity comprising a nucleotide sequence capable of hybridizing under salt and temperature conditions of 6.times. SSC at about 45.degree. C., followed by a wash of 0.2.times. SSC at 50.degree. C. to the complement of the nucleotide sequence of SEQ ID NO: 1, wherein the probe is of a length sufficient to hybridize with a complementary nucleic acid sequence thereto. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates generally to novel polynucleotides and the proteins encoded thereby and more particularly, to polynucleotides encoding a novel mammalian phosphatidylinositol-4-phosphate-5-kinase (PIP5K), and therapeutic, diagnostic and research methods employing same. BACKGROUND OF THE INVENTION Insulin action to recruit an intracellular pool of the glucose transporter protein GLUT4 to the fat/muscle cell surface has been established for more than a decade, yet the molecular details of this phenomenon are still elusive. Czech, M. P., Ann. Rev. Nutr. 15:441-471 (1995). Intriguingly, while GLUT4 appears to be a unique isoform for fat and muscle tissues, signaling element(s) specifically implicated in its sorting, directing and insulin-sensitive delivery to the cell surface are presently unknown. Phosphatidylinositol4-phosphate-5-kinase (PIP5K; EC2.7.1.68) has been implicated in membrane trafficking in yeast. Yamamoto, A. et al., Mol. Biol. Cell. 6:525-539 (1995). In particular, PIP5K synthesizes phosphatidylinositol 4,5-bisphosphate (Ptdlns[4,5]P.sub.2) from phosphatidylinositol-4-phosphate (Ptdlns[4]P). Loijens, J. C. et al., Advan. Enzyme Regul. 36:115-140 (1996). It has recently been reported that PIP5K also synthesizes Ptdlns[4,5]P.sub.2 from phosphatidylinositol-5-phosphate (Ptdlns[5]P). Hinchliffe, K. et al., Nature 390:123-124 (1997). The biosynthesis of Ptdlns[4,5]P.sub.2 has attracted increasing interest because of mounting evidence implicating metabolites of Ptdlns[4,5]P.sub.2 as important regulators of many cellular processes. Loijens, J. C. et al., Advan. Enzyme Regul. 36:115-140 (1996). In particular, Ptdlns[4,5]P.sub.2 is a key substrate of insulin-activated Pl 3-kinase, which enzyme, together with its Ptdlns[3,4]P.sub.2 and Ptdlns[3,4,5]P.sub.3 products, appear to be important elements in insulin action on GLUT4 membrane movements. Czech, M. P., Ann. Rev. Nutr. 15:441-471 (1995). The key role of activated Pl 3-kinase implies the presence of a large, easily available phosphoinositide substrate pool and suggests that the local production of Ptdlns[4]P and Ptdlns[4,5]P.sub.2 lipid substrates at key insulin-sensitive intracellular locations would aid an efficient Pl 3-kinase reaction and may be crucial for the Pl 3- kinase-mediated effect of insulin in GLUT4 directing and delivery to the fat/muscle cell surface. In addition, an alternative pathway of generating Ptdlns[3,4]P.sub.2 and Ptdlns[3,4,5]P.sub.3 has been recently suggested which utilizes Ptdlns[3]P substrates and concert action of phosphatidylinositol-4-phosphate-5-kinases. Zhang, X. et al., J. Biol. Chem. 272:17756-17761 (1997). Taken together, these data are consistent with the notion that the activity of PIP5K can contribute to the regulated pools of Ptdlns[3,4]P.sub.2 and Ptdlns[3,4,5]P.sub.3 stimulated by growth factors and insulin. Two distinct mammalian PlP5Ks, called type I (PIP5KI) and type II (PIP5KII), isolated from bovine and human erythrocytes, respectively, have been reported (Bazenet, C. E. et al., J. Biol. Chem. 265: 18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994)), as well as an isoform of PIP5KII (PIP5KIIa). Boronenkov, I. V. et al., J. Biol. Chem. 270:2881-2884 (1995). Yeast isozymes, specifically MSS4 and fab1, have also been isolated and studied. Yamamoto, A. et al., Mol. Biol. Cell 6:525-539 (1995); Yoshida, S. et al., Mol. Gen. Genet. 342:631-640 (1994); Yamamoto, A. et al., Mol. Biol. Cell 6:525-539 (1995). As mentioned above, the conversion from Ptdlns[4]P to Ptdlns[4,5]P.sub.2 is an important branchpoint in the phosphoinositide (PI) cycle, depicted in FIG. 1A. FIG. 1B depicts newly described inositol lipids, Ptdlns[5]P and Ptdlns[3,5]P.sub.2, and FIG. 1C includes the novel alternative pathway for Ptdlns[3,4,5]P.sub.3 production by PIP5Ks. The hydrolysis of Ptdlns[4,5]P.sub.2 by phosphoinositide-specific phospholipase C(PLC; EC 3.1.4.3) generates the second messengers, 1,2-diacylglycerol and inositol 1 ,4,5-triphosphate. 1,2-diacylglycerol activates several protein kinase C isoforms while inositol 1,4,5-triphosphate causes an increase in intracellular calcium. Rana, R. S. Physiol. Rev. 70:115-164 (1990). Ptdlns[4,5]P.sub.2 can also be phosphorylated by a PI 3-kinase (EC 2.7.1.137) to phosphatidylinositol 3,4,5-triphosphate (Ptdlns[3,4,5]P.sub.3), a second messenger whose targets are largely unknown but may include protein kinase C isoforms. Nakanishi, H. et al., J. Biol. Chem. 268:13-16 (1993); Toker, A. et al., J. Biol. Chem. 269:32358-32367 (1994). Furthermore, Ptdlns[4,5]P.sub.2 modulates the function of numerous enzymes including many actin-binding proteins (Janmey, P. A., Annu. Rev. Physiol. 56:169-191 (1994)), binds Ph domains found in some signaling proteins (Harlan, J. E. et al., Nature 371:168-170 (1994)), and appears to be involved in the secretory vesicle cycle. Eberhard, D. A. et al., Biochem. J. 268:15-25 (1990); Hay, J. C. et al., Nature 374:173-177 (1995); Liscovitch M. et al., Cell 81:659-662 (1995). PIP5Ks have been isolated from erythrocytes, brain, adrenal medulla, liver and other sources. Carpenter, C. L. et al., Biochemistry 29:11147-11156 (1990) (and references therein); Van Dongen, C. J. et al., Biochem. J. 233:859-864 (1986); Moritz, A et al., Biochim. Biophys. Acta 1168:79-86 (1993); Divecha, N. et al., Biochem. J. 288:637-642 (1992); Husebye, E. S. et al., Biochim. Biophys. Acta 1010:250-257 (1989); Urumow, T. et al., Biochim. Biophys. Acta 1052:152-158 (1990). In cells, PIP5K activity is found on the plasma membrane (Carpenter, C. L. et al., Biochemistry 29:11147-11156 (1990); Urumow, T. et al., Biochim. Biophys. Acta 1052:152-158 (1990); Ling, L. E. et al., J. Biol. Chem. 264:5080-5088 (1989); Smith, C. D. et al., J. Biol. Chem. 264:3206-3210 (1989); Bazenet, C. E. et al., J. Biol. Chem. 265:18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994)), associated with the cytoskeleton (Payrastre, B. et al., J. Cell Biol. 115:121-128 (1991); Grondin, P. et al., J. Biol. Chem. 266:15705-15709 (1991)), on the endoplasmic reticulum (Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991), and in nuclei (Divecha, N. et al., Biochem. J. 289:617-620 (1993); Payrastre, B. et al., J. Biol. Chem. 267:5078-5084 (1992); Divecha, N. et al., Cell 74:405-407 (1993)). There is also a soluble, cytosolic population of PIP5K. Ling, L. E. et al., J. Biol. Chem. 264:5080-5088 (1989); Bazenet, C. E. et al., J. Biol. Chem. 265:18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994); Moritz, A. et al., J. Neurochem. 54:351-354 (1990). The kinase's product, Ptdlns[4,5]P.sub.2, is primarily found in the plasma membrane but can be detected in isolated endoplasmic reticulum and nuclei. Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991); Tran, D. et al., Cell. Signal 5:565-581 (1993). Ptdlns[4]P is present in all of these fractions. Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991); Tran, D. et al., Cell. Signal 5:565-581 (1993). Hinchliffe, K. et al., Nature 390:123-124 (1997); Rameh, L. E. et al., Nature 390:192-196 (1997). One postulated reason for the large family of PIP5Ks is that many forms of regulation and cellular functions have been attributed to PIP5Ks, as summarized in FIG. 2. It would thus be desirable to provide a mechanism to further study the role of PIP5Ks. It would also be desirable to provide a novel mammalian PIP5K. It would further be desirable to provide a screening method for further studying the role of PIP5Ks, their substrates and products. It would still further be desirable to provide an animal model for further investigating the role of PlP5Ks. SUMMARY OF THE INVENTION A novel polynucleotide encoding a mammalian PIP5K referred to herein as p235, is provided. p235 is specifically expressed in adipocytes and myocytes and is believed to be involved in membrane trafficking, particularly, insulin-induced membrane trafficking of fat/muscle specific glucose transporter, GLUT4. The isolated cDNA for p235 set forth in SEQ ID NO: 1 is about 7.4 kbp long with an open reading frame extending from nucleotide 139 to 6294, encoding the novel protein. p235 is 2052 amino acids in length with Mr 233,040 and pl 6.34. The deduced polypeptide sequence is set forth in SEQ ID NO: 2. Thus, in one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, b) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, c) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2, having biological activity, d) a polynucleotide which is an allelic variant of the polynucleotide of a) and, e) a polynucleotide which encodes a species homologue of the protein of b) or c). In another embodiment, the present invention provides a gene corresponding to the cDNA of SEQ ID NO: 1. In yet another embodiment, the present invention provides a composition comprising a protein wherein the protein comprises an amino acid sequence selected from the group consisting of: a) the amino acid sequence of SEQ ID NO: 2, and b) fragments of the amino acid sequence of SEQ ID NO: 2. In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Processes are also provided for producing a protein, which comprise: (a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and (b) purifying the protein from the culture. The protein produced according to such methods is also provided by the present invention. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein. Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention. Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier. Methods of using the polynucleotide of the present invention and the protein encoded thereby to further study the role of PIP5Ks, their substrates and products, are also provided as well as research models including cell lines and transgenic and knockout animal models. Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and subjoined claims and by referencing the following drawings in which: FIGS. 1A-1C are schematics of the phosphoinositide cycle; FIG. 2 is a schematic of the regulation of the PIP5K isoforms and the cellular roles of Ptdlns[4,5]P.sub.2 synthesized by these enzymes; FIG. 3A shows the similarity between a subset of highly conserved motifs in the PIP5K domain of mouse p235, yeast Fab1 p, C. elegans C05E7.5 and human PIP5K Type I [Accession numbers: pirll556274 (Fab1p); pirllA57013 (EEA1); pirllS45129 (Vsp27); gill885385 (Hrs-2); gil065686 (C05E7.5) and, gi1743875 (PI(4) 5-kinase)]; and FIG. 3B shows the similarity between the FYVE motif in a conserved zinc-binding region. Potential Zn.sup.2+ -coordinating his/cis clusters are indicated below the alignment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A novel mammalian PIP5K referred to herein as p235, is provided. The isolated cDNA is about 7.4 kbp long with an open reading frame extending from nucleotide 139 to 6294, encoding the novel mammalian protein, p235. p235 is 2052 amino acids in length with estimated Mr 233,040 and pl 6.34. The predicted ATG initiation codon conforms well to the Kozak consensus sequence for the translation initiation start and is preceded by an in-frame terminator (nucleotide 78), supporting the notion that this ATG represents the translation initiator of the p235 gene product. Without intending to be bound by theory, it is believed that p235 may be involved in membrane trafficking, and in particular, GLUT4 translocation induced by insulin, that results in glucose transport into cells. Thus, by employing p235 and the polynucleotides encoding same, movement of GLUT4 onto the cell surface, e.g., adipocyte and myocyte surfaces, may be promoted. Likewise, by inhibiting p235 function, GLUT4 movement onto the cell surface may be inhibited. Increasing glucose uptake in cells by employing p235 and the polynucleotides encoding same may provide effective treatment of diseases involving a deficiency in glucose transport. For example, non-insulin-dependent diabetes mellitus (NIDDM) also known as Type 2 diabetes, is characterized by a decrease in the body's ability to utilize insulin. This resistance to insulin action is thought to be caused by either a significant reduction in the number of insulin receptors, a defect in the receptors preventing insulin binding, or a defect in the downstream signalling after insulin has bound to the receptor. Any of these defects result in a significant decrease in the amount of glucose taken up by the cells and an increase in the concentration of blood glucose. Increased cellular uptake of glucose by patients diagnosed with NIDDM may be obtained by providing increased levels of p235 to those cells. Inhibition of p235 function and sequence inhibition of glucose transporter movement into cells, particularly adipocytes, may provide a treatment for obesity. Glucose is the main source of energy for adipocytes as metabolism of glucose provides the building blocks for synthesis of triacylglycerols, the main components of adipocytes. Blocking the uptake of glucose would decrease the amount of adipose tissue. Inhibiting p235 function is therefore one method for blocking the uptake of glucose and decreasing the amount of adipose tissue. The nucleic acid sequence of the cDNA encoding p235 and its deduced amino acid sequence are set forth in SEQ ID NOS: 1 and 2, respectively. In a preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 1, or homologues therefore. In another preferred embodiment, the isolated and purified polypeptide of the invention comprises the amino acid sequence of SEQ ID NO: 2, as well as biological equivalents. Database analysis of the deduced amino acid sequence reveals that p235 contains, in order from its N-terminus, a zinc-binding motif, a large chaperonin-like region, and spread over the C-terminal portion, a putative catalytic domain of PIP5K. The overall architecture and size of p235 are thus very similar to the yeast Fab1p. Yamamoto, A. et al., Mol. Biol. Cell. 6:525-539 (1995). The putative catalytic region of p235 displays a high sequence similarity to those of human PIP5K Type I, Fab1p and C. elegans C05E7.5, and includes a predicted downstream nucleotide binding motif and sequences (FIG. 3A). This similarity suggests that p235 has a PIP5K activity. Intriguingly, p235 shares no homology with the mammalian PIP5K outside the kinase domain and is distinguished in having additional sequences on the N-terminal side of the catalytic domain. Thus, the very N-terminus of p235 shows a striking similarity to a domain denoted as FYVE finger, recently identified in eleven non-nuclear proteins such as EEA1, Fab1p, Vsp27, and Vac1, implicated in membrane trafficking. Stenmark, H. et al., J. Biol. Chem. 271:24048-24054 (1996). The FYVE finger has been defined as a genuine zinc-binding domain that determines specific endosomal localization and is characterized by 8 conserved cysteines and 2 histidines as potential coordinators of zinc (FIG. 3B). Taken together, these results are consistent with the idea that the characteristic FYVE finger localizes p235 to endosomes where it acts to increase the local production of Ptdlns[4,5]P.sub.2 and/or Ptdlns[3,4,5]P.sub.3, important elements in insulin signaling of GLUT4 translocation. To confirm p235 fat/muscle specific or enriched expression, Northern blot analysis (total RNA) of several cell types was performed. This analysis revealed that p235 mRNA is a single .about.9 kb transcript, highly abundant in insulin-serisitive L6 monocytes and 3T3-L1 adipocytes, while in COS, CHO, HeLa and MCF-7 cells the message is undetectable. Intriguingly, although highly enriched in insulin-sensitive adipocytes and myocytes, the p235 transcript exists in the fibroblastic lines. These data indicate that the transcript level of p235 increases in fully differentiated insulin-responsive cells. Fragments of the protein of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., BioTechnology 10:773-778 (1992) and in R. S. McDowell et al., J. Amer. Chem. Soc. 114:9245-9253 (1992). Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a protein--IgM fusion would generate a decavalent form of the protein of the invention. The present invention also provides a gene corresponding to the cDNA sequence disclosed herein. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. In another aspect, DNA sequence information provided by the present invention allows for the preparation of relatively short DNA (or RNA) sequences or probes that are identical to or hybridize to the nucleotide sequence disclosed herein. Nucleic acid probes (also referred to as oligonucleotide probes) of an appropriate length are prepared based on a consideration of the nucleotide sequence of SEQ ID NO: 1. The probes can be used in a variety of assays appreciated by those skilled in the art, for detecting the presence of complementary sequences in a given sample. The probes may be useful in research, prognostic and diagnostic applications. For example, the probes may be used to detect homologus nucleotide sequences, e.g., the human homolog. The design of the probe should preferably follow these parameters: a) it should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any; and b) it should be designed to have a T.sub.m of approximately 80.degree. C. (assuming 2 degrees for each A or T and 4 degrees for each G or C). The oligonucleotide should preferably be labeled with y-.sup.32 P ATP (specific activity 6000 Ci/mole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/mole. A further preferred nucleic acid sequence employed for hybridization studies or assays includes probe molecules that are complementary to at least a 10 to 70 or so long nucleotide stretch of the polynucleotide sequence shown in SEQ ID NO: 1. A size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. It will be appreciated that nucleic acid molecules having gene-complementary stretches of 25 to 40 nucleotides, 55 to 70 nucleotides, or even longer where desired, may be preferred. Such fragments can be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Pat. No. 4,603,102, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction enzyme sites. In certain embodiments, it is also advantageous to use oligonucleotide primers. The sequence of such primers is designed using the polynucleotide of the present invention and is used with PCR technology. The invention also encompasses allelic variants of the disclosed polynucleotide or protein; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotide. The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19:4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185:537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotidelexpression control sequence. A number of types of cells may act as suitable host cells for expression of the protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coil, Bacillus subtills, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods. The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac.RTM. kit) and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed." The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl.RTM. or Cibacrom blue 3GA Sepharose.RTM.; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST), hexahistidine or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, Conn.). Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein." The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein. The protein may also be produced by known conventional chemical synthesis. Methods for constructing the protein of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified protein in screening of therapeutic compounds and in immunological processes for the development of antibodies. The protein provided herein also include protein characterized by amino acid sequences similar to those of purified protein but into which modifications are naturally provided or deliberately engineered. For example, modifications in the peptide of DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequence may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Other fragments and derivatives of the sequence of the protein which would be expected to retain protein activity in whole or in part may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention. In one embodiment, the present invention provides an antibody immunoreactive with the p235 polypeptide. Also contemplated by the present invention are antibodies immunoreactive with homologues or biologically equivalent polynucleotides and polypeptides of the present invention. As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as polypeptide fragments of antibodies that retain a specific binding activity for p235. One skilled in the art will appreciate that anti-p235 antibody fragments such as Fab, F(ab').sub.2 and Fv fragments can retain specific binding activity for p235 and, thus, are included within the definition of an antibody. In addition, the term "antibody" as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies and fragments that retain binding activity. Methods of making antibodies are known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Press, 1988). As used herein, the term "nucleic acid" is intended to mean natural and synthetic linear and sequential arrays of nucleotides and nucleosides, e.g. in cDNA, genomic DNA (gDNA), mRNA, and RNA, oligonucleotides, oligonucleosides and derivatives thereof. It will also be appreciated that such nucleic acids can be incorporated into other nucleic acid chains referred to as "vectors" by recombinant-DNA techniques such as cleavage and ligation procedures. The terms "fragment" and "segment" are as used herein with reference to nucleic acids (e.g., cDNA, genomic DNA, i.e., gDNA) are used interchangeably to mean a portion of the subject nucleic acid such as constructed artificially (e.g. through chemical synthesis) or by cleaving a natural product into a multiplicity of pieces (e.g. with a nuclease or endonuclease to obtain restriction fragments). As used herein, "A" represents adenine; "T" represents thymine; "G" represents guanine; "C" represents cytosine; and "U" represents uracil. As referred to herein, the term "encoding" is intended to mean that the subject nucleic acid may be transcribed and translated into the subject protein in a cell, e.g. when the subject nucleic acid is linked to appropriate control sequences such as promoter and enhancer elements in a suitable vector (e.g. an expression vector) and the vector is introduced into a cell. The term "polypeptide" is used to mean three or more amino acids linked in a serial array. As referred to herein, the term "capable of hybridizing under high stringency conditions" means annealing a strand of DNA complementary to the DNA of interest under highly stringent conditions. Likewise, "capable of hybridizing under low stringency conditions" refers to annealing a strand of DNA complementary to the DNA of interest under low stringency conditions. In the present invention, hybridizing under either high or low stringency conditions would involve hybridizing a nucleic acid sequence (e.g., the complementary sequence to SEQ ID NO: 1 or portion thereof), with a second target nucleic acid sequence. "High stringency conditions" for the annealing process may involve, for example, high temperature and/or low salt content, which disfavor hydrogen bonding contacts among mismatched base pairs. "Low stringency conditions" would involve lower temperature, and/or lower salt concentration than that of high stringency conditions. Such conditions allow for two DNA strands to anneal if substantial, though not near complete complementarity exists between the two strands, as is the case among DNA strands that code for the same protein but differ in sequence due to the degeneracy of the genetic code. Appropriate stringency conditions which promote DNA hybridization, for example, 6.times. SSC at about 45.degree. C., followed by a wash of 2X SSC at 50.degree. C. are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.31-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.times. SSC at 50.degree. C. to a high stringency of about 0.2.times. SSC at 50.degree. C. In addition, the temperature in the wash step can be increased from low stringency at room temperature, about 22.degree. C., to high stringency conditions, at about 75.degree. C. Other stringency parameters are described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring N.Y., (1982), at pp. 387-389; see also Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Volume 2, Cold Spring Harbor Laboratory Press, Cold Spring, N.Y. at pp. 8.46-8.47 (1989). As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, for example between an antibody molecule immunoreacting with an antigen. The term "knockout" refers to partial or complete suppression of the expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell. The term "knockout construct" refers to a nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell. The nucleic acid sequence used as the knockout construct is typically comprised of 1) DNA from some portion of the gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and 2) a marker sequence used to detect the presence of the knockout construct in the cell. Typically, the knockout construct is inserted into an embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination. This ES cell is then injected into, and integrates with, the developing embryo. The phrases "disruption of the gene" and "gene disruption" refer to insertion of a nucleic acid sequence into one region of the native DNA sequence (usually one or more exons) and/or the promoter region of a gene so as to decrease or prevent expression of that gene in the cell as compared to the wild-type or naturally occurring sequence of the gene. By way of example, a nucleic acid construct can be prepared containing a DNA sequence encoding an antibiotic resistance gene which is inserted into the DNA sequence that is complementary to the DNA sequence (promoter and/or coding region) to be disrupted. When this nucleic acid construct is then transfected into a cell, the construct will integrate into the genomic DNA. Thus, many progeny of the cell will no longer express the gene at least in some cells, or will express it at a decreased level, as the DNA is now disrupted by the antibiotic resistance gene. The term "marker sequence" refers to a nucleic acid sequence that is 1) used as part of a nucleic acid construct (i.e., the "knockout construct") to disrupt the expression of the gene(s) of interest (e.g., p235), and 2) used as a means to identify those cells that have incorporated the knockout construct into the genome. The marker sequence may be any sequence that serves these purposes, although typically it will be a sequence encoding a protein that confers a detectable trait on the cell, such as an antibiotic resistance gene or an assayable enzyme not typically found in the cell. Where the marker sequence encodes a protein, the marker sequence will also typically contain a promoter that regulates its expression. The term "progeny" refers to any and all future generations derived and descending from a particular mammal, i.e., a mammal containing a knockout construct inserted into its genomic DNA. Thus, progeny of any successive generation are included herein such that the progeny, the F1, F2, F3, generations and so on indefinitely are included in this definition. The foregoing and other aspects of the invention may be better understood in connection with the following examples, which are presented for purposes of illustration and not by way of limitation. |
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