Abstract:
Novel Type III density enhanced protein tyrosine phosphatases are disclosed and exemplified by human DEP-1 enzyme. Polynucleotides encoding huDEP-1 are disclosed, along with methods and materials for production of the same by recombinant procedures. Binding molecules specific for DEP-1 are also disclosed as useful for modulating the biological activities of DEP-1.

Description:
This is a Continuation of U.S. application Ser. No. 08/237,940, filed May 3, 1994, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to purified and isolated protein tyrosine phosphatase enzymes (PTPs) and polynucleotides encoding the same. PTPs of the invention are characterized by upregulated mRNA transcription and/or translation, or post-translational modification leading to increased total cellular enzyme activity as a function of increased cellular contact with neighboring cells. Such density enhanced PTPs are referred to as DEPTPs. An illustrative human Type III receptor-like density-enhanced protein tyrosine phosphatase has been designated huDEP-1. 
     BACKGROUND OF THE INVENTION 
     Protein tyrosine phosphorylation is an essential element in signal transduction pathways which control fundamental cellular processes including growth and differentiation, cell cycle progression, and cytoskeletal function. Briefly, the binding of growth factors, or other ligands, to a cognate receptor protein tyrosine kinase (PTK) triggers autophosphorylation of tyrosine residues in the receptor itself and phosphorylation of tyrosine residues in the enzyme&#39;s target substrates. Within the cell, tyrosine phosphorylation is a reversible process; the phosphorylation state of a particular tyrosine residue in a target substrate is governed by the coordinated action of both PTKs, catalyzing phosphorylation, and protein tyrosine phosphatases (PTPs), catalyzing dephosphorylation. 
     The PTPs are a large and diverse family of enzymes found ubiquitously in eukaryotes [Charbonneau and Tonks, Ann. Rev. Cell Biol. 8:463-493 (1993)]. Structural diversity within the PTP family arises primarily from variation in non-catalytic (potentially regulatory) sequences which are linked to one or more highly conserved catalytic domains. In general, soluble cytoplasmic PTP forms are termed non-receptor PTPs and those with at least one non-catalytic region that traverses the cell membrane are termed receptor-like PTPs (RPTPs). 
     A variety of non-receptor PTPs have been identified which characteristically possess a single catalytic domain flanked by non-catalytic sequences. Such non-catalytic sequences have been shown to include, among others, sequences homologous to cytoskeletal-associated proteins [Yang and Tonks, Proc.Natl.Acad.Sci.(USA) 88:5949-5953 (1991)] or to lipid binding proteins [Gu, et al., Proc.Natl.Acad.Sci.(USA) 89:2980-2984 (1992)], and/or sequences that mediate association of the enzyme with specific intracellular membranes [Frangioni et al., Cell 68:545-560 (1992)], suggesting that subcellular localization may play a significant role in regulation of PTP activity. 
     Analysis of non-catalytic domain sequences of RPTPs suggests their involvement in signal transduction mechanisms. However, binding of specific ligands to the extracellular segment of RPTPs has been characterized in only a few instances. For example, homophilic binding has been demonstrated between molecules of PTPμ [Brady-Kalnay, et al., J.Cell. Biol. 122:961-972 (1993)] i.e., the ligand for PTPμ expressed on a cell surface is another PTPμ molecule on the surface of an adjacent cell. Little is otherwise known about ligands which specifically bind to, and modulate the activity of, the majority of RPTPs. 
     Many receptor-like PTPs comprise an intracellular carboxyl segment with two catalytic domains, a single transmembrane domain and an extracellular amino terminal segment [Krueger et al., EMBO J. 9:3241-3252 (1990)]. Subclasses of RPTPs are distinguished from one another on the basis of categories or &#34;types&#34; of extracellular domains [Fischer, et al., Science 253:401-406 (1991)]. Type I RPTPs have a large extracellular domain with multiple glycosylation sites and a conserved cysteine-rich region. CD45 is a typical Type I RPTP. The Type II RPTPs contain at least one amino terminal immunoglobulin (Ig)-like domain adjacent to multiple tandem fibronectin type III (FNIII)-like repeats. Similar repeated FNIII domains, believed to participate in protein:protein interactions, have been identified in receptors for IL2, IL4, IL6, GM-CSF, prolactin, erythropoietin and growth hormone [Patthy, Cell 61:13-14 (1992)]. The leukocyte common antigen-related PTP known as LAR exemplifies the Type II RPTP structure [Streuli et al., J.Exp.Med. 168:1523-1530 (1988)], and, like other Type II RPTPs, contains an extracellular region reminiscent of the NCAM class of cellular adhesion molecules [Edelman and Crossin, Ann.Rev.Biochem. 60:155-190 (1991)]. The Type III RPTPs, such as HPTPβ [Krueger et al., EMBO J. 9:3241-3252 (1990)], contain only multiple tandem FNIII repeats in the extracellular domain. The Type IV RPTPs, for example RPTPα [Krueger et al. (1990) supra], have relatively short extracellular sequences lacking cysteine residues but containing multiple glycosylation sites. A fifth type of RPTP, exemplified by PTPγ [Barnes, et al., Mol.Cell.Biol. 13:1497-1506 (1993)] and PTPζ [Krueger and Saito, Proc.Natl.Acad.Sci. (USA) 89:7417-7421 (1992)], is characterized by an extracellular domain containing a 280 amino acid segment which is homologous to carbonic anhydrase (CAH) but lacks essential histidine residues required for reversible hydration of carbon dioxide. 
     FNIII sequences characteristically found in the extracellular domains of Type II and Type III RPTPs comprise approximately ninety amino acid residues with a folding pattern similar to that observed for Ig-like domains [Bork and Doolittle, Proc.Natl.Acad.Sci(USA) 89:8990-8994 (1992)]. Highly conserved FNIII sequences have been identified in more than fifty different eukaryotic and prokaryotic proteins [Bork and Doolittle, Proc.Natl.Acad.Sci. (USA) 89:8990-8994 (1992)], but no generalized function has been established for these domains. Fibronectin itself contains fifteen to seventeen FNIII domain sequences, and it has been demonstrated that the second FNIII domain (FNIII 2 ) contains a binding site for heparin sulphate proteoglycan [Schwarzbauer, Curr.Opin.Cell Biol. 3:786-791 (1991)] and that FNIII 13  and FNIII 14  are responsible for heparin binding through ionic interactions [Schwarzbauer, Curr.Opin.Cell Biol. 3:786-791 (1991)]. Perhaps the best characterized interaction for a fibronectin FNIII domain involves FNIII 10  which is the major site for cell adhesion [Edelman and Crossin, Ann.Rev.Biochem 60:155-190 (1991); Leahy, et al., Science 258:987-991 (1992); Main, et al., Cell 71:671-678 (1992)]. FNIII 10  contains the amino acid sequence Arg-Gly-Asp (RGD) which is involved in promoting cellular adhesion through binding to particular members of the integrin superfamily of proteins. 
     Characteristics shared by both the soluble PTPs and the RPTPs include an absolute specificity for phosphotyrosine residues, a high affinity for substrate proteins, and a specific activity which is one to three orders of magnitude in excess of that of the PTKs in vitro [Fischer, et al., Science 253:401-406 (1991); Tonks, Curr.Opin.Cell.Biol. 2:1114-1124 (1990)]. This latter characteristic suggests that PTP activity may exert an antagonistic influence on the action of PTKs in vivo, the balance between these two thus determining the level of intracellular tyrosine phosphorylation. Supporting a dominant physiological role for PTP activity is the observation that treatment of NRK-1 cells with vanadate, a potent inhibitor of PTP activity, resulted in enhanced levels of phosphotyrosine and generation of a transformed cellular morphology [Klarlund, Cell 41:707-717 (1985)]. This observation implies potential therapeutic value for PTPs and agents which modulate PTP activity as indirect modifiers of PTK activity, and thus, levels of cellular phosphotyrosine. 
     Recent studies have highlighted aspects of the physiological importance of PTP activity. For example, mutations in the gene encoding a non-receptor hematopoietic cell protein tyrosine phosphatase, HCP, have been shown to result in severe immune dysfunction characteristic of the motheaten phenotype in mice [Schultz, et al., Cell 73:1445-1454 (1993)]. Under normal conditions HCP may act as a suppressor of PTK-induced signaling pathways, for example, the CSF-1 receptor [Schultz, et al., Cell 73:1445-1454 (1993)]. Some PTP enzymes may be the products of tumor suppressor genes and their mutation or deletion may contribute to the elevation in cellular phosphotyrosine associated with certain neoplasias [Brown-Shimer, et al., Cancer Res. 52:478-482 (1992); Wary, et al., Cancer Res. 53:1498-1502 (1993)]. Mutations observed in the gene for RPTPγ in murine L cells would be consistent with this hypothesis [Wary, et al., Cancer Res. 53:1498-1502 (1993)]. The observation that the receptor-like PTP CD45 is required for normal T cell receptor-induced signalling [Pingel and Thomas, Cell 58:1055-1065 (1989)] provides evidence implicating PTP activity as a positive mediator of cellular signalling responses. 
     Normal cells in culture exhibit contact inhibition of growth, i.e., as adjacent cells in a confluent monolayer touch each other, their growth is inhibited [Stoker and Rubin, Nature 215:171-172 (1967)]. Since PTKs promote cell growth, PTP action may underlie mechanisms of growth inhibition. In Swiss mouse 3T3 cells, a phosphatase activity associated with membrane fractions is enhanced eight-fold in confluent cells harvested at high density as compared to cells harvested from low or medium density cultures [Pallen and Tong, Proc.Natl.Acad.Sci. (USA) 88:6996-7000 (1991)]. This elevated activity was not observed in subconfluent cell cultures brought to quiescence by serum deprivation. The enhanced phosphatase activity was attributed to a 37 kD protein, as determined by gel filtration, but was not otherwise characterized. Similarly, PTPs have been directly linked to density arrest of cell growth; treatment of NRK-1 cells with vanadate was able to overcome density dependent growth inhibition and stimulate anchorage independent proliferation, a characteristic unique to transformed, or immortalized, cells [Klarland, Cell 41:707-717 (1985); Rijksen, et al., J.Cell Physiol. 154:343-401 (1993)]. 
     In contrast to these observations, PCT Publication No. WO 94/03610 discloses a transmembrane PTP, termed PTP35, the steady state mRNA level of which was observed to be at a maximum in actively growing cells. Little or no PTP35 mRNA expression was detected in confluent cell. This mode of regulation was also observed in mouse 3T3 cells. Thus, two RPTPs in the same cell type apparently participate in opposing processes, with one (PTP35) contributing to cellular growth and the other (the 35 kD PTP of Pallen and Tongs) contributing to cellular quiescence. 
     Interestingly, transcription of Type II RPTP LAR messenger RNA has been demonstrated to be upregulated in confluent fibroblast cell culture [Longo, et al., J.Biol.Chem. 268:26503-26511 (1993)]. LAR is proteolytically processed to generate a mature protein that is a complex of two non-covalently associated subunits, one containing the majority of the cell adhesion molecule-like extracellular domain [Yu, et al., Oncogene 7:1051-1057 (1992); Streuli, et al., EMBO J. 11:897-907 (1992)] and which is shed as cells approach confluence [Streuli, et al., EMBO J. 11:897-907 (1992)]. These observations lead to speculation regarding PTP involvement in modulation of cytoskeletal integrity, as well as other related cellular phenomena such as transformation, tumor invasion, metastasis, cell adhesion, and leukocyte movement along and passage through the endothelial cell layer in inflammation. The therapeutic implications are enormous for modulators of PTP activity which are capable of regulating any or all of these cellular events. 
     There thus exists a need in the art to identify members of the PTP family of enzymes and to characterize these proteins in terms of their amino acid and encoding DNA sequences. Such information would provide for the large scale production of the proteins, allow for identification of cells which express the phosphatases naturally and permit production of antibodies specifically reactive with the phosphatases. Moreover, elucidation of the substrates, regulatory mechanisms, and subcellular localization of these PTPs would contribute to an understanding of normal cell growth and provide information essential for the development of therapeutic agents useful for intervention in abnormal and/or malignant cell growth. 
     BRIEF DESCRIPTION OF THE INVENTION 
     As employed herein with respect to a protein tyrosine phosphatase, &#34;density enhanced&#34; denotes upregulated cellular mRNA transcription or translation and/or total cellular activity as a function of increased contact with neighboring cells. 
     In one aspect, the present invention provides purified and isolated polynucleotides (e.g., DNA and RNA transcripts, both sense and anti-sense strands) encoding a Type III density enhanced protein tyrosine phosphatase enzymatic activity exemplified by the human phosphatase huDEP-1 and variants, including fragments, thereof (i.e., fragments and deletion, addition or substitution analogs) which possess binding and/or immunological properties inherent to Type III density enhanced phosphatases. Preferred DNA molecules of the invention include cDNA, genomic DNA and wholly or partially chemically synthesized DNA molecules. A presently preferred polynucleotide is the DNA as set forth in SEQ ID NO: 1, encoding the human DEP-1 polypeptide of SEQ ID NO: 2. Also provided are recombinant plasmid and viral DNA constructions (expression constructs) which include Type III density enhanced phosphatase encoding sequences, especially constructions wherein the Type III density enhanced phosphatase encoding sequence is operatively linked to a homologous or heterologous transcriptional regulatory element or elements. 
     As another aspect of the invention, prokaryotic or eukaryotic host cells transformed or transfected with DNA sequences of the invention are provided which express a Type III density enhanced phosphatase polypeptide or variants thereof. Host cells of the invention are particularly useful for large scale production of Type III density enhanced phosphatase polypeptides, which can be isolated from either the host cell itself or the medium in which the host cell is grown. Host cells which express Type III density enhanced phosphatase polypeptides on the extracellular membrane surface are also useful as immunogens in the production of anti-Type III density enhanced phosphatase antibodies. 
     Also provided by the present invention are purified and isolated Type III density enhanced phosphatase polypeptides, including fragments and variants thereof. A preferred Type III density enhanced phosphatase polypeptide is set forth in SEQ ID NO: 2. Novel Type III density enhanced phosphatase polypeptides and variant polypeptides may be obtained as isolates from natural sources, but are preferably produced by recombinant procedures involving host cells of the invention. Completely glycosylated, partially glycosylated and wholly un-glycosylated forms of the Type III density enhanced phosphatase polypeptide may be generated by varying the host cell selected for recombinant production and/or post-isolation processing. Variant Type III density enhanced phosphatase polypeptides of the invention may comprise water soluble and insoluble polypeptides including analogs wherein one or more of the amino acids are deleted or replaced: (1) without loss, and preferably with enhancement, of one or more biological activities or immunological characteristics specific for Type III density enhanced phosphatases; or (2) with specific disablement of a particular ligand/receptor binding or signalling function. 
     Also comprehended by the present invention are peptides, polypeptides, and other non-peptide molecules which specifically bind to Type III density enhanced phosphatases of the invention. Preferred binding molecules include antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, anti-idiotype antibodies, CDR-grafted antibodies and the like), counterreceptors (e.g., membrane-associated and soluble forms) and other ligands (e.g., naturally occurring or synthetic molecules), including those which competitively bind Type III density enhanced phosphatases in the presence of anti-Type III density enhanced phosphatase monoclonal antibodies and/or specific counterreceptors. Binding molecules are useful for purification of Type III density enhanced phosphatase polypeptides of the invention and for identifying cell types which express the polypeptide. Binding molecules are also useful for modulating (i.e., inhibiting, blocking or stimulating) the in vivo binding and/or signal transduction activities of Type III density enhanced phosphatases. 
     Hybridoma cell lines which produce antibodies specific for Type III density enhanced phosphatases are also comprehended by the invention. Techniques for producing hybridomas which secrete monoclonal antibodies are well known in the art. Hybridoma cell lines may be generated after immunizing an animal with a purified Type III density enhanced phosphatase, or variants thereof, or cells which express a Type III density enhanced phosphatase or a variant thereof on the extracellular membrane surface. Immunogen cell types include cells which express a Type III density enhanced phosphatase in vivo, or transfected or transformed prokaryotic or eukaryotic host cells which normally do not express the protein in vivo. 
     The value of the information contributed through the disclosure of the DNA and amino acid sequences of human DEP-1 is manifest. In one series of examples, the disclosed human DEP-1 cDNA sequence makes possible the isolation of the human DEP-1 genomic DNA sequence, including transcriptional control elements. Transcriptional control elements comprehended by the invention include, for example, promoter elements and enhancer elements, as well as elements which contribute to repression, or downregulation, of mRNA transcription. Control elements of this type may be 5&#39; DNA sequences or 3&#39; DNA sequences with respect to the protein-encoding structural gene sequences, and/or DNA sequences located in introns. The 5&#39; and/or 3&#39; control elements may be proximal and/or distal the protein-encoding sequences of the structural gene. Identification of DNA sequences which modulate mRNA transcription in turn permits the identification of agents which are capable of effecting transcriptional modulation. 
     In another aspect, identification of polynucleotides encoding other Type III density enhanced phosphatases, huDEP-1 allelic variants and heterologous species (e.g., rat or mouse) DNAs is also comprehended. Isolation of the huDEP-1 genomic DNA and heterologous species DNAs may be accomplished by standard nucleic acid hybridization techniques, under appropriately stringent conditions, using all or part of the DEP-1 DNA or RNA sequence as a probe to screen an appropriate library. Alternatively, polymerase chain reaction (PCR) using oligonucleotide primers that are designed based on the known nucleotide sequence can be used to amplify and identify other cDNA and genomic DNA sequences. Synthetic DNAs encoding Type III density enhanced phosphatase polypeptide, including fragments and other variants thereof, may be synthesized by conventional methods. 
     DNA sequence information of the invention also makes possible the development, by homologous recombination or &#34;knockout&#34; strategies [see, e.g., Capecchi, Science 244:1288-1292 (1989)], of rodents that fail to express a functional Type III density enhanced phosphatase polypeptide or that express a variant Type III density enhanced phosphatase polypeptide. Such rodents are useful as models for studying the activities of Type III density enhanced phosphatases and modulators thereof in vivo. 
     DNA and amino acid sequences of the invention also make possible the analysis of Type III density enhanced phosphatase regions which actively participate in counterreceptor binding, as well as sequences which may regulate, rather than actively participate in, binding. Identification of motifs which participate in transmembrane signal transduction is also comprehended by the invention. Also comprehended is identification of motifs which determine subcellular localization of the immature and mature Type III density enhanced phosphatase proteins. 
     DNA of the invention is also useful for the detection of cell types which express Type III density enhanced phosphatase polypeptides. Identification of such cell types may have significant ramifications for development of therapeutic and prophylactic agents. Standard nucleic acid hybridization techniques which utilize e.g., huDEP-1 DNA to detect corresponding RNAs, may be used to determine the constitutive level of Type III density enhanced phosphatase transcription within a cell as well as changes in the level of transcription in response to internal or external agents. Identification of agents which modify transcription, translation, and/or activity of Type III density enhanced phosphatases can, in turn, be assessed for potential therapeutic or prophylactic value. DNA of the invention also makes possible in situ hybridization of e.g., huDEP-1 DNA to cellular RNA, to determine the cellular localization of Type III density enhanced phosphatase specific messages within complex cell populations and tissues. 
     Polynucleotides of the present invention also provide a method whereby substrate or other molecules which interact with Type III density enhanced phosphatases can be identified. A presently preferred method for identifying interacting molecules comprises the steps of: a) transforming or transfecting appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain; b) an optional step of cotranforming or co-transfecting the same host cells with a protein tyrosine kinase (e.g., v-src, c-src or the like) in order to phosphorylate potential interacting components and/or substrates introduced as in (d) below; c) expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of e.g., a huDEP-1 isoform and either the DNA-binding domain or the activating domain of the transcription factor; d) expressing in the host cells a library of second hybrid DNA sequences encoding second fusions of part or all of putative DEP-1 isoform-binding proteins and either the activating domain or DNA binding domain of the transcription factor which is not incorporated in the first fusion; e) detecting binding of DEP-1 isoform-binding proteins to the DEP-1 isoform in a particular host cell by detecting the production of reporter gene product in the host cell; and f) isolating second hybrid DNA sequences encoding DEP-1 isoform-binding protein from the particular host cell. Variations of the method altering the order in which e.g., the huDEP-1 isoforms and putative huDEP-1 isoform-binding proteins are fused to transcription factor domains, either at the amino terminal or carboxy terminal end of the transcription factor domains, are contemplated. In a preferred method, the promoter is the ADHI promoter, the DNA-binding domain is the lexA DNA-binding domain, the activating domain is the GAL4 transactivation domain, the reporter gene is the lacZ gene and the host cell is a yeast host cell. Those of ordinary skill in the art will readily envision that any of a number of other reporter genes and host cells are easily amenable to this technique. Likewise, any of a number of transcription factors with distinct DNA binding and activating domains can be utilized in this procedure, either with both the DNA binding and activating domains derived from the same transcription factor, or from different, but compatible transcription factors. As another variation of this method, mutant DEP-1 polypeptides, wherein a cysteine residue in the catalytic domain has been substituted with a serine residue, can be employed in this technique. Mutations of this type have been demonstrated with other phosphatases to recognize and bind substrates, but do not dephosphorylate the substrate since the phosphatase is inactive as a result of the mutation. 
     An alternative identification method contemplated by the invention for detecting proteins which bind to a Type III density enhanced phosphatase isoform comprises the steps of: a) transforming or transfecting appropriate host cells with a hybrid DNA sequence encoding a fusion between a putative Type III density enhanced phosphatase isoform-binding protein and a ligand capable of high affinity binding to a specific counterreceptor; b) expressing the hybrid DNA sequence in the host cells under appropriate conditions; c) immobilizing fusion protein expressed by the host cells by exposing the fusion protein to the specific counterreceptor in immobilized form; d) contacting a Type III density enhanced phosphatase isoform with the immobilized fusion protein; and e) detecting the Type III density enhanced phosphatase isoform bound to the fusion protein using a reagent specific for the Type III density enhanced phosphatase isoform. Presently preferred ligands/counterreceptor combinations for practice of the method are glutathione-S-transferase/glutathione, hemagglutinin/hemagglutinin-specific antibody, polyhistidine/nickel and maltose-binding protein/amylose. 
     Additional methods to identify proteins which specifically interact with Type III density enhanced phosphatase (i.e., substrates, ligands, modulators, etc.) are also contemplated by the invention. In one example, purified and isolated Type III density enhanced phosphatase polypeptide (e.g., huDEP-1 polypeptide) can be covalently coupled to an immobilized support (i.e., column resins, beads, etc.) and incubated with cell lysates to permit protein/protein interactions. Proteins which interact with the immobilized DEP-1 polypeptide can then be eluted from the support with gradient washing techniques which are standard in the art. 
     As another example, protein overlay techniques can be employed. DNA from cells which either express e.g., huDEP-1 or express polypeptides which can modulated or bind to huDEP-1, can be isolated and a library constructed by standard methods. This library can then be expressed in a heterologous cell line and resulting colonies transferred to an immobilizing support. Expressed proteins from these colonies are then contacted with DEP-1 and incubated under appropriate conditions to permit DEP-1/protein interactions. The resulting Type III density enhanced phosphatase/protein complexes formed can be detected by incubation with a specific Type III density enhanced phosphatase antibody. Colonies which interact with the specific antibody contain DNA encoding a protein which interacts with the Type III density enhanced phosphatase. Alternatively, cell or tissue lysates may be employed in this technique, using cells or tissues which normally express DEP-1, or cells which have been previously transfected or transformed with DEP-1 encoding DNA. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof, reference being made to the drawing wherein: 
     FIGS. 1A through 1B are photographs of Northern blot analysis autoradiograms; and 
     FIG. 2 shows the density-dependent expression of DEP-1. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is illustrated by the following examples relating to the isolation and characterization of genes encoding Type III density enhanced phosphatase polypeptides. Example 1 relates to the isolation of cDNA encoding human DEP-1. Example 2 discusses the tissue distribution of huDEP-1 as determined by Northern blot analysis. Example 3 addresses the generation of antibodies specific for DEP-1 and fragments thereof. Example 4 demonstrates expression of a huDEP-1 cDNA clone in COS cells. Example 5 relates to detection of endogenous expression of huDEP-1 in fibroblast cells. Example 6 addresses expression of huDEP-1 as a function of cell culture density. Example 7 relates to identification of ligands of huDEP-1. Example 8 discusses identification of modulators and substrates of huDEP-1 activity. Example 9 details characterization of the genomic huDEP-1 DNA. 
     EXAMPLE 1 
     Isolation and Characterization of huDEP-1 cDNA 
     In initial efforts to isolate cDNA encoding a novel human phosphatase regulated by a cell density-dependent mechanism, PCR primers were synthesized based on conserved amino acid sequences common to many previously identified phosphatases. These primers were then used to amplify polynucleotides from a cDNA library, the resulting amplification products were sequenced, and these sequences compared to previously reported DNA sequences. 
     Degenerate primers, corresponding to conserved PTP amino acid sequences set out in SEQ ID NO: 3 and SEQ ID NO: 4, were synthesized and used to prime a PCR with a HeLa cell cDNA library as template. 
     KCAQYWP SEQ ID NO: 3 
     HCSAGIG SEQ ID NO: 4 
     The corresponding primers used in the PCR reaction are set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, employing nucleotide symbols according to 37 U.S.C. § 1.882. 
     5&#39;-AARTGYGCNCARTAYTGGCC-3&#39; SEQ ID NO: 5 
     3&#39;-GTRACRTCRCGNCCITADCC-5&#39; SEQ ID NO: 6 
     Sequencing of seventy-seven independent subclones revealed seven distinct sequences, six of which corresponded to PTPs for which DNA sequences had previously been published, and included PTP1B [Tonks, et al., J.Biol.Chem 263:6722-6730 (1988)], TCPTP [Cool, et al., Proc.Natl.Acad.Sci(USA) 86:5257-5261 (1989)], RPTPα [Krueger, et al., EMBO J. 9:3241-3252 (1990)], LAR [Streuli, et al., J.Exp.Med. 168:1523-1530 (1988)], PTPH1 [Yang and Tonks, Proc.Natl.Acad.Sci. (USA) 88:5949-5953 (1991)], and PTPμ [Gebbink, et al., FEBS Lett. 290:123-130 (1991)]. The seventh clone was determined to comprise a unique 300 bp PCR fragment and was used to screen an oligo-dT-primed HeLa cell cDNA library (Stratagene, La Jolla, Calif.) in an effort to isolate a corresponding full-length cDNA. Approximately 1.8×10 6  phage plaques were screened as previously described [Yang and Tonks, Proc.Natl.Acad.Sci. (USA) 88:5949-5953 (1991)] and twenty-four positive clones were identified. The largest insert, a 5.1 kb cDNA, was cloned into pUC119, sequenced by the dideoxy chain termination method, and found to contain an open reading frame of 4011 nucleotides encoding a novel receptor-like PTP of 1337 amino acids. The DNA sequence of the 5.1 kb insert is set out in SEQ ID NO: 1, and its predicted amino acid sequence is set out in SEQ ID NO: 2. This human density-enhanced PTP was designated huDEP-1. 
     The proposed initiating ATG codon of the huDEP-1 gene is flanked by a purine (G) at the -3 position and is thus in agreement with the Kozak rules for initiation [Kozak, J.Cell Biol. 108:229-241 (1989)]. There is an in-frame stop codon approximately 290 bp upstream of the predicted initiation site, and the initiating ATG is followed by a hydrophobic region that may serve as a signal sequence. Based on the statistical analysis of known cleavage sites for the signal peptidase [von Heijne, Nuc.Acids Res. 14:4683-4690 (1986)], the amino terminus of the mature huDEP-1 polypeptide is assigned to Gly 37 . A second hydrophobic region is found between amino acids 977 and 996, and is followed by a stretch of predominantly basic residues, characteristic of a stop transfer sequence. Therefore, an extracellular region of 940 amino acids and an intracellular portion of 341 amino acids are predicted for the mature huDEP-1 protein. The extracellular domain comprises eight FNIII domains, and thirty-three potential sites for N-linked glycosylation are predicted. Thus, huDEP-1 conforms to the RPTP Type III topography according to the nomenclature of Fischer et al., supra. Unlike most RPTPs which possess a tandem repeat of catalytic domains, the cytoplasmic region contains a single catalytic domain spanning amino acid residues 1060 through 1296. Human DEP-1 is therefore representative of an expanding group of RPTPs with a single catalytic domain that includes PTPβ [Krueger, et al., EMBO J. 9:3241-3252 (1990)], DPTP1OD of Drosophila [Tian, et al., Cell 76:675-685 (1991); Yang, et al., Cell 67:661-673 (1991)], DPTP4E of Drosophila [Oon, et al., J.Biol.Chem. 268:23964-23971 (1993)], and the recently described SAP-I enzyme [Matozaki, et al., J.Biol. Chem. 269:2075-2081 (1994)]. Amino acid sequence comparison of the catalytic domain of huDEP-1 with other PTP domains revealed huDEP-1 is most closely related to PTPβ and SAP-1. The sequence includes several Ser-Pro motifs, as well as potential sites for phosphorylation by casein kinase II. 
     EXAMPLE 2 
     Northern Analysis of huDEP-1 Tissue Distribution 
     Because the expression of PTPs has previously been demonstrated to be ubiquitous in eukaryotes, various human tissues were analyzed in order to determine the relative degree of huDEP-1 mRNA expression. 
     RNA Multi Tissue Northern blot filters (Clontech, Palo Alto, Calif.), containing immobilized RNA from various human tissues, were probed with a 1.6 kb HindIII/EcoRI fragment of the huDEP-1 cDNA previously radiolabeled to a specific activity of 1.5×10 6  cpm/ng using a Megaprime DNA labeling kit (Amersham, Arlington Heights, Ill.). This probe represented the entire length of the isolated huDEP-1 cDNA. Hybridization was performed for 16 hours at 65° C. in a hybridization buffer containing 0.5 M Na 2  HPO 4  , 7% SDS, 1 mM EDTA, and labeled probe at a concentration of 10 6  cpm/ml. Filters were then washed 5 times at 65° C. in 40 mM Na 2  HPO 4 , 1% SDS, and 1 mM EDTA. The membrane was then subjected to autoradiography. The results are presented in FIGS. 1A and 1B, wherein the human tissue source of immobilized RNA is as follows. In FIG. 1A, RNA in lane 2 is from heart, lane 3 from brian, lane 4 from placenta, lane 5 from lung, lane 6 from liver, lane 7 from skeletal muscle, lane 8 from kidney, and lane 9 from pancreas. In FIG. 1B, RNA in lane 2 is from spleen, lane 3 from thymus, lane 4 from prostrate, lane 5 from testis, lane 6 from ovary, lane 7 from small intestines, lane 8 from colon, and lane 9 from peripheral blood leukocyte. 
     Northern analysis indicated that huDEP-1 is expressed in most tissues analyzed, with particularly high mRNA levels detected in placenta, kidney, spleen and peripheral blood leukocytes. 
     EXAMPLE 3 
     Generation of huDEP-1 Polyclonal Antibodies 
     Two peptides, unique to huDEP-1 and corresponding to amino acid residues 1297 through 1315 and residues 1321 through 1334 in SEQ ID NO: 2 (downstream from the catalytic region) were synthesized with an additional amino terminal cysteine residue and conjugated to rabbit serum albumin (RSA) with m-maleimido benzoic acid N-hydroxysuccinimide ester (MBS)(Pierce, Rockford, Ill.). Immunization protocols with these peptides were performed by Cocalico Biologicals (Reamstown, Pa.). Initially, a pre-bleed of the rabbits was performed prior to immunization. The first immunization included Freund&#39;s complete adjuvant and 500 μg conjugated peptide or 100 μg purified peptide. All subsequent immunizations, performed four weeks after the previous injection, included Freund&#39;s incomplete adjuvant with the same amount of protein. Bleeds were conducted seven to ten days after the immunizations. 
     For affinity purification of the antibodies, huDEP-1 peptide conjugated to RSA with MBS, was coupled to CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden). Antiserum was diluted 10-fold in 10 mM Tris-HCl, pH 7.5, and incubated overnight with the affinity matrix. After washing, bound antibodies were eluted from the resin with 100 mM glycine, pH 2.5. 
     The antibody generated against conjugated amino acid residues 1297 through 1315 was designated anti-CSH-241, and the antibody raised against the conjugated peptide corresponding to amino acid residues 1321 through 1334 was designated anti-CSH-243. 
     EXAMPLE 4 
     Expression of huDEP-1 by Transfected Host Cells 
     To study the protein product of the huDEP-1 cDNA, the 5.1 kb EcoRI insert was cloned into the expression vector pMT2 [Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) pp 16.17-16.22] and transfected into COS cells grown in DMEM supplemented with 10% FCS. Transfections were performed employing calcium phosphate techniques [Sambrook, et al (1989) pp. 16.32-16.40, supra] and cell lysates were prepared forty-eight hours after transfection from both transfected and untransfected COS cells. Lysates were subjected to analysis by immunoblotting using anti-CSH-243 antibody, and PTP assays of immune complexes as addressed below. 
     In immunoblotting experiments, preparation of cell lysates and electrophoresis were performed. Protein concentration was determined using BioRad protein assay solutions. After semi-dry electrophoretic transfer to nitro-cellulose, the membranes were blocked in 500 mM NaCl, 20 mM Tris, pH 7.5, 0.05% Tween-20 (TTBS) with 5% dry milk. After washing in TTBS and incubation with secondary antibodies (Amersham), enhanced chemiluminescence (ECL) protocols (Amersham) were performed as described by the manufacturer to facilitate detection. 
     For immune complex PTP assays, 60 μg of cell lysate were immunoprecipitated with 20 μl of anti-CSH-243 antisera or preimmune rabbit serum bound to 25 μl of Protein-A Sepharose (Pharmacia). After overnight incubation at 4° C., the immune complexes were washed three times in washing buffer (1% Triton X-100, 150 mM NaCl, 20 mM Hepes, pH 7.5, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mM benzamidine, and 1 mM DTT) and once in assay buffer (25 mM imidazole, pH 7.2, 0.5 mg/ml BSA, and 1 mM DTT). Protein-A Sepharose immune complexes were then resuspended in 150 μl of assay buffer and assayed for PTP activity as triplicates. Assays were performed for 6 minutes at 30° C. in a total volume of 60 μl using 3 μM [ 32  P-Tyr]-reduced carboxymethylated (RCM) lysozyme as substrate [Flint, et al., EMBO J. 12:1937-1946 (1993)]. 
     Affinity-purified anti-CSH-243 antibodies specifically detected a protein of 180 kD molecular weight in lysates from transfected cells. Furthermore, when immune complexes were analyzed for PTP activity, almost 10-fold higher activity was detected in anti-CSH-243 immune complexes from the transfected cells compared to the untransfected cells. This PTP activity was largely absent in immune complexes derived from immunoprecipitations with blocked antiserum or preimmune serum. It was concluded that the huDEP-1 cDNA encodes a 180 kD protein with intrinsic PTP activity. 
     EXAMPLE 5 
     Endogenous Expression of huDEP-1 
     To characterize endogenously expressed huDEP-1, lysates from different cell lines including CEM (ATCC CCL 119), HeLa (ATCC CCL 2), 293 (ATCC CRL 1573), Jurkat (ATCC TIB 152), K562 (ATCC CCL243), HL6O (ATCC CCL 240), WI38 (ATCC CCL 75) and AG 1518 (Coriell Cell Repositories, Camden, N.J.) were analyzed by immunoblotting with antibody anti-CSH-243 as described in Example 4. 
     WI38 cells, a diploid fetal lung fibroblast-like cell line with finite life span, showed the highest expression. Similar levels of expression were also detected in AG 1518 foreskin fibroblast cells. 
     To further examine the expression of huDEP-1, lysates from metabolically labeled cells were analyzed by immunoprecipitation and SDS-gel electrophoresis. Confluent cultures of WI38 and AG 1518 cells were metabolically labeled for four hours in methionine-free DMEM supplemented with 1 mg/ml bovine serum albumin (BSA) and 0.15 mCi/ml Translabel (ICN, Costa Mesa, Calif.). Cells were lysed in 0.5% DOC, 0.5% Triton X-100, 150 mM NaCl, 20 mM Hepes, pH 7.5, 5μg/ml aprotinin, 5 μg/ml leupeptin, 1 mM benzamidine, 1 mM DTT (lysis buffer) and lysates were centrifuged at 15,000×g for 15 minutes. Lysates corresponding to approximately 2×10 6  cells were then incubated with 20 μl of anti-CSH-243 or anti-CSH-243. After incubation for four hours at 4° C., 50 μl of a 1:1 Protein-A-Sepharose slurry was added to bind the protein/antibody complexes and incubation continued for 60 minutes. Immune complexes adsorbed to the Protein-A-Sepharose were collected by centrifugation and washed three times in 1% Triton X-100, 150 mM NaCl, 20 mM Hepes, pH 7.5, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mM benzamidine, 1 mM DTT (washing buffer) and once in 20 mM Tris, pH 7.5. Samples were eluted from the resin by incubation at 95° C. for 3 minutes in reducing SDS-sample buffer and analyzed by SDS-gel electrophoresis on 7% gels, followed by fluorography. 
     In both WI38 and AG 1518 cells, a protein of 180 kD was recognized specifically by the unblocked antisera. Anti-CSH-243 antisera immunoprecipitation with WI38 cell lysate also yielded significantly higher amounts (approximately 10 to 20 fold higher) of activity than precipitations with pre-immune serum or antiserum that had been previously incubated with 200 μg/ml of peptide-conjugate. 
     It appears that huDEP-1 is a phosphoprotein in vivo because the fact that the anti-CSH-243 antibody was capable of immunoprecipitating a 180 kD [ 32  P]-labeled protein from a cell lysate of WI38 cells which had been metabolically labelled with [ 32  P]-inorganic phosphate. 
     EXAMPLE 6 
     Cell Density-Dependent Expression and Activity of huDEP-1 
     WI38 cell lysates from sparse (less than 7,000 cells /cm 2 ) or dense (more than 25,000 cells/cm 2 ) cultures were compared for levels of expressed huDEP-1 protein by immunoblotting with anti-CSH-243 antibody as described in Example 4. A dramatic, ten- to twenty-fold increase in huDEP-1 expression was detected in dense cell cultures as shown in FIG. 2. Since 3 μg of total cell lysate from more confluent culture gave a relatively strong signal, and 15 μg of lysates from sparse cultures were below detection, it was estimated that at least 10-fold higher amounts of huDEP-1 are present in cells from dense cultures. Similar results were obtained with anti-CSH-241. When the amounts of PTP1B in cell lysates from sparse and dense cells were compared using an anti-PTP1B monoclonal antibody FG6 (Oncogene Science, Uniondale, N.Y.), no difference was observed. The observed effects on huDEP-1 expression are not restricted to WI38 cells as similar results were obtained in AG 1518 cells. 
     In order to determine if enzyme activity was also regulated by a density-dependent mechanism, huDEP-1 and PTB1B immune complexes and total cell lysates from both sparse and dense WI38 and AG 1518 cell cultures were also analyzed for phosphatase activity using the PTP assay. For immune complex PTP assays, 60 μg of cell lysate were immunoprecipitated with 20 μl of anti-CSH-243 antisera (with or without pretreatment with antigen) or preimmune serum bound to 25 μl of Protein-A Sepharose. After incubation overnight at 4° C., immune complexes were washed three times in washing buffer and once in 25 mM imidazole, pH 7.2, 0.5 mg/ml BSA, 1 mM DTT (assay buffer). Protein-A-Sepharose immune complexes were then suspended in 150 μl of assay buffer and assayed for PTP activity as triplicates. Assays were performed for 6 minutes at 30° C. in a total volume of 60 μl using 3 μM [ 32  P-Tyr] RCM lysozyme as substrate [Flint, et al., supra]. 
     In agreement with the increased huDEP-1 protein expression demonstrated in the immunoblotting experiments, huDEP-1 enzyme activity also increased in the dense cell cultures. The observed increase in activity in huDEP-1/CSH-243 immunoprecipitates from dense cultures (approximately two-to three-fold) was not as great as the observed increase in protein expression in dense cultures, most likely due to incomplete precipitation of all of the PTP using anti-CSH-243 antisera. No difference was observed in activity of PTP1B/FG6 immunoprecipitates or total cell lysates from sparse and dense cell cultures. 
     Finally, to investigate the kinetics of the density-dependent upregulation of huDEP-1 expression, lysates of WI38 and AG 1518 cells at intermediate cell densities were included in the immunoblotting analysis. The highest expression was found in cells at saturation density, however, at intermediate densities an increase in expression with respect to sparse cell cultures was also observed. Thus, the upregulation of huDEP-1 expression appears to be initiated prior to saturation density and not a result of growth arrest. 
     While the precise mechanism by which huDEP-1 expression is induced remains unclear, the demonstration that expression was induced in two distinct cell lines as cells approach confluence suggests involvement of huDEP-1 in promoting net dephosphorylation of proteins, countering the effects of growth promoting PTK activity. This possibility, in combination with the broad distribution of huDEP-1 expression, suggests that huDEP-1 may be involved in a general mechanism for contact inhibition of cell growth. 
     EXAMPLE 7 
     Identification of DEP-1 Ligands 
     The possibility that DEP-1 functions as an adhesion molecule will be tested using the Sf9 cell system [Brady-Kalnay, et al., J.Cell Biol. 122:961-972 (1993)] following transfection with DEP-1 cDNA. In addition to studies following transient expression, stable cell lines overexpressing DEP-1 will be generated. 
     If DEP-1 functions as an adhesion molecule, the extracellular counterreceptor(s) will be identified. One possibility is that, like PTPμ, DEP-1 binding is homophilic, where one DEP-1 molecule binds another DEP-1 molecule on an adjacent cell. Alternatively, DEP-1 specifically recognize a non-DEP-1 molecule in a heterophilic binding mechanism. 
     In addition, a number of deletion and site-directed mutagenesis strategies well known in the art will be applied to identify the important segments in the protein that confer binding specificity. Analysis of 2D gels of proteins that react with anti-phosphotyrosine antibodies, for example monoclonal antibody 4G10 (UBI, Lake Placid, N.Y.), will be used to initiate studies as to the effect on activity of engagement of the extracellular segment of the PTP in either homophilic binding interactions or antibody binding. 
     Use of &#34;epitope&#34; library technology [Scott and Smith, Science 249:386-390 (1990)] will be employed to identify peptide sequences that interact with DEP-1. This approach will prove particularly useful in the search for ligands for DEP-1 whose extracellular segment, comprising multiple FNIII repeats, may bind low M r  factors. 
     Protein:protein interactions have previously been reported for FNIII sequences and specific binding proteins, and this information will be utilized in several approaches to identify proteins which specifically interact with the extracellular domain of DEP-1. Specifically, protein:protein interactions will be investigated in cell &#34;panning&#34; experiments [Seed and Aruffo, Proc.Natl.Acad.Sci. (USA) 84:3365-3369 (1987)], gel overlay assays [Hirsch, et al., J.Biol.Chem. 267:2131-2134 (1992); Carr and Scott, Trends in Biochemical Sci. 17:246-249 (1992)], band shift analysis [Carr, et al., J.Biol.Chem. 267:13376-13382 (1992)], affinity chromatography, screening of expression libraries [Young and Davis, Proc.Natl.Acad.Sci. (USA) 80:1194-1198(1983)], etc. 
     EXAMPLE 8 
     Identification of Modulators/Substrates of DEP-1 
     Potential substrates of predicted physiological relevance will be tested for activity against the catalytic domain in vitro. 
     In addition, yeast screening systems [Fields and Song, Nature 340:245-246 (1989); Yang, et al., Science 257:6810682 (1992); Vojtek, et al., Cell 74:205-214 (1993)] will be utilized, particularly with reference to co-expression with a protein tyrosine kinase, for example, v-src or c-src, to isolate proteins with the capacity to regulate DEP-1 activity. 
     In a further attempt to identify substrates for DEP-1, a mutant form in which the cysteinyl residues of the active center has been replaced by serine will be expressed. Recent studies suggest that substrates bind to and remain complexed with the inactive phosphatase. The mutant PTP is capable of binding substrate molecules but traps them in a &#34;dead end&#34; complex that can be isolated by standard immunoprecipitation techniques [Sun, et al., Cell 75:487-493 (1993)]. Potential substrates may be co-immunoprecipitated with the mutant PTP from  35  S-labeled cells. Alternatively, wild-type, or native, DEP-1 enzyme may be utilized in this technique. Initial studies in this direction may make use of chimeric molecules, for which antibodies to the extracellular growth factor binding segment are commercially available, while antibodies are raised to the bona fide DEP-1 sequences. 
     EXAMPLE 9 
     Characterization of the Genomic DEP-1 Gene 
     Isolation of the cDNA sequences for DEP-1 will permit the isolation and purification of the corresponding genomic sequences for DEP-1. In preliminary work, it has been demonstrated that huDEP-1 mapped to human chromosome 11p, band 11.2 or the interface of 11.2 and 11.3. Isolation of these genomic DEP-1 sequences will permit the identification of putative regulatory sequences for DEP-1 transcription, and presumably identification of trans-acting transcriptional modulators of DEP-1 expression. In addition, isolation and purification of the human genomic clone will permit screening of libraries in other species to determine if homologous counterparts exist in the species. Identification of a homologous counterpart in mice will be of particular importance because of the possibility of generating a knockout strain. Mouse strains which do not express a particular protein are of considerable importance in that they permit determination of indications associated with absence of the protein in a living animal. 
     While the present invention has been described in terms of specific methods and compositions, it is understood that variations and modifications will occur to those skilled in the art. Therefore, only such limitations as appear in the claims should be placed on the invention. 
     
         __________________________________________________________________________#             SEQUENCE LISTING   - -  - - (1) GENERAL INFORMATION:   - -    (iii) NUMBER OF SEQUENCES: 6   - -  - - (2) INFORMATION FOR SEQ ID NO:1:   - -      (i) SEQUENCE CHARACTERISTICS:       (A) LENGTH: 5117 base - #pairs       (B) TYPE: nucleic acid       (C) STRANDEDNESS: single       (D) TOPOLOGY: linear   - -     (ii) MOLECULE TYPE: cDNA   - -     (ix) FEATURE:       (A) NAME/KEY: CDS       (B) LOCATION: 350..4364   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:   - - CCCCAGCCGC ATGACGCGCG GAGGAGGCAG CGGGACGAGC GCGGGAGCCG GG -#ACCGGGTA     60   - - GCCGCGCGCT GGGGGTGGGC GCCGCTCGCT CCGCCCCGCG AAGCCCCTGC GC -#GCTCAGGG    120   - - ACGCGGCCCC CCCGCGGCAG CCGCGCTAGG CTCCGGCGTG TGGCCGCGGC CG -#CCGCCGCG    180   - - CTGCCATGTC TCCGGGGAAG CCGGGGCGGG CGGAGCGGGG ACGAGGCGGA CC -#GGCTGGCG    240   - - GAGGAGGAGG CGAAGGAGAC GGCAGGAGGC GGCGACGACG GTGCCCGGGC TC -#GGGCGCAC    300   - - GGCGGGGCCC GATTCGCGCG TCCGGGGCAC GTTCCAGGGC GCGCGGGGC ATG - # AAG 355              - #                  - #                  - #Met Lys              - #                  - #                  - #  1  - - CCG GCG GCG CGG GAG GCG CGG CTG CCT CCG CG - #C TCG CCC GGG CTG CGC403 Pro Ala Ala Arg Glu Ala Arg Leu Pro Pro Ar - #g Ser Pro Gly Leu Arg      5        - #          10        - #          15  - - TGG GCG CTG CCG CTG CTG CTG CTG CTG CTG CG - #C CTG GGC CAG ATC CTG451 Trp Ala Leu Pro Leu Leu Leu Leu Leu Leu Ar - #g Leu Gly Gln Ile Leu 20             - #     25             - #     30  - - TGC GCA GGT GGC ACC CCT AGT CCA ATT CCT GA - #C CCT TCA GTA GCA ACT499 Cys Ala Gly Gly Thr Pro Ser Pro Ile Pro As - #p Pro Ser Val Ala Thr  35                 - # 40                 - # 45                 - # 50  - - GTT GCC ACA GGG GAA AAT GGC ATA ACG CAG AT - #C AGC AGT ACA GCA GAA547 Val Ala Thr Gly Glu Asn Gly Ile Thr Gln Il - #e Ser Ser Thr Ala Glu             55 - #                 60 - #                 65  - - TCC TTT CAT AAA CAG AAT GGA ACT GGA ACA CC - #T CAG GTG GAA ACA AAC595 Ser Phe His Lys Gln Asn Gly Thr Gly Thr Pr - #o Gln Val Glu Thr Asn         70     - #             75     - #             80  - - ACC AGT GAG GAT GGT GAA AGC TCT GGA GCC AA - #C GAT AGT TTA AGA ACA643 Thr Ser Glu Asp Gly Glu Ser Ser Gly Ala As - #n Asp Ser Leu Arg Thr     85         - #         90         - #         95  - - CCT GAA CAA GGA TCT AAT GGG ACT GAT GGG GC - #A TCT CAA AAA ACT CCC691 Pro Glu Gln Gly Ser Asn Gly Thr Asp Gly Al - #a Ser Gln Lys Thr Pro100              - #   105              - #   110  - - AGT AGC ACT GGG CCC AGT CCT GTG TTT GAC AT - #T AAA GCT GTT TCC ATC739 Ser Ser Thr Gly Pro Ser Pro Val Phe Asp Il - #e Lys Ala Val Ser Ile 115                 1 - #20                 1 - #25                 1 -#30   - - AGT CCA ACC AAT GTG ATC TTA ACT TGG AAA AG - #T AAT GAC ACA GCTGCT      787  Ser Pro Thr Asn Val Ile Leu Thr Trp Lys Se - #r Asn Asp Thr Ala Ala            135  - #               140  - #               145  - - TCT GAG TAC AAG TAT GTA GTA AAG CAT AAG AT - #G GAA AAT GAG AAG ACA835 Ser Glu Tyr Lys Tyr Val Val Lys His Lys Me - #t Glu Asn Glu Lys Thr        150      - #           155      - #           160  - - ATT ACT GTT GTG CAT CAA CCA TGG TGT AAC AT - #C ACA GGC TTA CGT CCA883 Ile Thr Val Val His Gln Pro Trp Cys Asn Il - #e Thr Gly Leu Arg Pro    165          - #       170          - #       175  - - GCG ACT TCA TAT GTA TTC TCC ATC ACT CCA GG - #A ATA GGC AAT GAG ACT931 Ala Thr Ser Tyr Val Phe Ser Ile Thr Pro Gl - #y Ile Gly Asn Glu Thr180              - #   185              - #   190  - - TGG GGA GAT CCC AGA GTC ATA AAA GTC ATC AC - #A GAG CCG ATC CCA GTT979 Trp Gly Asp Pro Arg Val Ile Lys Val Ile Th - #r Glu Pro Ile Pro Val 195                 2 - #00                 2 - #05                 2 -#10   - - TCT GAT CTC CGT GTT GCT CAC GGG TGT GAG GA - #A GGC TGC TCT CTCTCC     1027  Ser Asp Leu Arg Val Ala His Gly Cys Glu Gl - #u Gly Cys Ser Leu Ser            215  - #               220  - #               225  - - TGG AGC AAT GGC AAT GGC ACC GCC TCC TGC CG - #G GTT CTT CTT GAA AGC    1075 Trp Ser Asn Gly Asn Gly Thr Ala Ser Cys Ar - #g Val Leu Leu Glu Ser        230      - #           235      - #           240  - - ATT GGA AGC CAT GAG GAG TTG ACT CAA GAC TC - #A AGA CTT CAG GTC AAT    1123 Ile Gly Ser His Glu Glu Leu Thr Gln Asp Se - #r Arg Leu Gln Val Asn    245          - #       250          - #       255  - - ATC TCG GAC CTG AAG CCA GGG GTT CAA TAC AA - #C ATC AAC CCG TAT CTT    1171 Ile Ser Asp Leu Lys Pro Gly Val Gln Tyr As - #n Ile Asn Pro Tyr Leu260              - #   265              - #   270  - - CTA CAA TCA AAT AAG ACA AAG GGA GAC CCC TT - #G GCA CAG AAG GTG GCT    1219 Leu Gln Ser Asn Lys Thr Lys Gly Asp Pro Le - #u Ala Gln Lys Val Ala 275                 2 - #80                 2 - #85                 2 -#90   - - TGG ATG CCA GCA ATA CAG AGA GAA GCC GGG CA - #G GGA GCC CCA CCGCCC     1267  Trp Met Pro Ala Ile Gln Arg Glu Ala Gly Gl - #n Gly Ala Pro Pro Pro            295  - #               300  - #               305  - - CTG TGC ATG ATG AGT CCC TTC GTG GGA CCT GT - #G GAC CCA TCC TCC GGC    1315 Leu Cys Met Met Ser Pro Phe Val Gly Pro Va - #l Asp Pro Ser Ser Gly        310      - #           315      - #           320  - - CAG CAG TCC CGA GAC ACG GAA GTC CTG CTT GT - #C GGG TTA GAG CCT GGC    1363 Gln Gln Ser Arg Asp Thr Glu Val Leu Leu Va - #l Gly Leu Glu Pro Gly    325          - #       330          - #       335  - - ACC CGA TAC AAT GCC ACC GTT TAT TCC CAA GC - #A GCG AAT GGC ACA GAA    1411 Thr Arg Tyr Asn Ala Thr Val Tyr Ser Gln Al - #a Ala Asn Gly Thr Glu340              - #   345              - #   350  - - GGA CAG CCC CAG GCC ATA GAG TTC AGG ACA AA - #T GCT ATT CAG GTT TTT    1459 Gly Gln Pro Gln Ala Ile Glu Phe Arg Thr As - #n Ala Ile Gln Val Phe 355                 3 - #60                 3 - #65                 3 -#70   - - GAC GTC ACC GCT GTG AAC ATC AGT GCC ACA AG - #C CTG ACC CTG ATCTGG     1507  Asp Val Thr Ala Val Asn Ile Ser Ala Thr Se - #r Leu Thr Leu Ile Trp            375  - #               380  - #               385  - - AAA GTC AGC GAT AAC GAG TCG TCA TCT AAC TA - #T ACC TAC AAG ATA CAT    1555 Lys Val Ser Asp Asn Glu Ser Ser Ser Asn Ty - #r Thr Tyr Lys Ile His        390      - #           395      - #           400  - - GTG GCG GGG GAG ACA GAT TCT TCC AAT CTC AA - #C GTC AGT GAG CCT CGC    1603 Val Ala Gly Glu Thr Asp Ser Ser Asn Leu As - #n Val Ser Glu Pro Arg    405          - #       410          - #       415  - - GCT GTC ATC CCC GGA CTC CGC TCC AGC ACC TT - #C TAC AAC ATC ACA GTG    1651 Ala Val Ile Pro Gly Leu Arg Ser Ser Thr Ph - #e Tyr Asn Ile Thr Val420              - #   425              - #   430  - - TGT CCT GTC CTA GGT GAC ATC GAG GGC ACG CC - #G GGC TTC CTC CAA GTG    1699 Cys Pro Val Leu Gly Asp Ile Glu Gly Thr Pr - #o Gly Phe Leu Gln Val 435                 4 - #40                 4 - #45                 4 -#50   - - CAC ACC CCC CCT GTT CCA GTT TCT GAC TTC CG - #A GTG ACA GTG GTCAGC     1747  His Thr Pro Pro Val Pro Val Ser Asp Phe Ar - #g Val Thr Val Val Ser            455  - #               460  - #               465  - - ACG ACG GAG ATC GGC TTA GCA TGG AGC AGC CA - #T GAT GCA GAA TCA TTT    1795 Thr Thr Glu Ile Gly Leu Ala Trp Ser Ser Hi - #s Asp Ala Glu Ser Phe        470      - #           475      - #           480  - - CAG ATG CAT ATC ACA CAG GAG GGA GCT GGC AA - #T TCT CGG GTA GAA ATA    1843 Gln Met His Ile Thr Gln Glu Gly Ala Gly As - #n Ser Arg Val Glu Ile    485          - #       490          - #       495  - - ACC ACC AAC CAA AGT ATT ATC ATT GGT GGC TT - #G TTC CCT GGA ACC AAG    1891 Thr Thr Asn Gln Ser Ile Ile Ile Gly Gly Le - #u Phe Pro Gly Thr Lys500              - #   505              - #   510  - - TAT TGC TTT GAA ATA GTT CCA AAA GGA CCA AA - #T GGG ACT GAA GGG GCA    1939 Tyr Cys Phe Glu Ile Val Pro Lys Gly Pro As - #n Gly Thr Glu Gly Ala 515                 5 - #20                 5 - #25                 5 -#30   - - TCT CGG ACA GTT TGC AAT AGA ACT GTT CCC AG - #T GCA GTG TTT GACATC     1987  Ser Arg Thr Val Cys Asn Arg Thr Val Pro Se - #r Ala Val Phe Asp Ile            535  - #               540  - #               545  - - CAC GTG GTC TAC GTC ACC ACC ACG GAG ATG TG - #G CTG GAC TGG AAG AGC    2035 His Val Val Tyr Val Thr Thr Thr Glu Met Tr - #p Leu Asp Trp Lys Ser        550      - #           555      - #           560  - - CCT GAC GGT GCT TCC GAG TAT GTC TAC CAT TT - #A GTC ATA GAG TCC AAG    2083 Pro Asp Gly Ala Ser Glu Tyr Val Tyr His Le - #u Val Ile Glu Ser Lys    565          - #       570          - #       575  - - CAT GGC TCT AAC CAC ACA AGC ACG TAT GAC AA - #A GCG ATT ACT CTC CAG    2131 His Gly Ser Asn His Thr Ser Thr Tyr Asp Ly - #s Ala Ile Thr Leu Gln580              - #   585              - #   590  - - GGC CTG ATT CCG GGC ACC TTA TAT AAC ATC AC - #C ATC TCT CCA GAA GTG    2179 Gly Leu Ile Pro Gly Thr Leu Tyr Asn Ile Th - #r Ile Ser Pro Glu Val 595                 6 - #00                 6 - #05                 6 -#10   - - GAC CAC GTC TGG GGG GAC CCC AAC TCC ACT GC - #A CAG TAC ACA CGGCCC     2227  Asp His Val Trp Gly Asp Pro Asn Ser Thr Al - #a Gln Tyr Thr Arg Pro            615  - #               620  - #               625  - - AGC AAT GTG TCC AAC ATT GAT GTA AGT ACC AA - #C ACC ACA GCA GCA ACT    2275 Ser Asn Val Ser Asn Ile Asp Val Ser Thr As - #n Thr Thr Ala Ala Thr        630      - #           635      - #           640  - - TTA AGT TGG CAG AAC TTT GAT GAC GCC TCT CC - #C ACG TAC TCC TAC TGC    2323 Leu Ser Trp Gln Asn Phe Asp Asp Ala Ser Pr - #o Thr Tyr Ser Tyr Cys    645          - #       650          - #       655  - - CTT CTT ATT GAG AAG GCT GGA AAT TCC AGC AA - #C GCA ACA CAA GTA GTC    2371 Leu Leu Ile Glu Lys Ala Gly Asn Ser Ser As - #n Ala Thr Gln Val Val660              - #   665              - #   670  - - ACG GAC ATT GGA ATT ACT GAC GCT ACA GTC AC - #T GAA TTA ATA CCT GGC    2419 Thr Asp Ile Gly Ile Thr Asp Ala Thr Val Th - #r Glu Leu Ile Pro Gly 675                 6 - #80                 6 - #85                 6 -#90   - - TCA TCA TAC ACA GTG GAG CTC TTT GCA CAA GT - #A GGG GAT GGG ATCAAG     2467  Ser Ser Tyr Thr Val Glu Leu Phe Ala Gln Va - #l Gly Asp Gly Ile Lys            695  - #               700  - #               705  - - TCA CTG GAA CCT GGC CGG AAG TCA TTC TGT AC - #A GAT CCT GCG TCC ATG    2515 Ser Leu Glu Pro Gly Arg Lys Ser Phe Cys Th - #r Asp Pro Ala Ser Met        710      - #           715      - #           720  - - GCC TCC TTC GAC TGC GAA GTG GTC CCC AAA GA - #G CCA GCC CTG GTT CTC    2563 Ala Ser Phe Asp Cys Glu Val Val Pro Lys Gl - #u Pro Ala Leu Val Leu    725          - #       730          - #       735  - - AAA TGG ACC TGC CCT CCT GGC GCC AAT GCA GG - #C TTT GAG CTG GAG GTC    2611 Lys Trp Thr Cys Pro Pro Gly Ala Asn Ala Gl - #y Phe Glu Leu Glu Val740              - #   745              - #   750  - - AGC AGT GGA GCC TGG AAC AAT GCG ACC CAC CT - #G GAG AGC TGC TCC TCT    2659 Ser Ser Gly Ala Trp Asn Asn Ala Thr His Le - #u Glu Ser Cys Ser Ser 755                 7 - #60                 7 - #65                 7 -#70   - - GAG AAT GGC ACT GAG TAT AGA ACG GAA GTC AC - #G TAT TTG AAT TTTTCT     2707  Glu Asn Gly Thr Glu Tyr Arg Thr Glu Val Th - #r Tyr Leu Asn Phe Ser            775  - #               780  - #               785  - - ACC TCG TAC AAC ATC AGC ATC ACC ACT GTG TC - #C TGT GGA AAG ATG GCA    2755 Thr Ser Tyr Asn Ile Ser Ile Thr Thr Val Se - #r Cys Gly Lys Met Ala        790      - #           795      - #           800  - - GCC CCC ACC CGG AAC ACC TGC ACT ACT GGC AT - #C ACA GAT CCC CCT CCT    2803 Ala Pro Thr Arg Asn Thr Cys Thr Thr Gly Il - #e Thr Asp Pro Pro Pro    805          - #       810          - #       815  - - CCA GAT GGA TCC CCT AAT ATT ACA TCT GTC AG - #T CAC AAT TCA GTA AAG    2851 Pro Asp Gly Ser Pro Asn Ile Thr Ser Val Se - #r His Asn Ser Val Lys820              - #   825              - #   830  - - GTC AAG TTC AGT GGA TTT GAA GCC AGC CAC GG - #A CCC ATC AAA GCC TAT    2899 Val Lys Phe Ser Gly Phe Glu Ala Ser His Gl - #y Pro Ile Lys Ala Tyr 835                 8 - #40                 8 - #45                 8 -#50   - - GCT GTC ATT CTC ACC ACC GGG GAA GCT GGT CA - #C CCT TCT GCA GATGTC     2947  Ala Val Ile Leu Thr Thr Gly Glu Ala Gly Hi - #s Pro Ser Ala Asp Val            855  - #               860  - #               865  - - CTG AAA TAC ACG TAT GAC GAT TTC AAA AAG GG - #A GCC TCA GAT ACT TAT    2995 Leu Lys Tyr Thr Tyr Asp Asp Phe Lys Lys Gl - #y Ala Ser Asp Thr Tyr        870      - #           875      - #           880  - - GTG ACA TAC CTC ATA AGA ACA GAA GAA AAG GG - #A CGT TCT CAG AGC TTG    3043 Val Thr Tyr Leu Ile Arg Thr Glu Glu Lys Gl - #y Arg Ser Gln Ser Leu    885          - #       890          - #       895  - - TCT GAA GTT TTG AAA TAT GAA ATT GAC GTT GG - #G AAT GAG TCA ACC ACA    3091 Ser Glu Val Leu Lys Tyr Glu Ile Asp Val Gl - #y Asn Glu Ser Thr Thr900              - #   905              - #   910  - - CTT GGT TAT TAC AAT GGG AAG CTG GAA CCT CT - #G GGC TCC TAC CGG GCT    3139 Leu Gly Tyr Tyr Asn Gly Lys Leu Glu Pro Le - #u Gly Ser Tyr Arg Ala 915                 9 - #20                 9 - #25                 9 -#30   - - TGT GTG GCT GGC TTC ACC AAC ATT ACC TTC CA - #C CCT CAA AAC AAGGGG     3187  Cys Val Ala Gly Phe Thr Asn Ile Thr Phe Hi - #s Pro Gln Asn Lys Gly            935  - #               940  - #               945  - - CTC ATT GAT GGG GCT GAG AGC TAT GTG TCC TT - #C AGT CGC TAC TCA GAT    3235 Leu Ile Asp Gly Ala Glu Ser Tyr Val Ser Ph - #e Ser Arg Tyr Ser Asp        950      - #           955      - #           960  - - GCT GTT TCC TTG CCC CAG GAT CCA GGT GTC AT - #C TGT GGA GCG GTT TTT    3283 Ala Val Ser Leu Pro Gln Asp Pro Gly Val Il - #e Cys Gly Ala Val Phe    965          - #       970          - #       975  - - GGC TGT ATC TTT GGT GCC CTG GTT ATT GTG AC - #T GTG GGA GGC TTC ATC    3331 Gly Cys Ile Phe Gly Ala Leu Val Ile Val Th - #r Val Gly Gly Phe Ile980              - #   985              - #   990  - - TTC TGG AGA AAG AAG AGG AAA GAT GCA AAG AA - #T AAT GAA GTG TCC TTT    3379 Phe Trp Arg Lys Lys Arg Lys Asp Ala Lys As - #n Asn Glu Val Ser Phe 995                 1 - #000                1005 - #                1010  - - TCT CAA ATT AAA CCT AAA AAA TCT AAG TTA AT - #C AGA GTG GAG AAT TTT    3427 Ser Gln Ile Lys Pro Lys Lys Ser Lys Leu Il - #e Arg Val Glu Asn Phe            1015 - #               1020  - #              1025  - - GAG GCC TAC TTC AAG AAG CAG CAA GCT GAC TC - #C AAC TGT GGG TTC GCA    3475 Glu Ala Tyr Phe Lys Lys Gln Gln Ala Asp Se - #r Asn Cys Gly Phe Ala        1030     - #           1035      - #          1040  - - GAG GAA TAC GAA GAT CTG AAG CTT GTT GGA AT - #T AGT CAA CCT AAA TAT    3523 Glu Glu Tyr Glu Asp Leu Lys Leu Val Gly Il - #e Ser Gln Pro Lys Tyr    1045         - #       1050          - #      1055  - - GCA GCA GAA CTG GCT GAG AAT AGA GGA AAG AA - #T CGC TAT AAT AAT GTT    3571 Ala Ala Glu Leu Ala Glu Asn Arg Gly Lys As - #n Arg Tyr Asn Asn Val1060             - #   1065              - #  1070  - - CTG CCC TAT GAT ATT TCC CGT GTC AAA CTT TC - #G GTC CAG ACC CAT TCA    3619 Leu Pro Tyr Asp Ile Ser Arg Val Lys Leu Se - #r Val Gln Thr His Ser 1075                1080 - #                1085 - #               1090  - - ACG GAT GAC TAC ATC AAT GCC AAC TAC ATG CC - #T GGC TAC CAC TCC AAG    3667 Thr Asp Asp Tyr Ile Asn Ala Asn Tyr Met Pr - #o Gly Tyr His Ser Lys            1095 - #               1100  - #              1105  - - AAA GAT TTT ATT GCC ACA CAA GGA CCT TTA CC - #G AAC ACT TTG AAA GAT    3715 Lys Asp Phe Ile Ala Thr Gln Gly Pro Leu Pr - #o Asn Thr Leu Lys Asp        1110     - #           1115      - #          1120  - - TTT TGG CGT ATG GTT TGG GAG AAA AAT GTA TA - #T GCC ATC ATT ATG TTG    3763 Phe Trp Arg Met Val Trp Glu Lys Asn Val Ty - #r Ala Ile Ile Met Leu    1125         - #       1130          - #      1135  - - ACT AAA TGT GTT GAA CAG GGA AGA ACC AAA TG - #T GAG GAG TAT TGG CCC    3811 Thr Lys Cys Val Glu Gln Gly Arg Thr Lys Cy - #s Glu Glu Tyr Trp Pro1140             - #   1145              - #  1150  - - TCC AAG CAG GCT CAG GAC TAT GGA GAC ATA AC - #T GTG GCA ATG ACA TCA    3859 Ser Lys Gln Ala Gln Asp Tyr Gly Asp Ile Th - #r Val Ala Met Thr Ser 1155                1160 - #                1165 - #               1170  - - GAA ATT GTT CTT CCG GAA TGG ACC ATC AGA GA - #T TTC ACA GTG AAA AAT    3907 Glu Ile Val Leu Pro Glu Trp Thr Ile Arg As - #p Phe Thr Val Lys Asn            1175 - #               1180  - #              1185  - - ATC CAG ACA AGT GAG AGT CAC CCT CTG AGA CA - #G TTC CAT TTC ACC TCC    3955 Ile Gln Thr Ser Glu Ser His Pro Leu Arg Gl - #n Phe His Phe Thr Ser        1190     - #           1195      - #          1200  - - TGG CCA GAC CAC GGT GTT CCC GAC ACC ACT GA - #C CTG CTC ATC AAC TTC    4003 Trp Pro Asp His Gly Val Pro Asp Thr Thr As - #p Leu Leu Ile Asn Phe    1205         - #       1210          - #      1215  - - CGG TAC CTC GTT CGT GAC TAC ATG AAG CAG AG - #T CCT CCC GAA TCG CCG    4051 Arg Tyr Leu Val Arg Asp Tyr Met Lys Gln Se - #r Pro Pro Glu Ser Pro1220             - #   1225              - #  1230  - - ATT CTG GTG CAT TGC AGT GCT GGG GTC GGA AG - #G ACG GGC ACT TTC ATT    4099 Ile Leu Val His Cys Ser Ala Gly Val Gly Ar - #g Thr Gly Thr Phe Ile 1235                1240 - #                1245 - #               1250  - - GCC ATT GAT CGT CTC ATC TAC CAG ATA GAG AA - #T GAG AAC ACC GTG GAT    4147 Ala Ile Asp Arg Leu Ile Tyr Gln Ile Glu As - #n Glu Asn Thr Val Asp            1255 - #               1260  - #              1265  - - GTG TAT GGG ATT GTG TAT GAC CTT CGA ATG CA - #T AGG CCT TTA ATG GTG    4195 Val Tyr Gly Ile Val Tyr Asp Leu Arg Met Hi - #s Arg Pro Leu Met Val        1270     - #           1275      - #          1280  - - CAG ACA GAG GAC CAG TAT GTT TTC CTC AAT CA - #G TGT GTT TTG GAT ATT    4243 Gln Thr Glu Asp Gln Tyr Val Phe Leu Asn Gl - #n Cys Val Leu Asp Ile    1285         - #       1290          - #      1295  - - GTC AGA TCC CAG AAA GAC TCA AAA GTA GAT CT - #T ATC TAC CAG AAC ACA    4291 Val Arg Ser Gln Lys Asp Ser Lys Val Asp Le - #u Ile Tyr Gln Asn Thr1300             - #   1305              - #  1310  - - ACT GCA ATG ACA ATC TAT GAA AAC CTT GCG CC - #C GTG ACC ACA TTT GGA    4339 Thr Ala Met Thr Ile Tyr Glu Asn Leu Ala Pr - #o Val Thr Thr Phe Gly 1315                1320 - #                1325 - #               1330  - - AAG ACC AAT GGT TAC ATC GCC TAATTCCAAA GGAATAACC - #T TTCT    4384 Lys Thr Asn Gly Tyr Ile Ala            1335  - - GGAGTGAACC AGACCGTCGC ACCCACAGCG AAGGCACATG CCCCGATGTC GA -#CATGTTTT   4444   - - TATATGTCTA ATATCTTAAT TCTTTGTTCT GTTTTGTGAG AACTAATTTT GA -#GGGCATGA   4504   - - AGCTGCATAT GATAGATGAC AAATTGGGGC TGTCGGGGGC TGTGGATGGG TG -#GGGAGCAA   4564   - - ATCATCTGCA TTCCTGATGA CCAATGGGAT GAGGTCACTT TTTTTTTTTT CC -#CCCTTGAG   4624   - - GATTGCGGAA AACCAGGAAA AGGGATCTAT GATTTTTTTT TCCAAAACAA TT -#TCTTTTTT   4684   - - AAAAAGACTA TTTTATATGA TTCACATGCT AAAGCCAGGA TTGTGTTGGG TT -#GAATATAT   4744   - - TTTAAGTATC AGAGGTCTAT TTTTACCTAC TGTGTCTTGG AATCTAGCCG AT -#GGAAAATA   4804   - - CCTAATTGTG GATGATGATT GCGCAGGGAG GGGTACGTGG CACCTCTTCC GA -#ATGGGTTT   4864   - - TCTATTTGAA CATGTGCCTT TTCTGAATTA TGCTTCCACA GGCAAAACTC AG -#TAGAGATC   4924   - - TATATTTTTG TACTGAATCT CATAATTGGA ATATACGGAA TATTTAAACA GT -#AGCTTAGC   4984   - - ATCAGAGGTT TGCTTCCTCA GTAACATTTC TGTTCTCATT TGATCAGGGG AG -#GCCTCTTT   5044   - - GCCCCGGCCC CGCTTCCCCT GCCCCCGTGT GATTTGTGCT CCATTTTTTC TT -#CCCTTTTC   5104   - - CCTCCCAGTT TTC              - #                  - #   - #    5117  - -  - - (2) INFORMATION FOR SEQ ID NO:2:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 1337 amino - #acids      (B) TYPE: amino acid      (D) TOPOLOGY: linear  - -     (ii) MOLECULE TYPE: protein  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:  - - Met Lys Pro Ala Ala Arg Glu Ala Arg Leu Pr - #o Pro Arg Ser Pro Gly   1               5 - #                 10 - #                 15  - - Leu Arg Trp Ala Leu Pro Leu Leu Leu Leu Le - #u Leu Arg Leu Gly Gln         20     - #             25     - #             30  - - Ile Leu Cys Ala Gly Gly Thr Pro Ser Pro Il - #e Pro Asp Pro Ser Val     35         - #         40         - #         45  - - Ala Thr Val Ala Thr Gly Glu Asn Gly Ile Th - #r Gln Ile Ser Ser Thr 50             - #     55             - #     60  - - Ala Glu Ser Phe His Lys Gln Asn Gly Thr Gl - #y Thr Pro Gln Val Glu  65                 - # 70                 - # 75                 - # 80  - - Thr Asn Thr Ser Glu Asp Gly Glu Ser Ser Gl - #y Ala Asn Asp Ser Leu             85 - #                 90 - #                 95  - - Arg Thr Pro Glu Gln Gly Ser Asn Gly Thr As - #p Gly Ala Ser Gln Lys        100      - #           105      - #           110  - - Thr Pro Ser Ser Thr Gly Pro Ser Pro Val Ph - #e Asp Ile Lys Ala Val    115          - #       120          - #       125  - - Ser Ile Ser Pro Thr Asn Val Ile Leu Thr Tr - #p Lys Ser Asn Asp Thr130              - #   135              - #   140  - - Ala Ala Ser Glu Tyr Lys Tyr Val Val Lys Hi - #s Lys Met Glu Asn Glu 145                 1 - #50                 1 - #55                 1 -#60   - - Lys Thr Ile Thr Val Val His Gln Pro Trp Cy - #s Asn Ile Thr GlyLeu             165  - #               170  - #               175  - - Arg Pro Ala Thr Ser Tyr Val Phe Ser Ile Th - #r Pro Gly Ile Gly Asn        180      - #           185      - #           190  - - Glu Thr Trp Gly Asp Pro Arg Val Ile Lys Va - #l Ile Thr Glu Pro Ile    195          - #       200          - #       205  - - Pro Val Ser Asp Leu Arg Val Ala His Gly Cy - #s Glu Glu Gly Cys Ser210              - #   215              - #   220  - - Leu Ser Trp Ser Asn Gly Asn Gly Thr Ala Se - #r Cys Arg Val Leu Leu 225                 2 - #30                 2 - #35                 2 -#40   - - Glu Ser Ile Gly Ser His Glu Glu Leu Thr Gl - #n Asp Ser Arg LeuGln             245  - #               250  - #               255  - - Val Asn Ile Ser Asp Leu Lys Pro Gly Val Gl - #n Tyr Asn Ile Asn Pro        260      - #           265      - #           270  - - Tyr Leu Leu Gln Ser Asn Lys Thr Lys Gly As - #p Pro Leu Ala Gln Lys    275          - #       280          - #       285  - - Val Ala Trp Met Pro Ala Ile Gln Arg Glu Al - #a Gly Gln Gly Ala Pro290              - #   295              - #   300  - - Pro Pro Leu Cys Met Met Ser Pro Phe Val Gl - #y Pro Val Asp Pro Ser 305                 3 - #10                 3 - #15                 3 -#20   - - Ser Gly Gln Gln Ser Arg Asp Thr Glu Val Le - #u Leu Val Gly LeuGlu             325  - #               330  - #               335  - - Pro Gly Thr Arg Tyr Asn Ala Thr Val Tyr Se - #r Gln Ala Ala Asn Gly        340      - #           345      - #           350  - - Thr Glu Gly Gln Pro Gln Ala Ile Glu Phe Ar - #g Thr Asn Ala Ile Gln    355          - #       360          - #       365  - - Val Phe Asp Val Thr Ala Val Asn Ile Ser Al - #a Thr Ser Leu Thr Leu370              - #   375              - #   380  - - Ile Trp Lys Val Ser Asp Asn Glu Ser Ser Se - #r Asn Tyr Thr Tyr Lys 385                 3 - #90                 3 - #95                 4 -#00   - - Ile His Val Ala Gly Glu Thr Asp Ser Ser As - #n Leu Asn Val SerGlu             405  - #               410  - #               415  - - Pro Arg Ala Val Ile Pro Gly Leu Arg Ser Se - #r Thr Phe Tyr Asn Ile        420      - #           425      - #           430  - - Thr Val Cys Pro Val Leu Gly Asp Ile Glu Gl - #y Thr Pro Gly Phe Leu    435          - #       440          - #       445  - - Gln Val His Thr Pro Pro Val Pro Val Ser As - #p Phe Arg Val Thr Val450              - #   455              - #   460  - - Val Ser Thr Thr Glu Ile Gly Leu Ala Trp Se - #r Ser His Asp Ala Glu 465                 4 - #70                 4 - #75                 4 -#80   - - Ser Phe Gln Met His Ile Thr Gln Glu Gly Al - #a Gly Asn Ser ArgVal             485  - #               490  - #               495  - - Glu Ile Thr Thr Asn Gln Ser Ile Ile Ile Gl - #y Gly Leu Phe Pro Gly        500      - #           505      - #           510  - - Thr Lys Tyr Cys Phe Glu Ile Val Pro Lys Gl - #y Pro Asn Gly Thr Glu    515          - #       520          - #       525  - - Gly Ala Ser Arg Thr Val Cys Asn Arg Thr Va - #l Pro Ser Ala Val Phe530              - #   535              - #   540  - - Asp Ile His Val Val Tyr Val Thr Thr Thr Gl - #u Met Trp Leu Asp Trp 545                 5 - #50                 5 - #55                 5 -#60   - - Lys Ser Pro Asp Gly Ala Ser Glu Tyr Val Ty - #r His Leu Val IleGlu             565  - #               570  - #               575  - - Ser Lys His Gly Ser Asn His Thr Ser Thr Ty - #r Asp Lys Ala Ile Thr        580      - #           585      - #           590  - - Leu Gln Gly Leu Ile Pro Gly Thr Leu Tyr As - #n Ile Thr Ile Ser Pro    595          - #       600          - #       605  - - Glu Val Asp His Val Trp Gly Asp Pro Asn Se - #r Thr Ala Gln Tyr Thr610              - #   615              - #   620  - - Arg Pro Ser Asn Val Ser Asn Ile Asp Val Se - #r Thr Asn Thr Thr Ala 625                 6 - #30                 6 - #35                 6 -#40   - - Ala Thr Leu Ser Trp Gln Asn Phe Asp Asp Al - #a Ser Pro Thr TyrSer             645  - #               650  - #               655  - - Tyr Cys Leu Leu Ile Glu Lys Ala Gly Asn Se - #r Ser Asn Ala Thr Gln        660      - #           665      - #           670  - - Val Val Thr Asp Ile Gly Ile Thr Asp Ala Th - #r Val Thr Glu Leu Ile    675          - #       680          - #       685  - - Pro Gly Ser Ser Tyr Thr Val Glu Leu Phe Al - #a Gln Val Gly Asp Gly690              - #   695              - #   700  - - Ile Lys Ser Leu Glu Pro Gly Arg Lys Ser Ph - #e Cys Thr Asp Pro Ala 705                 7 - #10                 7 - #15                 7 -#20   - - Ser Met Ala Ser Phe Asp Cys Glu Val Val Pr - #o Lys Glu Pro AlaLeu             725  - #               730  - #               735  - - Val Leu Lys Trp Thr Cys Pro Pro Gly Ala As - #n Ala Gly Phe Glu Leu        740      - #           745      - #           750  - - Glu Val Ser Ser Gly Ala Trp Asn Asn Ala Th - #r His Leu Glu Ser Cys    755          - #       760          - #       765  - - Ser Ser Glu Asn Gly Thr Glu Tyr Arg Thr Gl - #u Val Thr Tyr Leu Asn770              - #   775              - #   780  - - Phe Ser Thr Ser Tyr Asn Ile Ser Ile Thr Th - #r Val Ser Cys Gly Lys 785                 7 - #90                 7 - #95                 8 -#00   - - Met Ala Ala Pro Thr Arg Asn Thr Cys Thr Th - #r Gly Ile Thr AspPro             805  - #               810  - #               815  - - Pro Pro Pro Asp Gly Ser Pro Asn Ile Thr Se - #r Val Ser His Asn Ser        820      - #           825      - #           830  - - Val Lys Val Lys Phe Ser Gly Phe Glu Ala Se - #r His Gly Pro Ile Lys    835          - #       840          - #       845  - - Ala Tyr Ala Val Ile Leu Thr Thr Gly Glu Al - #a Gly His Pro Ser Ala850              - #   855              - #   860  - - Asp Val Leu Lys Tyr Thr Tyr Asp Asp Phe Ly - #s Lys Gly Ala Ser Asp 865                 8 - #70                 8 - #75                 8 -#80   - - Thr Tyr Val Thr Tyr Leu Ile Arg Thr Glu Gl - #u Lys Gly Arg SerGln             885  - #               890  - #               895  - - Ser Leu Ser Glu Val Leu Lys Tyr Glu Ile As - #p Val Gly Asn Glu Ser        900      - #           905      - #           910  - - Thr Thr Leu Gly Tyr Tyr Asn Gly Lys Leu Gl - #u Pro Leu Gly Ser Tyr    915          - #       920          - #       925  - - Arg Ala Cys Val Ala Gly Phe Thr Asn Ile Th - #r Phe His Pro Gln Asn930              - #   935              - #   940  - - Lys Gly Leu Ile Asp Gly Ala Glu Ser Tyr Va - #l Ser Phe Ser Arg Tyr 945                 9 - #50                 9 - #55                 9 -#60   - - Ser Asp Ala Val Ser Leu Pro Gln Asp Pro Gl - #y Val Ile Cys GlyAla             965  - #               970  - #               975  - - Val Phe Gly Cys Ile Phe Gly Ala Leu Val Il - #e Val Thr Val Gly Gly        980      - #           985      - #           990  - - Phe Ile Phe Trp Arg Lys Lys Arg Lys Asp Al - #a Lys Asn Asn Glu Val    995          - #       1000          - #      1005  - - Ser Phe Ser Gln Ile Lys Pro Lys Lys Ser Ly - #s Leu Ile Arg Val Glu1010             - #   1015              - #  1020  - - Asn Phe Glu Ala Tyr Phe Lys Lys Gln Gln Al - #a Asp Ser Asn Cys Gly 1025                1030 - #                1035 - #               1040  - - Phe Ala Glu Glu Tyr Glu Asp Leu Lys Leu Va - #l Gly Ile Ser Gln Pro            1045 - #               1050  - #              1055  - - Lys Tyr Ala Ala Glu Leu Ala Glu Asn Arg Gl - #y Lys Asn Arg Tyr Asn        1060     - #           1065      - #          1070  - - Asn Val Leu Pro Tyr Asp Ile Ser Arg Val Ly - #s Leu Ser Val Gln Thr    1075         - #       1080          - #      1085  - - His Ser Thr Asp Asp Tyr Ile Asn Ala Asn Ty - #r Met Pro Gly Tyr His1090             - #   1095              - #  1100  - - Ser Lys Lys Asp Phe Ile Ala Thr Gln Gly Pr - #o Leu Pro Asn Thr Leu 1105                1110 - #                1115 - #               1120  - - Lys Asp Phe Trp Arg Met Val Trp Glu Lys As - #n Val Tyr Ala Ile Ile            1125 - #               1130  - #              1135  - - Met Leu Thr Lys Cys Val Glu Gln Gly Arg Th - #r Lys Cys Glu Glu Tyr        1140     - #           1145      - #          1150  - - Trp Pro Ser Lys Gln Ala Gln Asp Tyr Gly As - #p Ile Thr Val Ala Met    1155         - #       1160          - #      1165  - - Thr Ser Glu Ile Val Leu Pro Glu Trp Thr Il - #e Arg Asp Phe Thr Val1170             - #   1175              - #  1180  - - Lys Asn Ile Gln Thr Ser Glu Ser His Pro Le - #u Arg Gln Phe His Phe 1185                1190 - #                1195 - #               1200  - - Thr Ser Trp Pro Asp His Gly Val Pro Asp Th - #r Thr Asp Leu Leu Ile            1205 - #               1210  - #              1215  - - Asn Phe Arg Tyr Leu Val Arg Asp Tyr Met Ly - #s Gln Ser Pro Pro Glu        1220     - #           1225      - #          1230  - - Ser Pro Ile Leu Val His Cys Ser Ala Gly Va - #l Gly Arg Thr Gly Thr    1235         - #       1240          - #      1245  - - Phe Ile Ala Ile Asp Arg Leu Ile Tyr Gln Il - #e Glu Asn Glu Asn Thr1250             - #   1255              - #  1260  - - Val Asp Val Tyr Gly Ile Val Tyr Asp Leu Ar - #g Met His Arg Pro Leu 1265                1270 - #                1275 - #               1280  - - Met Val Gln Thr Glu Asp Gln Tyr Val Phe Le - #u Asn Gln Cys Val Leu            1285 - #               1290  - #              1295  - - Asp Ile Val Arg Ser Gln Lys Asp Ser Lys Va - #l Asp Leu Ile Tyr Gln        1300     - #           1305      - #          1310  - - Asn Thr Thr Ala Met Thr Ile Tyr Glu Asn Le - #u Ala Pro Val Thr Thr    1315         - #       1320          - #      1325  - - Phe Gly Lys Thr Asn Gly Tyr Ile Ala1330             - #   1335  - -  - - (2) INFORMATION FOR SEQ ID NO:3:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 7 amino - #acids      (B) TYPE: amino acid      (C) STRANDEDNESS: single      (D) TOPOLOGY: linear  - -     (ii) MOLECULE TYPE: peptide  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:  - - Lys Cys Ala Gln Tyr Trp Pro 1               5  - -  - - (2) INFORMATION FOR SEQ ID NO:4:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 7 amino - #acids      (B) TYPE: amino acid      (C) STRANDEDNESS: single      (D) TOPOLOGY: linear  - -     (ii) MOLECULE TYPE: peptide  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:  - - His Cys Ser Ala Gly Ile Gly 1               5  - -  - - (2) INFORMATION FOR SEQ ID NO:5:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 20 base - #pairs      (B) TYPE: nucleic acid      (C) STRANDEDNESS: single      (D) TOPOLOGY: linear  - -     (ii) MOLECULE TYPE: DNA  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:  - - AARTGYGCNC ARTAYTGGCC            - #                  - #  - # 20  - -  - - (2) INFORMATION FOR SEQ ID NO:6:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 20 base - #pairs      (B) TYPE: nucleic acid      (C) STRANDEDNESS: single      (D) TOPOLOGY: linear  - -     (ii) MOLECULE TYPE: DNA  - -     (ix) FEATURE:      (D) OTHER INFORMATION: - #/note= &#34;Base designated N at           postion 6 - # is Inosine.&#34;  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:  - - CCDATNCCNG CRCTRCARTG            - #                  - #  - # 20__________________________________________________________________________