WO1998044111A1

WO1998044111A1 - Thymus receptor tyrosine kinase (trtk) and methods of use

Info

Publication number: WO1998044111A1
Application number: PCT/US1998/006021
Authority: WO
Inventors: Daniel R. Soppet; Steven M. Ruben
Original assignee: Human Genome Sciences Inc
Current assignee: Human Genome Sciences Inc
Priority date: 1997-03-28
Filing date: 1998-03-27
Publication date: 1998-10-08
Anticipated expiration: 1999-09-28
Also published as: AU6588298A

Abstract

The present invention relates to human thymus receptor tyrosine kinase (TRTK) receptors. More specifically, isolated nucleic acid molecules are provided encoding human TRTK proteins. TRTK polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Further provided are diagnostic methods for detecting disease states associated with the aberrant expression of TRTK and therapeutic methods for treating such disease states.

Description

Thymus Receptor Tyrosine Kinase (TRTK) and Methods of Use

Background of the Invention

Field of the Invention

The present invention relates to novel human thymus receptor tyrosine kinase (TRTK) receptors. More specifically, isolated nucleic acid molecules are provided encoding human TRTK proteins. TRTK polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Further provided are diagnostic methods for detecting disease states associated with the aberrant expression of TRTK and therapeutic methods for treating such disease states.

Related Art

A number of viral oncogenes and cellular proto-oncogenes have been shown to comprise a gene family whose products are plasma membrane associated and exhibit tyrosine-specific protein kinase activity. Hunter, T. and Cooper, J.,

Ann. Rev. Biochem. 54:891 (1985). These proteins generally consist of an intracellular domain with tyrosine kinase activity, a single pass transmembrane domain, and an extracellular domain that generally bind ligands which stimulate the receptor. Studies using antibodies prepared with synthetic peptides have shown that regions of receptor molecules with homology to the v-Src protein are normally necessary for biological activity. Reviewed in Pimentel, E. HANDBOOK OF GROWTH FACTORS, Volume 1, CRC Press, Inc. (1994), 208.

Protein phosphorylation plays an important role in the regulation of cellular metabolism, differentiation and proliferation. Reviewed in Pimentel, supra at 207- 265. For example, the activation of mitogen-activated protein kinases (MAPK) in blastula-stage cells from Xenopus embryos which normally gives rise to epidermal tissue is necessary and sufficient to induce mesoderm formation. Mutational analysis has demonstrated that the product of the c-ret proto- oncogene, Ret, is a member of the RTK superfamily and is required for normal development of the enteric nervous system and kidney. Durbec et al, Nature 381:189 (1996). Thus, this RTK has been implicated in both tissue differentiation and organ development.

RTKs normally respond to an extracellular stimulus by activation of their intracellular tyrosine kinase activity . This process generally occurs by dimerization of RTK protein molecules followed by autophosphorylation. Reviewed in Alberts, B . et al. , Molecular Biology of the Cell, Garland Publishing, Inc. ( 1994), 759-771. Dominant-negative inhibition of RTKs has been demonstrated by the overexpression of mutant forms of receptors containing an inactive kinase domain. Alberts et al, supra.

A number of RTKs have also been shown to be involved in oncogenesis. One such RTK is encoded by the eph gene. The eph gene is expressed in several normal tissues and is overexpressed in colon carcinoma, lung adenocarcinoma, mammary carcinoma and heptacellular carcinoma. Hirai et al. , Science 238:\1\1 (1987). The Eph receptor is a transmembrane glycoprotein with tyrosine kinase activity which is believed to respond to growth factor ligands. The nucleotide sequence of the eph gene encodes a primary translation product of 984 amino acids containing intracellular, transmembrane and extracellular domains . Hirai et al, supra.

Inhibitors of RTKs have been identified. K-252a, for example, is a compound isolated from a microorganism in the genus Nocardiopsis and initially identified as an inhibitor of protein kinase C. Pimentel, supra at 178. K-252awas later found to inhibit NGF -induced cellular effects and neurite outgrowth in PC 12 cells. Cho et al, Mol. Cell. Biol. 9: 135-143 (1989). Berg et al. demonstrated that K-252a mediated inhibition of NGF-specific signal transduction results from the ability of K-252a to inhibit the kinase activity of the product of the tr&proto- oncogene. Berg, M. et al, J. Biol Chem. 267:13 (1992). Summary of the Invention

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the TRTK proteins having the amino acid sequences shown in FIG. 1A-1D (SEQ ID NO:2 and SEQ ID NO:3) or encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 97830 on

December 20, 1996. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TRTK polypeptides or peptides by recombinant techniques.

The invention further provides isolated TRTK polypeptides having an amino acid sequences encoded by a polynucleotide described herein.

In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on the ability of an LERK family to bind to the TRTK receptors. In particular, the method involves contacting a TRTK receptor with an LERK ligand polypeptide and a candidate compound and determining whether ligand binding to the TRTK receptor is increased or decreased due to the presence of the candidate compound.

The invention further provides a diagnostic method useful during diagnosis or prognosis of a disorder resulting from aberrant cell proliferation {e.g., colon carcinoma, lung adenocarcinoma, mammary carcinoma and heptacellular carcinoma).

An additional aspect of the invention is related to a method for treating an individual in need of an increased level of TRTK activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated TRTK polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of TRTK receptor activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a TRTK antagonist.

Brief Description of the Figures

FIG. 1A-1D shows the nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequences (SEQ ID NO:2 and SEQ ID NO:3) of two TRTK receptors.

One translation product is initiated from the AUG codon located at positions 481 to 483 (SEQ ID NO:2) and encodes a protein with an amino terminus corresponding to amino acid residues number 16 in FIG. 1 A- 1 D. This protein has a predicted leader sequence of about 16 amino acid residues (double underlined) and a deduced molecular weight of about 109.3 kilodaltons (kDa). It is further predicted that amino acid residues from about 16 to about 594 (amino acid residues from about -16 to about 563 in SEQ ID NO:2) constitute the extracellular domain; amino acid residues from about 594 to about 622 (amino acid residues from about 563 to about 591 in SEQ ID NO:2) the transmembrane domain; and amino acid residues from about 623 to about 1021 (amino acid residues from about 592 to about 990 in SEQ ID NO:2) the intracellular domain.

The same nucleotide sequence shown in FIG. 1A-1D (SEQ ID NO:l) also encodes a TRTK receptor initiated from the AUG codon located at positions 436 to 438 (SEQ ID NO:3). This protein has a predicted leader sequence of about 14 amino acid residues (single underlined) and a deduced molecular weight of about

111.0 kDa. It is further predicted that amino acid residues from about 1 to about 594 (amino acid residues from about -14 to about 580 in SEQ ID NO:3) constitute the extracellular domain; amino acid residues from about 594 to about 622 (amino acid residues from about 580 to about 608 in SEQ ID NO:3) the transmembrane domain; and amino acid residues from about 623 to about 1021

(amino acid residues from about 609 to about 1007 in SEQ ID NO:3) the intracellular domain. FIG. 2 shows a schematic representation of the pHE4a expression vector (SEQ ID NO:32) and the subcloned TRTK cDNA coding sequence. The locations of the kanamycin resistance marker gene, the TRTK coding sequence, the oriC sequence, and the lacϊq coding sequence are indicated. FIG. 3 shows the nucleotide sequence of the regulatory elements of the pHE4a promoter (SEQ ID NO:33). The two lac operator sequences, the Shine- Delgarno sequence (S/D), and the terminal Hindlϊl and Ndel restriction sites (italicized) are indicated.

Detailed Description

The present invention provides a new member of the eph gene family and two products of that gene, thymus receptor tyrosine kinases (TRTK). The DNA sequence encoding the TRTK receptors of the present invention was initially isolated from a 16 week old human fetal cell cDNA library and encodes two putative receptors of the LERK family of ligands. Further, these receptors are believed to be involved in hematopoietic development. As member of the Eph family of RTKs, aberrant expression of TRTK is potentially involved in a number of diseases resulting from alterations in normal cellular proliferation, e.g. , cancer.

The present invention provides isolated nucleic acid molecules comprising polynucleotides encoding TRTK polypeptides having the amino acid sequences shown in FIG. 1 A-1D (SEQ ID NO:2 and SEQ ID NO:3), which were determined by sequencing a cloned cDNA. The TRTK proteins of the present invention shares sequence homology with cek9, a member of the EPH family of receptor tyrosine kinases. The nucleotide sequence shown in SEQ ID NO: 1 was obtained by sequencing a cDNA clone, which was deposited on December 20, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland

20852, and given accession number 97830. The cDNA is inserted in the EcoRI restriction endonuclease site in the polylinker of the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA). Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. Using the information provided herein, such as the nucleotide sequence in

SEQ ID NO: 1 , a nucleic acid molecule of the present invention encoding a TRTK polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in SEQ ID NO:l was discovered in a cDNA library derived from 16 week old human fetal cells. The gene was also identified in cDNA libraries from the following tissues and cell types: thymus, Jurkat cells, activated T cells, chronic synovitis, infant brain, fetal kidney, fetal epithelium, fetal lung, both eight and sixteen week embryos, pineal gland, kidney cortex, epididymis, testes, testicular tumor, Wilms tumor, hemangiopericytoma and pancreatic tumor. The determined nucleotide sequence of the TRTK cDNA shown in SEQ ID NO:l is believed to contain two open reading frames (ORF). One open reading frame encodes a protein of about 1006 amino acid residues, with a predicted leader sequence of about 16 amino acid residues, and a deduced molecular weight of about 109.3 kDa. The amino acid sequence of this predicted mature TRTK receptor is shown in FIG. 1 A- ID (SEQ ID NO:2) from amino acid residue about 32 to residue about 1021 (amino acid residues from about 1 to about 990 in SEQ ID NO:2). The TRTK protein shown in FIG. 1A-1D (SEQ ID NO:2) is about 60% identical and about 76% similar to cek9.

The second open reading frame, shown in SEQ ID NO:3, encodes a protein of about 1021 amino acid residues, with a predicted leader sequence of about 14 amino acid residues, and a deduced molecular weight of about 111.0 kDa. The amino acid sequence of this predicted mature TRTK receptor is shown in FIG. 1A-1D from amino acid residue about 15 to residue about 1021 (amino acid residues from about 1 to about 1007 in SEQ ID NO:3).

It is also possible that the translation product shown in SEQ ID NO:3 contains a 31 amino acid leader sequence which is cleavage between the same amino acids as the protein shown in shown in SEQ ID NO:2. Thus, the TRTK protein shown in SEQ ID NO : 3 may be initially translated as a product containing

1021 amino acids and may be processed to a product which corresponds to the same mature form of the protein shown in SEQ ID NO:2.

A number of eukaryotic mRNAs have been shown to encode more than one translation product based on the utilization of different AUG codons. In eukaryotic cells the selection of an AUG codon as the translational start point is determined by both the location of that codon in relationship to the cap at the 5' end of the mRNA molecule and the nucleotides surrounding the codon. If the first AUG codon is poorly situated for the initiation of transcription, many ribosomes will skip over this codon and proceed to another AUG codon in the mRNA molecule. As a result, multiple translation products can be produced from the same mRNA molecule. Similarly, the cDNA encoding the TRTK receptors of the present invention is believed to produce a mRNA molecule from which more than one translation product is generated. These translation products are shown in SEQ ID NO:2 and SEQ ID NO:3. As indicated, the present invention also provides the mature form(s) of the

TRTK receptor of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein. Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide. Therefore, the present invention provides a nucleotide sequence encoding the mature TRTK polypeptides having the amino acid sequences encoded by the cDNA clone contained in the host identified as ATCC Deposit No. 97830. By the mature TRTK proteins having the amino acid sequences encoded by the cDNA clone contained in the host identified as ATCC Deposit No. 97830 are meant the mature forms of the TRTK receptors produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frames encoded by the human DNA sequence of the clone contained in the vector in the deposited host. As indicated below, the mature TRTK receptors having the amino acid sequences encoded by the cDNA clone contained in ATCC Deposit No. 97830 may or may not differ from the predicted

"mature" TRTK proteins shown in SEQ ID NO:2 (amino acids from about 1 to about 990) or SEQ ID NO: 3 (amino acids from about 1 to about 1007) depending on the accuracy of the predicted cleavage site based on computer analysis.

Methods for predicting whether a protein has a secretory leader as well as the cleavage point for that leader sequence are available. For instance, the methods of McGeoch (Virus Res. 5:271-286 (1985)) and von Heinje (Nucleic Acids Res. 74:4683-4690 (1986)) can be used. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. von Heinje, supra. However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequences of the complete TRTK polypeptides of the present invention were analyzed by a computer program ("PSORT") (K. Nakai and M. Kanehisa, Genomics 14:891-911 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis by the PSORT program predicted the cleavage sites between amino acids -1 and 1 in SEQ ID NO:2 and between amino acids amino acids -1 and 1 in SEQ ID NO:3. Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (-1,-3) rule of von Heine, von Heinje, supra. Thus, the leader sequence for the TRTK receptor protein of SEQ ID NO:2 is predicted to consist of amino acid residues -16 to - 1 in SEQ ID NO:2, while the predicted mature TRTK protein consists of residues 1 to 990 in SEQ ID NO:2. Further, the leader sequence for the TRTK receptor protein of SEQ ID NO: 3 is predicted to consist of amino acid residues -14 to -1 in SEQ ID NO:3, while the predicted mature TRTK protein consists of residues 1 to 1007 in SEQ ID NO:3.

As one of ordinary skill would appreciate, however, due to the possibilities of sequencing errors, as well as the variability of cleavage sites for leaders in different known proteins, the TRTK polypeptides encoded by the deposited cDNA may vary from the description provided herein. Further, the deposited cDNA is believed to comprise two open reading frames. The first open reading frame encodes a protein of about 1006 amino acids (SEQ ID NO:2), but may be anywhere in the range of 980-1032 amino acids. The leader sequence of this protein is about 16 amino acids, but may be anywhere in the range of about 5 to about 27 amino acids. The second open reading frame encodes a protein of about 1021 amino acids (SEQ IDNO:3), but may be anywhere in the range of 995-1047 amino acids. The leader sequence of this protein is about 14 amino acids, but may be anywhere in the range of about 5 to about 41 amino acids.

As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule,

DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNA molecules comprising the open reading frames shown in SEQ ID NO:l; DNA molecules comprising the coding sequence for the mature TRTK receptors shown in SEQ ID NO:2 (last 990 amino acids) and SEQ ID NO:3 (last 1007 amino acids); and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TRTK receptors. Of course, the genetic code is well known in the art.

Thus, it would be routine for one skilled in the art to generate such degenerate variants.

In another aspect, the invention provides isolated nucleic acid molecules encoding the TRTK polypeptides having the amino acid sequences encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97830 on December 20, 1996. Preferably, this nucleic acid molecule will encode the mature polypeptides encoded by the above-described deposited cDNA clone. In a further embodiment, nucleic acid molecules are provided encoding the mature TRTK polypeptides or the full-length TRTK polypeptides lacking the N-terminal methionine. The invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence of the cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TRTK receptor gene in human tissue, for instance, by Northern blot analysis.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDN A or the nucleotide sequence shown in SEQ ID NO:l is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO: 1. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 1.

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising a complete TRTK receptor extracellular domain (predicted to constitute amino acid residues from about -16 to about 563 in SEQ ID NO:2 and amino acid residues from about -14 to about 580 in SEQ ID NO:3); a polypeptide comprising a TRTK receptor extracellular domain minus the leader sequence (predicted to constitute amino acid residues from about 1 to about 563 in SEQ ID NO: 2 and amino acid residues from about 1 to about 580 in SEQ ID NO:3); a polypeptide comprising a TRTK receptor transmembrane domain (predicted to constitute amino acid residues from about 563 to about 591 in SEQ ID NO:2 and amino acid residues from about 580 to about 608 in SEQ ID NO: 3); a polypeptide comprising a TRTK receptor intracellular domain (predicted to constitute amino acid residues from about 592 to about 990 in SEQ ID NO:2 and amino acid residues from about 609 to about 1007 in SEQ ID NO:3); and a polypeptide comprising TRTK receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted. As above with the leader sequence, the amino acid residues constituting TRTK receptor extracellular, transmembrane and intracellular domains have been predicted by computer analysis. Thus, as one of ordinary skill would appreciate, the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain.

Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of a TRTK receptor protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 19 to about 184 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 229 to about 414 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 439 to about 563 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 591 to about 684 in SEQ ID NO:2; and a polypeptide comprising amino acid residues from about 784 to about 990 in SEQ ID NO:2. The inventors have determined that the above polypeptide fragments are antigenic regions of the TRTK receptor shown in SEQ ID NO:2. Similar antigenic polypeptides exist for the TRTK protein shown in SEQ ID NO:3 and comprise the same amino acid sequences described above. Methods for determining other such epitope-bearing portions of the TRTK proteins are described in detail below.

In addition, the present inventors have identified the following cDNA clones related to extensive portions of SEQ ID NO:l : HKFBA76R (SEQ ID NO:9), HTTCR36R (SEQ ID NO: 10), HPBAA66R (SEQ ID NO: 11), HJPBJ20RB (SEQ

ID NO:12), and HTTCR63R (SEQ ID NO:13).

The following public ESTs, which relate to portions of SEQ ID NO: 1 , have also been identified: AA351300 (SEQ ID NO: 14), T09277 (SEQ ID NO: 15), AA609284 (SEQ ID NO.16), T08512 (SEQ ID NO:17), AA171336 (SEQ ID NO:18), R85150 (SEQ ID NO:19), AA297321 (SEQ ID NO.20), T08511 (SEQ

ID NO.21), R61179 (SEQ ID NO:22), AA310877 (SEQ ID NO:23), T09276 (SEQ ID NO:24), T03387 (SEQ ID NO:25), H19908 (SEQ ID NO:26), AA158493 (SEQ ID NO:27), AA298297 (SEQ ID NO:28), R52792 (SEQ ID NO:29), H20300 (SEQ ID NO:30), and R87294 (SEQ ID NO:31). In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the cDNA clone contained in ATCC Deposit 97830. By "stringent hybridization conditions" is intended overnight incubation at 42 °C in a solution comprising: 50% formamide, 5x SSC (150 mM

NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 °C.

By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below. By a portion of a polynucleotide of "at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: 1). Of course, a polynucleotide which hybridizes only to a poly A sequence of a

TRTK receptor cDNA, or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g. , practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention which encode a TRTK polypeptide may include, but are not limited to those encoding the amino acid sequences of the mature polypeptides, by themselves; the coding sequence for the mature polypeptides and additional sequences, such as those encoding the about 16 amino acid leader for the TRTK receptor shown in SEQ ID NO:2, the about 14 amino acid leader for the TRTK receptor shown in SEQ ID NO:3, or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptides, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example-ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 55:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:161 (1984). As discussed below, other such fusion proteins include the TRTK receptor fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the TRTK receptors. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the TRTK receptors or portions thereof. Also especially preferred in this regard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID

NO:2; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:3; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:2, but lacking the N-terminal methionine; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:3, but lacking the N-terminal methionine; (e) a nucleotide sequence encoding the TRTK polypeptide having the amino acid sequence at positions from about 1 to about 990 in SEQ ID NO:2; (f) a nucleotide sequence encoding the TRTK polypeptide having the amino acid sequence at positions from about 1 to about 1007 in SEQ ID NO:3; (g) a nucleotide sequence encoding TRTK polypeptides having the amino acid sequences encoded by the cDNA clone contained in ATCC Deposit No. 97830; (h) a nucleotide sequence encoding mature TRTK polypeptides having the amino acid sequences encoded by the cDNA clone contained in ATCC Deposit No. 97830; (i) a nucleotide sequence encoding the amino acid sequence at positions from about -16 to about 563 in SEQ ID NO:2; (j) a nucleotide sequence encoding the amino acid sequence at positions from about -14 to about 580 in SEQ ID NO:3; (k) a nucleotide sequence encoding the amino acid sequence at positions from about 1 to about 563 in SEQ ID NO:2; (1) anucleotide sequence encoding the amino acid sequence at positions from about 563 to about 591 in SEQ ID NO:2; (m) a nucleotide sequence encoding the amino acid sequence at positions from about 592 to about

990 in SEQ ID NO:2; (n) a nucleotide sequence encoding TRTK receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted; and (o) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), 0), (k), (1), (m) or (n).

By a polynucleotide having anucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a TRTK polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the TRTK receptor. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in SEQ ID NO:l or to the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix,

Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

The present application is directed to nucleic acid molecules at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:l or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having TRTK receptor activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TRTK receptor activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having TRTK receptor activity include, ter alia, ( 1 ) isolating the TRTK receptor gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TRTK receptor gene, as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting TRTK receptor mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:l or to the nucleic acid sequence of the deposited cDNA which do, in fact, encode a polypeptide having TRTK receptor activity. By "a polypeptide having TRTK receptor activity" is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TRTK receptors of the invention (either the full-length protein or, preferably, the mature protein), as measured in a particular biological assay. For example, TRTK receptor activity can be measured using an in vitro kinase assay. One such assay involves transfecting human cells, such as fibroblasts, with the TRTK gene of the present invention and contacting those cells with a ligand known to elicit a TRTK response, such as an LERK family ligand. After stimulation, the cells are lysed and the lysate is treated with anti-TRTK antibodies followed by immunoprecipitation with protein A- Sepharose beads. The kinase activity of the immunoprecipitation complex is then measured by resuspension and incubation of the beads in a reaction buffer containing ³²P-ATP followed by analysis by SDS polyacrylamide gel electrophoresis. A similar in vitro kinase assay system has been described for measuring the activity of the c-ret proto-oncogene. Berg et al, J. Biol. Chem. 267: 13-16 (1996). Phosphorylation of tyrosine residues of either the receptor or a target molecule can also be evaluated using Western blot analysis as described in Soppet et al, Cell 55:895-903 (1991).

Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in SEQ ID NO:l will encode "a polypeptide having TRTK receptor activity." In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having TRTK receptor activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid). For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. etal, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306- 1310 ( 1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.

Vectors and Host Cells

The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of TRTK polypeptides or fragments thereof by recombinant techniques. The polynucleotides may be j oined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retro viral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated. As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQΕ70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL5-receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D.

Bennett et al, Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K. Johanson et α/., J Biol. Chem., Yo\. 270, No. 16:9459-9471 (1995).

In addition to the use of expression vectors in the practice of the present invention, the present invention further includes novel expression vectors comprising operator and promoter elements operatively linked to nucleotide sequences encoding a protein of interest. One example of such a vector is pHE4a which is described in detail below.

As summarized in FIGs. 2 and 3, components of the pHE4a vector (SEQ ID NO:32) include: 1 ) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, 6) the lactose operon repressor gene (laclq) and 7) a multiple cloning site linker region. The origin of replication (oriC) is derived from pUC 19 (LTI, Gaithersburg, MD). The promoter sequence and operator sequences were made synthetically. Synthetic production of nucleic acid sequences is well known in the art. CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto, CA 94303. The pHE4a vector was deposited with the ATCC on February 25, 1998 and given accession number 209645.

A nucleotide sequence encoding TRTK (SEQ ID NO: 1 ), is operatively linked to the promoter and operator of pHE4a by restricting the vector with Ndel and either Xbal, BamHl, Xhol, or Aspl 18, and isolating the larger fragment (the multiple cloning site region is about 310 nucleotides) on a gel. The nucleotide sequence encoding TRTK (SEQ ID NO: 1 ) having the appropriate restriction sites is generated, for example, according to the PCR protocol described in Example 1 , using PCR primers having restriction sites for Ndel (as the 5' primer) and either

Xbal, BamHl, Xhol, or Aspl 18 (as the 3' primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

As noted above, the pHE4a vector contains a laclq gene. Laclq is an allele of the lacl gene which confers tight regulation of the lac operator. Amann,

E. et al, Gene 59:301-315 (1988); Stark, M., Gene 51:255-261 (1987). The laclq gene encodes a repressor protein which binds to lac operator sequences and blocks transcription of down-stream (i. e., 3') sequences. However, the laclq gene product dissociates from the lac operator in the presence of either lactose or certain lactose analogs, e.g. , isopropyl B-D-thiogalactopyranoside (IPTG). TRTK thus is not produced in appreciable quantities in uninduced host cells containing the pHE4a vector. Induction of these host cells by the addition of an agent such as IPTG, however, results in the expression of the TRTK coding sequence.

The promoter/operator sequences of the pHE4a vector (SEQ ID NO:32) comprise a T5 phage promoter and two lac operator sequences. One operator is located 5' to the transcriptional start site and the other is located 3' to the same site. These operators, when present in combination with the laclq gene product, confer tight repression of down-stream sequences in the absence of a lac operon inducer, e.g., IPTG. Expression of operatively linked sequences located down-stream from the lac operators may be induced by the addition of a lac operon inducer, such as IPTG. Binding of a lac inducer to the laclq proteins results in their release from the lac operator sequences and the initiation of transcription of operatively linked sequences. Lac operon regulation of gene expression is reviewed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807.

The pHE4 series of vectors contain all of the components of the pHE4a vector except for the TRTK coding sequence. Features of the pHE4a vectors include optimized synthetic T5 phage promoter, lac operator, and Shine-Delgarno sequences. Further, these sequences are also optimally spaced so that expression of an inserted gene may be tightly regulated and high level of expression occurs upon induction.

Among known bacterial promoters suitable for use in the production of proteins of the present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retro viral LTRs, such as those of the Rous Sarcoma Virus (RS V), and metallothionein promoters, such as the mouse metallothionein-I promoter. The pHE4a vector also contains a Shine-Delgarno sequence 5' to the AUG initiation codon. Shine-Delgarno sequences are short sequences generally located about 10 nucleotides up-stream (i.e., 5') from the AUG initiation codon. These sequences essentially direct prokaryotic ribosomes to the AUG initiation codon. Thus, the present invention is also directed to expression vector useful for the production of the proteins of the present invention. This aspect of the invention is exemplified by the pHE4a vector (SEQ ID NO:32).

The TRTK receptor can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

TRTK Polypeptides and Fragments

The invention further provides isolated TRTK polypeptides having the amino acid sequences encoded by the deposited cDNA, the amino acid sequence in SEQ ID NO:2, the amino acid sequence in SEQ ID NO:3, or a peptide or polypeptide comprising a portion of the above polypeptides.

It will be recognized in the art that some amino acid sequences of the TRTK receptors can be varied without significant effect of the structure or function of the proteins. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Thus, the invention further includes variations of the TRTK receptors which show substantial TRTK receptor activity or which include regions of the TRTK receptors such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the

Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247. 1306-1310 (1990).

Thus, the fragment, derivative or analog of the polypeptide of SEQ ID NO:2, SEQ ID NO:3, or that encoded by the deposited cDNA, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide

(for example, polyethylene gly col), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids with other charged amino acids and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the TRTK protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al, Clin Exp. Immunol. 2:331-340 (1967); Robbins et al, Diabetes 55:838-845 (1987); Cleland et al, Crit. Rev. Therapeutic Drug Carrier Systems 10:301-311 (1993)). The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al, Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-α to only one of the two known types of TNF receptors. Thus, the TRTK receptors of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.

As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of protein (see Table 1).

Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions for any given TRTK polypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3. Amino acids in the TRTK proteins of the present invention that are essential for function can be identified by methods known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vivo proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith etal, J. Mol. Biol. 224:899-904 (1992) and de Vos et al Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in an isolated form. By "isolated polypeptide" is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an "isolated polypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of a TRTK polypeptide of the present invention can be substantially purified by the one-step method described in Smith and Johnson, Gene 57:31-40 (1988). The polypeptides of the present invention include the polypeptides encoded by the deposited cDNA including their leaders; the mature polypeptides encoded by the deposited the cDNA minus the leaders (i. e. , the mature proteins) a polypeptide comprising amino acids about -16 to about 990 in SEQ ID NO:2 apolypeptide comprising amino acids about -14 to about 1007 in SEQ ID NO:3 a polypeptide comprising amino acids about -15 to about 990 in SEQ ID NO: 2 a polypeptide comprising amino acids about - 13 to about 1007 in SEQ ID NO:3 a polypeptide comprising amino acids about 1 to about 990 in SEQ ID NO:2; a polypeptide comprising amino acids about 1 to about 1007 in SEQ ID NO:3; a polypeptide comprising the complete TRTK receptor extracellular domain (predicted to constitute amino acid residues from about -16 to about 563 in SEQ ID NO:2 and amino acid residues from about -14 to about 580 in SEQ ID NO:3); a polypeptide comprising the complete TRTK receptor extracellular domain minus the leader sequence (predicted to constitute amino acid residues from about 1 to about 563 in SEQ ID NO:2 and amino acid residues from about 1 to about 580 in SEQ ID NO:3); a polypeptide comprising the TRTK receptor transmembrane domain (predicted to constitute amino acid residues from about 563 to about 591 in SEQ ID NO:2 and amino acid residues from about 580 to about 608 in SEQ ID NO:3); a polypeptide comprising the TRTK receptor intracellular domain (predicted to constitute amino acid residues from about 592 to about 990 in SEQ ID NO:2 and amino acid residues from about 609 to about 1007 in SEQ ID

NO:3); and a polypeptide comprising the TRTK receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted; as well as polypeptides which are at least 95% identical, more preferably at least 96% identical, still more preferably at least 98% or 99% identical to those described above and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

By apolypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TRTK polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the TRTK receptor. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence shown in SEQ ID NO:3, or to the amino acid sequences encoded by deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program

(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

The polypeptides of the present invention could be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.

In another aspect, the invention provides peptides or polypeptides comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al, Proc. Natl. Acad. Sci. USA 81:3998-

4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenic epitope (i. e. , that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R.A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i. e. , immunogenic epitopes) nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson etal, Cell

37:767-778 (1984) at 777. Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TRTK receptor-specific antibodies include: a polypeptide comprising amino acid residues from about 19 to about 184 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 229 to about 414 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 439 to about 563 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about

591 to about 684 in SEQ ID NO:2; and a polypeptide comprising amino acid residues from about 784 to about 990 in SEQ ID NO:2. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TRTK receptor protein. Similar antigenic polypeptides exist for the TRTK protein shown in SEQ ID NO: 3 and comprise the same amino acid sequences described above.

The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl Acad. Sci. USA 52:5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).

As one of skill in the art will appreciate, TRTK polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al, Nature 331:84- 86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TRTK protein or protein fragment alone (Fountoulakis et al, J. Biochem. 270:3958-3964 (1995)).

Cancer Diagnosis and Prognosis

It is believed that certain tissues in mammals with cancer (e.g., colon carcinoma, lung adenocarcinoma, mammary carcinoma and hepatocellular carcinoma) express significantly enhanced levels of TRTK receptor and mRNA encoding TRTK receptor when compared to a corresponding " standard" mammal, i.e., a mammal of the same species not having the cancer. Further, it is believed that enhanced levels of TRTK receptor can be detected in certain body fluids (e. g. , sera, plasma, urine, and spinal fluid) from mammals with cancer when compared to sera from mammals of the same species not having the cancer. Thus, the invention provides a diagnostic method useful during tumor diagnosis, which involves assaying the expression level of the gene encoding a TRTK receptor of the present invention in mammalian cells or body fluid and comparing the gene expression level with a standard TRTK receptor gene expression level, whereby an increase in the gene expression level over the standard is indicative of certain tumors.

Where a tumor diagnosis has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced TRTK gene expression will experience a worse clinical outcome relative to patients expressing the gene at a lower level.

By "assaying the expression level of the gene encoding a TRTK receptor" is intended qualitatively or quantitatively measuring or estimating the level of a

TRTK protein or the level of the mRNA encoding a TRTK receptor in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the TRTK protein level or mRNA level in a second biological sample).

Preferably, the TRTK protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard TRTK protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the cancer. As will be appreciated in the art, once a standard TRTK protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains TRTK protein or mRNA. Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain mature TRTK protein, and ovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breast and umbilical tissue. The present invention is useful for detecting cancer in mammals. In particular the invention is useful during diagnosis of the of following types of cancers in mammals: breast, liver, lung and colon. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans. Total cellular RNA can be isolated from a biological sample using the single-step guanidinium-thiocyanate-phenol-chloroform method described in ChomczynskiandSacchi,_4«α/. Biochem. 752:156-159 (1987). LevelsofmRNA encoding the TRTK receptors are then assayed using any appropriate method. These include Northern blot analysis (Haradaetal, Cell 55:303-312 (1990)), SI nuclease mapping (Fujita et al, Cell 49:351- 367 (1987)), the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Fujita et al, Cell 49:351-361 (1987)), and reverse transcription in combination with the ligase chain reaction (RT-LCR). Assaying TRTK protein levels in a biological sample can occur using antibody-based techniques. For example, TRTK protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al, J. Cell. Biol 101:916-985 (1985); Jalkanen, M., et al, J. Cell . Biol. 105:3081-3096 (1987)). Other antibody-based methods useful for detecting TRTK receptor gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels, such as glucose oxidase, and radioisotopes, such as iodine (^I251, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

Therapeutics

TRTK is believed to be involved in a number of disease states such as cancers (e.g., testicular carcinoma, pancreatic carcinoma, colon carcinoma, lung adenocarcinoma, mammary carcinoma and heptacellular carcinoma) and other diseases associated with the aberrant proliferation of cells. Induction of a disease state by TRTK resulting in the increased proliferation of cells may result from either overexpression of a TRTK receptor or increased stimulation of the receptor by a ligand. Thus, an important target for an inhibitor of TRTK function is the extracellular domain of the receptor protein which is both exposed on the cell surface and is the site where extracellular ligands bind. Therefore, included within the scope of the invention are molecules capable of antagonizing the binding of ligands which stimulate TRTK receptors. Such molecules include antibodies specific for the TRTK proteins which prevent the stimulation of the receptor by other ligands.

It is also believed that TRTK proteins are associated with the cytoplasmic membrane and stimulation of these receptors is associated with cancer and other disease states resulting from aberrant cell proliferation. Therefore, soluble derivatives of TRTK receptors which disrupt the response of these receptors by competing for the stimulatory ligand are also within the scope of the present invention. Suitable soluble derivatives of the TRTK receptors include the entire extracellular domains of these receptors or fragments thereof.

Induction of a disease state related to decreased proliferation of cells may result from either the underexpression of TRTK receptors or decreased stimulation by a ligand. In such an instance, the present invention includes antibodies and exogenously added ligands which stimulate TRTK receptors.

Modes of Administration

It will be appreciated that conditions caused by a decrease in the standard or normal level of TRTK receptor activity in an individual, can be treated by administration of TRTK protein. Thus, the invention further provides a method of treating an individual in need of an increased level of TRTK receptor activity comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated TRTK polypeptide of the present invention, particularly a mature form of a TRTK receptor, effective to increase

TRTK receptor activity level in such an individual.

As a general proposition, the total pharmaceutically effective amount of TRTK polypeptide administered parenterally per dose will be in the range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the TRTK polypeptide is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.

Pharmaceutical compositions containing one or more TRTK polypeptides of the present invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease. In certain preferred embodiments in this regard, the cDNA herein disclosed is used to clone genomic DNA of a TRTK receptor gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA then is used for in situ chromosome mapping using well known techniques for this purpose. In addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.

Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with probes from the cDNA as short as 50 or 60 bp. For a review of this technique, see Verma et al, Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance In Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting. Examples

Example 1: Expression and Purification of TRTK in E. coli

The bacterial expression vector pQE9 (pDIO) is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311). pQE9 encodes ampicillin antibiotic resistance ("Amp^r") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., supra, and suitable single restriction enzyme cleavage sites. These elements are arranged such that an inserted DNA fragment encoding a polypeptide expresses that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the amino terminus of that polypeptide.

The DNA sequence encoding the desired portion of a TRTK protein lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers. These primers anneal to the amino terminal sequences of the desired portion of the TRTK protein and to sequences in the deposited construct 3' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE9 vector are added to the 5' and 3' primer sequences, respectively. For cloning the mature TRTK protein of SEQ ID NO:2, the 5' primer has the sequence:

5' CAGTTGGATCCCTGGAAGAGGTATTGCTGGAC 3' (SEQ ID NO:4 containing the underlined BamHl restriction site followed by 21 nucleotides complementary to the nucleotide sequence encoding the amino terminus of the mature TRTK sequence shown in SEQ ID NO: 2. One of ordinary skill in the art would appreciate, of course, that the point in the protein coding sequence where the 5' primer begins may be varied to amplify a DNA segment encoding any desired portion of either complete TRTK protein shown in SEQ ID NO:2 or SEQ ID NO: 3 shorter or longer than the mature forms. The 3' primer has the sequence: 5'TGAGTGTCGACTATCGTCATTATCAGACCTCCACTGAGCCCTGCTG 3' (SEQ ID NO:5) containing the underlined Hindlll restriction site followed by 24 nucleotides complementary to the carboxy terminal coding sequence of the

DNA sequence in SEQ ID NO: 1. Of course, as with the 5' primer, the point in the protein coding sequence where the 3' primer begins may also be varied to amplify a DNA segment encoding any desired portion of the complete TRTK proteins shorter or longer than the mature forms. The amplified TRTK DNA fragment and the vector pQE9 are digested with BamHl and Hindlll and the digested DNAs are then ligated together. Insertion of the TRTK DNA into the restricted pQE9 vector places the TRTK protein coding region downstream from the IPTG-inducible promoter and in- frame with an initiating AUG and the six histidine codons. The ligation mixture is transformed into competent E. coli cells using standard procedures such as those described in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan^r"), is used in carrying out the illustrative example described herein. This strain, which is only one of many that are suitable for expressing TRTK protein, is available commercially from QIAGEN, Inc., supra. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is used to inoculate a large culture, at a dilution of approximately 1 :25 to 1 :250. The cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. Isopropyl-b-D- thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.

The cells are then stirred for 3-4 hours at 4°C in 6 M guanidine-HCl, pH 8. The cell debris is removed by centrifugation, and the supernatant containing the TRTK is loaded onto a nickel-nitrilo-tri-acetic acid ("NiNTA") affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the NI-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the TRTK is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it against phosphate buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6 M-1 M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins can be eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4°C or frozen at -80°C.

Example 2: Cloning and Expression of TRTK protein in a Baculovirus Expression System

In this illustrative example, the plasmid shuttle vector p A2 is used to insert the cloned DNA encoding the complete protein TRTK protein shown in SEQ ID NO:2, including its naturally associated secretary signal (leader) sequence, into a baculovirus to express the mature TRTK protein, using standard methods as described in Summers et al, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of ^'the Autographa californica nuclear polyhedrosis virus ( AcMNPV) followed by convenient restriction sites such as BamHl anάAsp!18. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide. Many other baculovirus vectors could be used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et αl, Virology 170:31 -39.

The cDNA sequence encoding the full length TRTK protein of SEQ ID NO:2, including the AUG initiation codon and the naturally associated leader sequence, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the sequence encoding the TRTK protein shown in SEQ ID NO:2. The 5' primer has the sequence:

5' GGGAACGGATCCGCCATCATGGTGTGTAGCCTATGG 3' (SEQ ID NO : 6) containing the underlined BαmHl restriction enzyme site, an efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J Mol. Biol 196:941-950 (1987), followed by 18 bases of the sequence encoding the TRTK protein shown in SEQ ID NO:2, beginning with the AUG initiation codon. The 3' primer has the sequence:

5' CCCTCTAGACCCTTCTGGTACCAGTGTCCAGGGCTGAGTC 3' (SEQ ID NO: 7) containing the underlined Xbal restriction site followed by 13 nucleotides complementary to the 3' non-coding sequence in SEQ ID NO: 1.

In order to express a portion of the protein containing essentially only the extracellular domain of the TRTK protein shown in SEQ ID NO:2, the same 5' primer shown above is used in conjunction with a different 3' primer. One suitable 3' primer has the sequence: 5' GGCTCTAGATCACCGCTGCATGGCCACCAGC 3' (SEQ ID NO:8) containing the underlined Xbal restriction site followed by 19 nucleotides complementary to sequences shown in SEQ ID NO: 1.

In either instance, the amplified fragment is isolated from a 1 % agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc.,LaJolla, Ca.). The fragment then is digested with BamHl and Xbal and again is purified on a 1% agarose gel. This fragment is designated herein "FI".

The plasmid is digested with the restriction enzymes BαmHl andXbαl and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La

Jolla, Ca.). This vector DNA is designated herein "VI".

Fragment FI and the dephosphorylated plasmid VI are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates. Bacteria are identified that contain the plasmid with the human TRTK gene using the PCR method, in which one of the primers that is used to amplify the gene and the second primer is from well within the vector so that only those bacterial colonies containing the TRTK gene fragment will show amplification of the DNA. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBacTRTK.

Five μg of the plasmid pBacTRTK is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA ("BaculoGold™ baculovirus DNA" , Pharmingen, San Diego, C A.), using the lipofection method described by

Feigner et al, Proc. Natl. Acad. Sci. USA 54:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacTRTK are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27 °C for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10). After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e. g. , Eppendorf) . The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C. The recombinant virus is called V-TRTK. To verify the expression of the TRTK gene, Sf9 cells are grown in Grace's medium supplemented with 10% heat inactivated FBS. The cells are infected with the recombinant baculovirus V-TRTK at a multiplicity of infection ("MOI") of about 2. Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc.,

Rockville, MD). If radiolabeled proteins are desired, 42 hours later, 5 μCi of ³⁵S- methionine and 5 μCi ³⁵S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the mature protein and thus the cleavage point and length of the secretory signal peptide.

Example 3: Cloning and Expression of TRTK in Mammalian Cells

A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g. , RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human HeLa 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy etal, Biochem J. 227:211-219 (1991); Bebbingtoneto/., Bio/Technology 10: 169- 175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

The expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology, 438- 447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al, Cell 47:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHl, Xbal and Aspl 18, facilitate the cloning of the gene of interest. The vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.

Example 3(a): Cloning and Expression in COS Cells

The expression plasmid, pTRTK HA, is made by cloning a cDNA encoding a TRTK into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.). The expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37:161 (1984). The fusion of the HA tag to the target protein allows easy detection and recovery of the recombinant protein with an antibody that recognizes the HA epitope. pcDNAIII contains, in addition, the selectable neomycin marker. A DNA fragment encoding the TRTK protein shown in SΕQ ID NO:2 is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter. The plasmid construction strategy is as follows. The TRTK cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of TRTK in E. coli. Suitable primers include the following, which are used in this example. The 5' primer, containing the underlined BamHl site, a Kozak sequence, an AUG start codon and 5 codons of the 5' coding region of the complete TRTK protein shown in SΕQ ID NO:2 has the following sequence: 5' GGGAACGGATCCGCCATCATGGTGTGTAGCCTATGG 3' (SΕQ ID

NO: 6). The 3' primer, containing the underlined Xbal site and 13 bp of 3' non- coding sequence, has the following sequence (at the 3' end): 5' CCCTCTAGACCCTTCTGGTACCAGTGTCCAGGGCTGAGTC 3' (SΕQ ID NO:7). The PCR amplified DNA fragment and the vector, pcDNAIII, are digested with BamHl and Xbal and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the TRTK-encoding fragment.

For expression of recombinant TRTK, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). Cells are incubated under conditions for expression of TRTK by the vector.

Expression of the TRTK-HA fusion protein is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlo w etal,

Antibodies: A Laboratory Manual, 2nd Ed. ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing ³⁵S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1 % NP-40, 0.1%

SDS, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.

Example 3(b): Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of TRTK protein. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J Biol. Chem. 255:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. Biophys. Ada, 1097:101-143, Page, M. j. and Sydenham, M.A. 1991, Biotechnology 9:64-68). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co- amplified and over-expressed. It is known in the art that this approach may be used to develop cell lines carrying more than 1 ,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained which contain the amplified gene integrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985:438-447) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al, Cell 47:521-530 (1985)). Downstream of the promoter are BamHl, Xbal, and Aspl 18 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene. Other high efficiency promoters can also be used for the expression, e.g., the human β-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the TRTK in a regulated way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Nαtl Acad. Sci. USA 89: 5547-5551). For the polyadenylation of the mRNA other signals, e.g. , from the human growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with the restriction enzymes BamHl and Xbal and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel. The DNA sequence encoding the complete TRTK protein shown in SEQ

ID NO:2, including its leader sequence is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene. The 5' primer has the sequence: 5' GGGAACGGATCCGCCATCATGGTGTGTAGCCTATGG 3' (SEQ ID NO: 6) containing the underlined BamHl restriction enzyme site followed by an efficient signal for initiation of translation in eukaryotes, as described by Kozak, M., J Mol. Biol. 196:941-950 (1987), and 18 bases of the coding sequence of TRTK shown in FIG. 1A-1D (SEQ ID NO:l). The 3' primer has the sequence: 5' CCCTCTAGACCCTTCTGGTACCAGTGTCCAGGGCTGAGTC 3' (SEQ ID NO:7) containing the underlined Xbal restriction site and 13 nucleotides complementary to the non-translated region of the TRTK gene shown in SEQ ID NO:l.

The amplified fragment is digested with the endonucleases BamHl and Xbal and then purified again on a 1 % agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB 101 or

XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. 5 μg of the expression plasmid pC4 is cotransfected with 0.5 μg of the plasmid pSV2-neo using lipofectin (Feigner etal, supra). The plasmid pSV2- neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate

(1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 4: Tissue distribution of TRTK mRNA expression

Northern blot analysis is carried out to examine TRTK gene expression in human tissues, using methods described by, among others, Sambrook et al, cited above. A cDNA probe containing the entire nucleotide sequence encoding a TRTK protein (SEQ ID NO:l) is labeled with ³²P using the reef/prime™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100™ column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT 1200-1. The purified labeled probe is then used to examine various human tissues for TRTK mRNA. Multiple Tissue Northern (MTN) blots containing various human tissues

(H) or human immune system tissues (IM) are obtained from Clontech and are examined with the labeled probe using ExpressHyb™ hybridization solution (Clontech) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at -70 °C overnight, and films developed according to standard procedures.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: HUMAN GENOME SCIENCES, INC. 9410 KEY WEST AVENUE ROCKVILLΞ, MD 20850

APPLICANTS/INVENTORS: SOPPET, DANIEL R.

RUBEN, STEVEN M.

(ii) TITLE OF INVENTION: THYMUS RECEPTOR TYROSINE KINASE (TRTK) PROTEIN AND METHODS OF USE

(iii) NUMBER OF SEQUENCES: 33

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.

(B) STREET: 1100 NEW YORK AVENUE, SUITE 600

(C) CITY: WASHINGTON

(D) STATE: D.C.

(E) COUNTRY: US

(F) ZIP: 20005-3934

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: TO BE ASSIGNED

(B) FILING DATE: HEREWITH

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/042,856

(B) FILING DATE: 28-MAR-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: STΞFFE, ERIC K.

(B) REGISTRATION NUMBER: 36,688

(C) REFERENCE/DOCKET NUMBER: 1488.069PC01/EKS/SGW

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 371-2600

(B) TELEFAX: (202) 371-2540

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3673 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 436..3498

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 436..477

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 478..3498

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

GGAAGAAATA AAGGGATTAT AGTCCACCCA ATTCACAGAC TTCTGAGACT CAGACACGAG 60

GAGAGATAGA GAACCGCCAA TCTCTAGATC AACAAGCAAA GGAGGTGCCA AGCCTGTTTG 120

TCTTCATTGT GACACTGGAG TCTAGATGCT GGGAAGTCCA AGATCAGGGT GCCGGCATGG 180

TCAGTTCCTG GCGAAGCTCT CTTCTAGGTT TCAGACTGCC CTCTTCTTTG TTGTGTCCTC 240

GAATGGCAGA AAAAGGGGTG GCTGTTGGAG GAAGGGAGGA GAGTAAATGA AGAGAAAGAA 300

CTGGAATAAC CCCTTGCAGA AAAAAAAAAA AAGGGAAGCA AGCTTAGCTG TACACCCTGA 360

GTCTTGCAAA AGCTGCAGCC CCACCCAGGA GCAGGGTGGT GGCTGGGGCG ATGGTGGACG 420

CCCTGAAGAT GTCCC ATG GCT ACT GAA GGG GCT GCC CAG TTA GGG AAC AGA 471 Met Ala Thr Glu Gly Ala Ala Gin Leu Gly Asn Arg -14 -10 -5

GTG GCG GGC ATG GTG TGT AGC CTA TGG GTG CTG CTC CTG GTG TCT TCA 519 Val Ala Gly Met Val Cys Ser Leu Trp Val Leu Leu Leu Val Ser Ser 1 5 10

GTT CTG GCT CTG GAA GAG GTA TTG CTG GAC ACC ACC GGA GAG ACA TCT 567 Val Leu Ala Leu Glu Glu Val Leu Leu Asp Thr Thr Gly Glu Thr Ser 15 20 25 30

GAG ATT GGC TGG CTC ACC TAC CCA CCA GGG GGG TGG GAC GAG GTG AGT 615 Glu lie Gly Trp Leu Thr Tyr Pro Pro Gly Gly Trp Asp Glu Val Ser 35 40 45

GTT CTG GAC GAC CAG CGA CGC CTG ACT CGG ACC TTT GAG GCA TGT CAT 663 Val Leu Asp Asp Gin Arg Arg Leu Thr Arg Thr Phe Glu Ala Cys His 50 55 60 GTG GCA GGG GCC CCT CCA GGC ACC GGG CAG GAC AAT TGG TTG CAG ACA 711 Val Ala Gly Ala Pro Pro Gly Thr Gly Gin Asp Asn Trp Leu Gin Thr 65 70 75

CAC TTT GTG GAG CGG CGC GGG GCC CAG AGG GCG CAC ATT CGA CTC CAC 759 His Phe Val Glu Arg Arg Gly Ala Gin Arg Ala His lie Arg Leu His 80 85 90

TTC TCT GTG CGG GCA TGC TCC AGC CTG GGT GTG AGC GGC GGC ACC TGC 807 Phe Ser Val Arg Ala Cys Ser Ser Leu Gly Val Ser Gly Gly Thr Cys 95 100 105 110

CGG GAG ACC TTC ACC CTT TAC TAC CGT CAG GCT GAG GAG CCC GAC AGC 855 Arg Glu Thr Phe Thr Leu Tyr Tyr Arg Gin Ala Glu Glu Pro Asp Ser 115 120 125

CCT GAC AGC GTT TCC TCC TGG CAC CTC AAA CGC TGG ACC AAG GTG GAC 903 Pro Asp Ser Val Ser Ser Trp His Leu Lys Arg Trp Thr Lys Val Asp 130 135 140

ACA ATT GCA GCA GAC GAG AGC TTT CCC TCC TCC TCC TCC TCC TCC TCC 951 Thr lie Ala Ala Asp Glu Ser Phe Pro Ser Ser Ser Ser Ser Ser Ser 145 150 155

TCT TCT TCC TCT GCA GCG TGG GCT GTG GGA CCC CAC GGG GCT GGG CAG 999 Ser Ser Ser Ser Ala Ala Trp Ala Val Gly Pro His Gly Ala Gly Gin 160 165 170

CGG GCT GGA CTG CAA CTG AAC GTC AAA GAG CGG AGC TTT GGG CCT CTC 1047 Arg Ala Gly Leu Gin Leu Asn Val Lys Glu Arg Ser Phe Gly Pro Leu 175 180 185 190

ACC CAA CGC GGC TTC TAC GTG GCC TTC CAG GAC ACG GGG GCC TGC CTG 1095 Thr Gin Arg Gly Phe Tyr Val Ala Phe Gin Asp Thr Gly Ala Cys Leu 195 200 205

GCC CTG GTC GCT GTC AGG CTC TTC TCC TAC ACC TGC CCT GCC GTG CTC 1143 Ala Leu Val Ala Val Arg Leu Phe Ser Tyr Thr Cys Pro Ala Val Leu 210 215 220

CGA TCC TTT GCT TCC TTT CCA GAG ACG CAG GCC AGT GGG GCT GGG GGG 1191 Arg Ser Phe Ala Ser Phe Pro Glu Thr Gin Ala Ser Gly Ala Gly Gly 225 230 235

GCC TCC CTG GTG GCA GCT GTG GGC ACC TGT GTG GCT CAT GCA GAG CCA 1239 Ala Ser Leu Val Ala Ala Val Gly Thr Cys Val Ala His Ala Glu Pro 240 245 250

GAG GAG GAT GGA GTA GGG GGC CAG GCA GGA GGC AGC CCC CCC AGG CTG 12-87 Glu Glu Asp Gly Val Gly Gly Gin Ala Gly Gly Ser Pro Pro Arg Leu 255 260 265 270

CAC TGC AAC GGG GAG GGC AAG TGG ATG GTA GCT GTC GGG GGC TGC CGC 1335 His Cys Asn Gly Glu Gly Lys Trp Met Val Ala Val Gly Gly Cys Arg 275 280 285 TGC CAG CCT GGA TAC CAA CCA GCA CGA GGA GAC AAG GCC TGC CAA GCC 1383 Cys Gin Pro Gly Tyr Gin Pro Ala Arg Gly Asp Lys Ala Cys Gin Ala 290 295 300

TGC CCA CGG GGG CTC TAT AAG TCT TCT GCT GGG AAT GCT CCC TGC TCA 1431 Cys Pro Arg Gly Leu Tyr Lys Ser Ser Ala Gly Asn Ala Pro Cys Ser 305 310 315

CCA TGC CCT GCC CGC AGT CAC GCT CCC AAC CCA GCA GCC CCC GTT TGC 1479 Pro Cys Pro Ala Arg Ser His Ala Pro Asn Pro Ala Ala Pro Val Cys 320 325 330

CCC TGC CTG GAG GGC TTC TAC CGG GCC AGT TCC GAC CCA CCA GAG GCC 1527 Pro Cys Leu Glu Gly Phe Tyr Arg Ala Ser Ser Asp Pro Pro Glu Ala 335 340 345 350

CCC TGC ACT GGT CCT CCA TCG GCT CCC CAG GAG CTT TGG TTT GAG GTG 1575 Pro Cys Thr Gly Pro Pro Ser Ala Pro Gin Glu Leu Trp Phe Glu Val 355 360 365

CAA GGC TCA GCA CTC ATG CTA CAC TGG CGC CTG CCT CGG GAG CTG GGG 1623 Gin Gly Ser Ala Leu Met Leu His Trp Arg Leu Pro Arg Glu Leu Gly 370 375 380

GGT CGA GGG GAC CTG CTC TTC AAT GTC GTG TGC AAG GAG TGT GAA GGC 1671 Gly Arg Gly Asp Leu Leu Phe Asn Val Val Cys Lys Glu Cys Glu Gly 385 390 395

CGC CAG GAA CCT GCC AGC GGT GGT GGG GGC ACT TGT CAC CGC TGC AGG 1719 Arg Gin Glu Pro Ala Ser Gly Gly Gly Gly Thr Cys His Arg Cys Arg 400 405 410

GAT GAG GTC CAC TTC GAC CCT CGC CAG AGA GGC CTG ACT GAG AGC CGA 1767 Asp Glu Val His Phe Asp Pro Arg Gin Arg Gly Leu Thr Glu Ser Arg 415 420 425 430

GTG TTA GTG GGG GGA CTC CGG GCA CAC GTA CCC TAC ATC TTA GAG GTG 1815 Val Leu Val Gly Gly Leu Arg Ala His Val Pro Tyr lie Leu Glu Val 435 440 445

CAG GCT GTT AAT GGG GTG TCT GAG CTC AGC CCT GAC CCT CCT CAG GCT 1863 Gin Ala Val Asn Gly Val Ser Glu Leu Ser Pro Asp Pro Pro Gin Ala 450 455 460

GCA GCC ATC AAT GTC AGC ACC AGC CAT GAA GTG CCC TCT GCT GTC CCT 1911 Ala Ala lie Asn Val Ser Thr Ser His Glu Val Pro Ser Ala Val Pro 465 470 475

GTG GTG CAC CAG GTG AGC CGG GCA TCC AAC AGC ATC ACG GTG TCC TGG 1959 Val Val His Gin Val Ser Arg Ala Ser Asn Ser lie Thr Val Ser Trp 480 485 490

CCG CAG CCC GAC CAG ACC AAT GGG AAC ATC CTG GAC TAT CAG CTC CGC 2007 Pro Gin Pro Asp Gin Thr Asn Gly Asn lie Leu Asp Tyr Gin Leu Arg 495 500 505 510 TAC TAT GAC CAG GCA GAA GAC GAA TCC CAC TCC TTC ACC CTG ACC AGC 2055 Tyr Tyr Asp Gin Ala Glu Asp Glu Ser His Ser Phe Thr Leu Thr Ser 515 520 525

GAG ACC AAC ACT GCC ACC GTG ACA CAG CTG AGC CCT GGC CAC ATC TAT 2103 Glu Thr Asn Thr Ala Thr Val Thr Gin Leu Ser Pro Gly His lie Tyr 530 535 540

AGT TTC CAG GTG CGG GCC CGG ACT GCT GCC GGC CAC GGC CCC TAC GGG 2151 Ser Phe Gin Val Arg Ala Arg Thr Ala Ala Gly His Gly Pro Tyr Gly 545 550 555

GGC AAA GTC TAT TTC CAG ACA CTT CCT CAA GGG GAG CTG TCT TCC CAG 2199 Gly Lys Val Tyr Phe Gin Thr Leu Pro Gin Gly Glu Leu Ser Ser Gin 560 565 570

CTT CCG GAA AGA CTC TCC TTG GTG ATC GGC TCC ATC CTG GGG GCT TTG 2247 Leu Pro Glu Arg Leu Ser Leu Val lie Gly Ser lie Leu Gly Ala Leu 575 580 585 590

GCC TTC CTC CTG CTG GCA GCC ATC ACC GTG CTG GCG GTC GTC TTC CAG 2295 Ala Phe Leu Leu Leu Ala Ala He Thr Val Leu Ala Val Val Phe Gin 595 600 605

CGG AAG CGG CGT GGG ACT GGC TAC ACG GAG CAG CTG CAG CAA TAC AGC 2343 Arg Lys Arg Arg Gly Thr Gly Tyr Thr Glu Gin Leu Gin Gin Tyr Ser 610 615 620

AGC CCA GGA CTC GGG GTG AAG TAT TAC ATC GAC CCC TCC ACC TAC GAG 2391 Ser Pro Gly Leu Gly Val Lys Tyr Tyr He Asp Pro Ser Thr Tyr Glu 625 630 635

GAC CCC TGT CAG GCC ATC CGA GAA CTT GCC CGG GAA GTC GAT CCT GCT 2439 Asp Pro Cys Gin Ala He Arg Glu Leu Ala Arg Glu Val Asp Pro Ala 640 645 650

TAT ATC AAG ATT GAG GAG GTC ATT GGG ACA GGC TCT TTT GGA GAA GTG 2487 Tyr He Lys He Glu Glu Val He Gly Thr Gly Ser Phe Gly Glu Val 655 660 665 670

CGC CAG GGC CGC CTG CAG CCA CGG GGA CGG AGG GAG CAG ACT GTG GCC 2535 Arg Gin Gly Arg Leu Gin Pro Arg Gly Arg Arg Glu Gin Thr Val Ala 675 680 685

ATC CAG GCC CTG TGG GCC GGG GGC GCC GAA AGC CTG CAG ATG ACC TTC 2583 He Gin Ala Leu Trp Ala Gly Gly Ala Glu Ser Leu Gin Met Thr Phe 690 695 700

CTG GGC CGG GCC GCA GTG CTG GGT CAG TTC CAG CAC CCC AAC ATC CTG 2631 Leu Gly Arg Ala Ala Val Leu Gly Gin Phe Gin His Pro Asn He Leu 705 710 715

CGG CTG GAG GGC GTG GTC ACC AAG AGC CGA CCC CTC ATG GTG CTG ACG 2679 Arg Leu Glu Gly Val Val Thr Lys Ser Arg Pro Leu Met Val Leu Thr 720 725 730 GAG TTC ATG GAG CTT GGC CCC CTG GAC AGC TTC CTC AGG CAG CGG GAG 2727 Glu Phe Met Glu Leu Gly Pro Leu Asp Ser Phe Leu Arg Gin Arg Glu 735 740 745 750

GGC CAG TTC AGC AGC CTG CAG CTG GTG GCC ATG CAG CGG GGA GTG GCT 2775 Gly Gin Phe Ser Ser Leu Gin Leu Val Ala Met Gin Arg Gly Val Ala 755 760 765

GCT GCC ATG CAG TAC CTG TCC AGC TTT GCC TTC GTC CAT CGC TCG CTG 2823 Ala Ala Met Gin Tyr Leu Ser Ser Phe Ala Phe Val His Arg Ser Leu 770 775 780

TCT GCC CAC AGC GTG CTG GTG AAT AGC CAC TTG GTG TGC AAG GTG GCC 2871 Ser Ala His Ser Val Leu Val Asn Ser His Leu Val Cys Lys Val Ala 785 790 795

CGT CTT GGC CAC AGT CCT CAG GGC CCA AGT TGT TTG CTT CGC TGG GCA 2919 Arg Leu Gly His Ser Pro Gin Gly Pro Ser Cys Leu Leu Arg Trp Ala 800 805 810

GCC CCA GAG GTC ATT GCA CAT GGA AAG CAT ACA ACA TCC AGT GAT GTC 2967 Ala Pro Glu Val He Ala His Gly Lys His Thr Thr Ser Ser Asp Val 815 820 825 830

TGG AGC TTT GGG ATA CTC ATG TGG GAA GTG ATG AGT TAT GGA GAA CGG 3015 Trp Ser Phe Gly He Leu Met Trp Glu Val Met Ser Tyr Gly Glu Arg 835 840 845

CCT TAC TGG GAC ATG AGT GAG CAG GAG GTA CTA AAT GCA ATA GAG CAG 3063 Pro Tyr Trp Asp Met Ser Glu Gin Glu Val Leu Asn Ala He Glu Gin 850 855 860

GAG TTC CGG CTG CCC CCG CCT CCA GGC TGT CCT CCT GGA TTA CAT CTA 3111 Glu Phe Arg Leu Pro Pro Pro Pro Gly Cys Pro Pro Gly Leu His Leu 865 870 875

CTT ATG TTG GAC ACT TGG CAG AAG GAC CGT GCC CGG CGG CCT CAT TTT 3159 Leu Met Leu Asp Thr Trp Gin Lys Asp Arg Ala Arg Arg Pro His Phe 880 885 890

GAC CAG CTG GTG GCT GCA TTT GAC AAG ATG ATC CGC AAG CCA GAT ACC 3207 Asp Gin Leu Val Ala Ala Phe Asp Lys Met He Arg Lys Pro Asp Thr 895 900 905 910

CTG CAG GCT GGC GGG GAC CCA GGG GAA AGG CCT TCC CAG GCC CTT CTG 3255 Leu Gin Ala Gly Gly Asp Pro Gly Glu Arg Pro Ser Gin Ala Leu Leu 915 920 925

ACC CCT GTG GCC CTG GAC TTT CCT TGT CTG GAC TCA CCC CAG GCC TGG 33O3 Thr Pro Val Ala Leu Asp Phe Pro Cys Leu Asp Ser Pro Gin Ala Trp 930 935 940

CTT TCA GCC ATT GGA CTG GAG TGC TAC CAG GAC AAC TTC TCC AAG TTT 3351 Leu Ser Ala He Gly Leu Glu Cys Tyr Gin Asp Asn Phe Ser Lys Phe 945 950 955 GGC CTC TGT ACC TTC AGT GAT GTG GCT CAG CTC AGC CTA GAA GAC CTG 3399 Gly Leu Cys Thr Phe Ser Asp Val Ala Gin Leu Ser Leu Glu Asp Leu 960 965 970

CCT GCC CTG GGC ATC ACC CTG GCT GGC CAC CAG AAG AAG CTG CTG CAC 3447 Pro Ala Leu Gly He Thr Leu Ala Gly His Gin Lys Lys Leu Leu His 975 980 985 990

CAC ATC CAG CTC CTT CAG CAA CAC CTG AGG CAG CAG GGC TCA GTG GAG 3495 His He Gin Leu Leu Gin Gin His Leu Arg Gin Gin Gly Ser Val Glu 995 1000 1005

GTC TGAGAATGAC GATACCCGTG ACTCAGCCCT GGACACTGGT CCGAGAAGGG 3548

Val

ACATGTGGGA CGTGAGCCGG GCTCCAACAG CCTCTGTGAG AGATGCCCCA CACCAAACCC 3608 AACCCTCCCG ATGGCTGCAT TCCCTGGTCC TCCGCCTCTC CACCAGCCCC CTCCTCATTA 3668 AAGGG 3673

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1006 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Val Cys Ser Leu Trp Val Leu Leu Leu Val Ser Ser Val Leu Ala -16 -15 -10 -5

Leu Glu Glu Val Leu Leu Asp Thr Thr Gly Glu Thr Ser Glu He Gly 1 5 10 15

Trp Leu Thr Tyr Pro Pro Gly Gly Trp Asp Glu Val Ser Val Leu Asp 20 25 30

Asp Gin Arg Arg Leu Thr Arg Thr Phe Glu Ala Cys His Val Ala Gly 35 40 45

Ala Pro Pro Gly Thr Gly Gin Asp Asn Trp Leu Gin Thr His Phe Val 50 55 60

Glu Arg Arg Gly Ala Gin Arg Ala His He Arg Leu His Phe Ser Val 65 70 75 80

Arg Ala Cys Ser Ser Leu Gly Val Ser Gly Gly Thr Cys Arg Glu Thr Phe Thr Leu Tyr Tyr Arg Gin Ala Glu Glu Pro Asp Ser Pro Asp Ser 100 105 110

Val Ser Ser Trp His Leu Lys Arg Trp Thr Lys Val Asp Thr He Ala 115 120 125

Ala Asp Glu Ser Phe Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 130 135 140

Ser Ala Ala Trp Ala Val Gly Pro His Gly Ala Gly Gin Arg Ala Gly 145 150 155 160

Leu Gin Leu Asn Val Lys Glu Arg Ser Phe Gly Pro Leu Thr Gin Arg 165 170 175

Gly Phe Tyr Val Ala Phe Gin Asp Thr Gly Ala Cys Leu Ala Leu Val 180 185 190

Ala Val Arg Leu Phe Ser Tyr Thr Cys Pro Ala Val Leu Arg Ser Phe 195 200 205

Ala Ser Phe Pro Glu Thr Gin Ala Ser Gly Ala Gly Gly Ala Ser Leu 210 215 220

Val Ala Ala Val Gly Thr Cys Val Ala His Ala Glu Pro Glu Glu Asp 225 230 235 240

Gly Val Gly Gly Gin Ala Gly Gly Ser Pro Pro Arg Leu His Cys Asn 245 250 255

Gly Glu Gly Lys Trp Met Val Ala Val Gly Gly Cys Arg Cys Gin Pro 260 265 270

Gly Tyr Gin Pro Ala Arg Gly Asp Lys Ala Cys Gin Ala Cys Pro Arg 275 280 285

Gly Leu Tyr Lys Ser Ser Ala Gly Asn Ala Pro Cys Ser Pro Cys Pro 290 295 300

Ala Arg Ser His Ala Pro Asn Pro Ala Ala Pro Val Cys Pro Cys Leu 305 310 315 320

Glu Gly Phe Tyr Arg Ala Ser Ser Asp Pro Pro Glu Ala Pro Cys Thr 325 330 335

Gly Pro Pro Ser Ala Pro Gin Glu Leu Trp Phe Glu Val Gin Gly Ser 340 345 350

Ala Leu Met Leu His Trp Arg Leu Pro Arg Glu Leu Gly Gly Arg Gly 355 360 365

Asp Leu Leu Phe Asn Val Val Cys Lys Glu Cys Glu Gly Arg Gin Glu 370 375 380

Pro Ala Ser Gly Gly Gly Gly Thr Cys His Arg Cys Arg Asp Glu Val 385 390 395 400

His Phe Asp Pro Arg Gin Arg Gly Leu Thr Glu Ser Arg Val Leu Val 405 410 415

Gly Gly Leu Arg Ala His Val Pro Tyr He Leu Glu Val Gin Ala Val 420 425 430

Asn Gly Val Ser Glu Leu Ser Pro Asp Pro Pro Gin Ala Ala Ala He 435 440 445

Asn Val Ser Thr Ser His Glu Val Pro Ser Ala Val Pro Val Val His 450 455 460

Gin Val Ser Arg Ala Ser Asn Ser He Thr Val Ser Trp Pro Gin Pro 465 470 475 480

Asp Gin Thr Asn Gly Asn He Leu Asp Tyr Gin Leu Arg Tyr Tyr Asp 485 490 495

Gin Ala Glu Asp Glu Ser His Ser Phe Thr Leu Thr Ser Glu Thr Asn 500 505 510

Thr Ala Thr Val Thr Gin Leu Ser Pro Gly His He Tyr Ser Phe Gin 515 520 525

Val Arg Ala Arg Thr Ala Ala Gly His Gly Pro Tyr Gly Gly Lys Val 530 535 540

Tyr Phe Gin Thr Leu Pro Gin Gly Glu Leu Ser Ser Gin Leu Pro Glu 545 550 555 560

Arg Leu Ser Leu Val He Gly Ser He Leu Gly Ala Leu Ala Phe Leu 565 570 575

Leu Leu Ala Ala He Thr Val Leu Ala Val Val Phe Gin Arg Lys Arg 580 585 590

Arg Gly Thr Gly Tyr Thr Glu Gin Leu Gin Gin Tyr Ser Ser Pro Gly 595 600 605

Leu Gly Val Lys Tyr Tyr He Asp Pro Ser Thr Tyr Glu Asp Pro Cys 610 615 620

Gin Ala He Arg Glu Leu Ala Arg Glu Val Asp Pro Ala Tyr He Lys 625 630 635 640

He Glu Glu Val He Gly Thr Gly Ser Phe Gly Glu Val Arg Gin Gly 645 650 655

Arg Leu Gin Pro Arg Gly Arg Arg Glu Gin Thr Val Ala He Gin Ala 660 665 670

Leu Trp Ala Gly Gly Ala Glu Ser Leu Gin Met Thr Phe Leu Gly Arg 675 680 685 Ala Ala Val Leu Gly Gin Phe Gin His Pro Asn He Leu Arg Leu Glu 690 695 700

Gly Val Val Thr Lys Ser Arg Pro Leu Met Val Leu Thr Glu Phe Met 705 710 715 720

Glu Leu Gly Pro Leu Asp Ser Phe Leu Arg Gin Arg Glu Gly Gin Phe 725 730 735

Ser Ser Leu Gin Leu Val Ala Met Gin Arg Gly Val Ala Ala Ala Met 740 745 750

Gin Tyr Leu Ser Ser Phe Ala Phe Val His Arg Ser Leu Ser Ala His 755 760 765

Ser Val Leu Val Asn Ser His Leu Val Cys Lys Val Ala Arg Leu Gly 770 775 780

His Ser Pro Gin Gly Pro Ser Cys Leu Leu Arg Trp Ala Ala Pro Glu 785 790 795 800

Val He Ala His Gly Lys His Thr Thr Ser Ser Asp Val Trp Ser Phe 805 810 815

Gly He Leu Met Trp Glu Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp 820 825 830

Asp Met Ser Glu Gin Glu Val Leu Asn Ala He Glu Gin Glu Phe Arg 835 840 845

Leu Pro Pro Pro Pro Gly Cys Pro Pro Gly Leu His Leu Leu Met Leu 850 855 860

Asp Thr Trp Gin Lys Asp Arg Ala Arg Arg Pro His Phe Asp Gin Leu 865 870 875 880

Val Ala Ala Phe Asp Lys Met He Arg Lys Pro Asp Thr Leu Gin Ala 885 890 895

Gly Gly Asp Pro Gly Glu Arg Pro Ser Gin Ala Leu Leu Thr Pro Val 900 905 910

Ala Leu Asp Phe Pro Cys Leu Asp Ser Pro Gin Ala Trp Leu Ser Ala 915 920 925

He Gly Leu Glu Cys Tyr Gin Asp Asn Phe Ser Lys Phe Gly Leu Cys 930 935 940

Thr Phe Ser Asp Val Ala Gin Leu Ser Leu Glu Asp Leu Pro Ala Leu 945 950 955 960

Gly He Thr Leu Ala Gly His Gin Lys Lys Leu Leu His His He Gin 965 970 975

Leu Leu Gin Gin His Leu Arg Gin Gin Gly Ser Val Glu Val 980 985 990

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1021 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Met Ala Thr Glu Gly Ala Ala Gin Leu Gly Asn Arg Val Ala Gly Met -14 -10 -5 1

Val Cys Ser Leu Trp Val Leu Leu Leu Val Ser Ser Val Leu Ala Leu 5 10 15

Glu Glu Val Leu Leu Asp Thr Thr Gly Glu Thr Ser Glu He Gly Trp 20 25 30

Leu Thr Tyr Pro Pro Gly Gly Trp Asp Glu Val Ser Val Leu Asp Asp 35 40 45 50

Gin Arg Arg Leu Thr Arg Thr Phe Glu Ala Cys His Val Ala Gly Ala 55 60 65

Pro Pro Gly Thr Gly Gin Asp Asn Trp Leu Gin Thr His Phe Val Glu 70 75 80

Arg Arg Gly Ala Gin Arg Ala His He Arg Leu His Phe Ser Val Arg 85 90 95

Ala Cys Ser Ser Leu Gly Val Ser Gly Gly Thr Cys Arg Glu Thr Phe 100 105 110

Thr Leu Tyr Tyr Arg Gin Ala Glu Glu Pro Asp Ser Pro Asp Ser Val 115 120 125 130

Ser Ser Trp His Leu Lys Arg Trp Thr Lys Val Asp Thr He Ala Ala 135 140 145

Asp Glu Ser Phe Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 150 155 160

Ala Ala Trp Ala Val Gly Pro His Gly Ala Gly Gin Arg Ala Gly Leu 165 170 175

Gin Leu Asn Val Lys Glu Arg Ser Phe Gly Pro Leu Thr Gin Arg Gly 180 185 190

Phe Tyr Val Ala Phe Gin Asp Thr Gly Ala Cys Leu Ala Leu Val Ala 195 200 205 210 Val Arg Leu Phe Ser Tyr Thr Cys Pro Ala Val Leu Arg Ser Phe Ala 215 220 225

Ser Phe Pro Glu Thr Gin Ala Ser Gly Ala Gly Gly Ala Ser Leu Val 230 235 240

Ala Ala Val Gly Thr Cys Val Ala His Ala Glu Pro Glu Glu Asp Gly 245 250 255

Val Gly Gly Gin Ala Gly Gly Ser Pro Pro Arg Leu His Cys Asn Gly 260 265 270

Glu Gly Lys Trp Met Val Ala Val Gly Gly Cys Arg Cys Gin Pro Gly 275 280 285 290

Tyr Gin Pro Ala Arg Gly Asp Lys Ala Cys Gin Ala Cys Pro Arg Gly 295 300 305

Leu Tyr Lys Ser Ser Ala Gly Asn Ala Pro Cys Ser Pro Cys Pro Ala 310 315 320

Arg Ser His Ala Pro Asn Pro Ala Ala Pro Val Cys Pro Cys Leu Glu 325 330 335

Gly Phe Tyr Arg Ala Ser Ser Asp Pro Pro Glu Ala Pro Cys Thr Gly 340 345 350

Pro Pro Ser Ala Pro Gin Glu Leu Trp Phe Glu Val Gin Gly Ser Ala 355 360 365 370

Leu Met Leu His Trp Arg Leu Pro Arg Glu Leu Gly Gly Arg Gly Asp 375 380 385

Leu Leu Phe Asn Val Val Cys Lys Glu Cys Glu Gly Arg Gin Glu Pro 390 395 400

Ala Ser Gly Gly Gly Gly Thr Cys His Arg Cys Arg Asp Glu Val His 405 410 415

Phe Asp Pro Arg Gin Arg Gly Leu Thr Glu Ser Arg Val Leu Val Gly 420 425 430

Gly Leu Arg Ala His Val Pro Tyr He Leu Glu Val Gin Ala Val Asn 435 440 445 450

Gly Val Ser Glu Leu Ser Pro Asp Pro Pro Gin Ala Ala Ala He Asn 455 460 465

Val Ser Thr Ser His Glu Val Pro Ser Ala Val Pro Val Val His Gin 470 475 480

Val Ser Arg Ala Ser Asn Ser He Thr Val Ser Trp Pro Gin Pro Asp 485 490 495

Gin Thr Asn Gly Asn He Leu Asp Tyr Gin Leu Arg Tyr Tyr Asp Gin 500 505 510

Ala Glu Asp Glu Ser His Ser Phe Thr Leu Thr Ser Glu Thr Asn Thr 515 520 525 530

Ala Thr Val Thr Gin Leu Ser Pro Gly His He Tyr Ser Phe Gin Val 535 540 545

Arg Ala Arg Thr Ala Ala Gly His Gly Pro Tyr Gly Gly Lys Val Tyr 550 555 560

Phe Gin Thr Leu Pro Gin Gly Glu Leu Ser Ser Gin Leu Pro Glu Arg 565 570 575

Leu Ser Leu Val He Gly Ser He Leu Gly Ala Leu Ala Phe Leu Leu 580 585 590

Leu Ala Ala He Thr Val Leu Ala Val Val Phe Gin Arg Lys Arg Arg 595 600 605 610

Gly Thr Gly Tyr Thr Glu Gin Leu Gin Gin Tyr Ser Ser Pro Gly Leu 615 620 625

Gly Val Lys Tyr Tyr He Asp Pro Ser Thr Tyr Glu Asp Pro Cys Gin 630 635 640

Ala He Arg Glu Leu Ala Arg Glu Val Asp Pro Ala Tyr He Lys He 645 650 655

Glu Glu Val He Gly Thr Gly Ser Phe Gly Glu Val Arg Gin Gly Arg 660 665 670

Leu Gin Pro Arg Gly Arg Arg Glu Gin Thr Val Ala He Gin Ala Leu 675 680 685 690

Trp Ala Gly Gly Ala Glu Ser Leu Gin Met Thr Phe Leu Gly Arg Ala 695 700 705

Ala Val Leu Gly Gin Phe Gin His Pro Asn He Leu Arg Leu Glu Gly 710 715 720

Val Val Thr Lys Ser Arg Pro Leu Met Val Leu Thr Glu Phe Met Glu 725 730 735

Leu Gly Pro Leu Asp Ser Phe Leu Arg Gin Arg Glu Gly Gin Phe Ser 740 745 750

Ser Leu Gin Leu Val Ala Met Gin Arg Gly Val Ala Ala Ala Met Gin ^' 755 760 765 770

Tyr Leu Ser Ser Phe Ala Phe Val His Arg Ser Leu Ser Ala His Ser 775 780 785

Val Leu Val Asn Ser His Leu Val Cys Lys Val Ala Arg Leu Gly His 790 795 800 Ser Pro Gin Gly Pro Ser Cys Leu Leu Arg Trp Ala Ala Pro Glu Val 805 810 815

He Ala His Gly Lys His Thr Thr Ser Ser Asp Val Trp Ser Phe Gly 820 825 830

He Leu Met Trp Glu Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp 835 840 845 850

Met Ser Glu Gin Glu Val Leu Asn Ala He Glu Gin Glu Phe Arg Leu 855 860 865

Pro Pro Pro Pro Gly Cys Pro Pro Gly Leu His Leu Leu Met Leu Asp 870 875 880

Thr Trp Gin Lys Asp Arg Ala Arg Arg Pro His Phe Asp Gin Leu Val 885 890 895

Ala Ala Phe Asp Lys Met He Arg Lys Pro Asp Thr Leu Gin Ala Gly 900 905 910

Gly Asp Pro Gly Glu Arg Pro Ser Gin Ala Leu Leu Thr Pro Val Ala 915 920 925 930

Leu Asp Phe Pro Cys Leu Asp Ser Pro Gin Ala Trp Leu Ser Ala He 935 940 945

Gly Leu Glu Cys Tyr Gin Asp Asn Phe Ser Lys Phe Gly Leu Cys Thr 950 955 960

Phe Ser Asp Val Ala Gin Leu Ser Leu Glu Asp Leu Pro Ala Leu Gly 965 970 975

He Thr Leu Ala Gly His Gin Lys Lys Leu Leu His His He Gin Leu 980 985 990

Leu Gin Gin His Leu Arg Gin Gin Gly Ser Val Glu Val 995 1000 1005

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : CAGTTGGATC CCTGGAAGAG GTATTGCTGG AC 32 (2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : TGAGTGTCGA CTATCGTCAT TATCAGACCT CCACTGAGCC CTGCTG 46

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GGGAACGGAT CCGCCATCAT GGTGTGTAGC CTATGG 36

(2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : CCCTCTAGAC CCTTCTGGTA CCAGTGTCCA GGGCTGAGTC 40

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGCTCTAGAT CACCGCTGCA TGGCCACCAG C 31

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 448 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

TGNCAGTTCG ACATGATGGG TTCCAAANAA TTCGGNCACA GGNAGGCCCC CTGCACTGGT 60

CCTCCATCGG CTCCCCAGGA CTTTTGGTTT GAGGTGCAAG GCTCAGCACT CATGCTACAC 120

TGGCGCCTGC CTCGGGAGCT GGGGGGTCGA GGGGACCTGC TCTTCAATGT CGTGTGCAAG 180

GAGTGTGAAG GCCGCCAGAA CCTGCCAGCG GTGGTGGGGG CACTTGTCAC CGCTGCAGGG 240

ATGAGGTCCA CTTCGACCCT CGCCANAAGG CCTGACTGAG AGCCGAGTGT TAGTGGGGGG 300

ACTCCGGGCA CACGTACCCT AACATCTTAG AGGTGCAGGC TGTTAATGGG GTGTCTGAGC 360

TCAGCCCTGG ACCCTCNTCA NGCTGCAGCA TCAATGTTCA GCANCAGCCA TGAANTGCCC 420

TNTGCTGTTC CTGTGGTTGC AACAAGTT 448 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 413 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GGCAGAGCTG CCACCGTGAC ACAGCTGAGC CCTGGCCACA TCTATGGTTT CCAGGTGCGG 60

GNNNGGACTG CTGCCGGCCA CGCGNCTNAC GGGGCAANAG TCTATTTCCA GACACTTCCT 120

CAAGGGGAGC TGTCTTCCCA GCTTCCGGAA AGACTCTCCT TGGTGAATCG GNTCCATCCT 180

GGGGGCTTTG GGCCTTCCTC CTGCTGGCAG CCATCACCGT GCTGGGCGGT TCGTTTTTCC 240

AGCGGANGGG NCGGTGGGGA CTGGNTTACA CGGNGGCAGC TGCAGCAATN ACAGCAGCCC 300 AGGGATTCGG GGTGAAGTTT TTACATCGAC CCTTNCAANT ACGAGGGACC CTNTTAAGGC 360 CATTCNGGGA TTTTNCCNGG AAGTCGGTTC CGGTTTATTT TCNAGTTTGG GGG 413

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 201 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CCATGCAGTA CCTGTCCAGC TTTGCCTTCG TCCATCGCTC GCTGTCTGCC CACAGCGTGC 60

TGGTGAATAG CCACTTNGTG TGCAAGGTGG CCCGTNTTGG CCACAGTCCT CAGGGCCCAA 120

GTTGTTTGCT TCGCTTGGGC AGCCCCAAGA GGTCATTNCA CANGGANAAG CATACAACAT 180

CCAGTGATGT CTGGGAGCTT T 201 (2) INFORMATION FOR SEQ ID NO : 12 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 195 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GGCACAGCTG CACTGCAACG GGGAGGGCAA GTGGATGGTA GCTGTCGGGG GCTGCCGCTG 60

CCAGCCTGGA TACCAACCAG CACGAGGAGA CAAGGCCTGC CAAGCCTGCC CACGGGGGCT 120

CTATNAAGTC TTCTGCTGGG AATGCTCCCT GCTNCACCAT GCCCTGCTGC CGGCCACGNC 180

NCCTNACGGG GGCCA 195 (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 496 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

CACGTGGAGA TGGGCAGGAG GGCCAGGCTG TCGTCCCCCC TCCACAGACC GACTAAAGAG 60

CAGTCTGGAG GGTGACAAGG GGGCAGCAAG GGGGTGGAAA TGGGAGCNTC ATCCCCAGTC 120

ACCGTTTTGT TCCTCAGGAA GCGGCGTGGG ACTGGCTACA CGGAGCAGCT GCAGCAATAC 180

AGCAGCCCAG GACTCGGGGT GAAGTATTAC ATCGACCCCT CCACCTACGA AGGACCCCTG 240

TTCAGGCCAT CCGAGGAACT TGCCCGGGAA AGTCGATCCT GCTTNNTNTC CAAGGTTTGA 300

GGGAGGTTCA TTGGGGACCA GGTTCTTTTT GGGNGAAGTT NCGNCCAAGG NCCGNCTTGC 360

ANNCCACGGG GGNCCGNAGG GAGCCANATT TTTGGGCCNT CCAGGCCTTT GGGCCCNNGN 420

NCGCCCGAAA GCTTTNNAAT TAACCTTTCT TGGGCCCGGC CCNATTTTTG GGTNAATTTC 480

CAGNNCCCAA AATTTT 496 (2) INFORMATION FOR SEQ ID NO : 14 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 433 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 :

ATCCGCAAGC CAGATACCNT GCAGGCTGGC GGGGACCCAG GGGAAAGGCC TTCCCAGGCC 60

CTTCTGACCC CTGTGGCCNT GGACTTTCCT TGTCTGGACT CACCCCAGGC CTGGCTTTCA 120

GCCATTGGAC TGGAGTNCTA CCAGGACAAC TTCTCCAAGT TTGGCCTCTG TACCTTCAGT 180

GATGTGGCTC AGCTCAGCNT AGAAGACCTG CCTGCCCTGG GCATCACCNT GGCTGGCCAC 240

CAGAAGAAGC TGCTGCACCA CATCCAGCTC CTTCAGCAAC ACCTGAGGCA GCAGGGCTCA 300

GTGGAGGTCT GAGAATGACG ATACCCGTGA CTCAGCCCTG GACACTGGTC CGAGAAGGGA 360

CATGTGGGAC GTGAGCCGGG CTTCAACAGC CTCTTTGAGA GATGCCCCAC AACAAAACCA 420

ACCTTCCGAT GGG 433 (2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 398 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

AGGCCATCCG AGAACTTGCC CGGGAAGTCG ATCCTGCTTA TATCAAGATT GAGGAGGTCA 60

TTGGGACAGG CTCTTTTGGA GAAGTGCGCC AGGGCCGCCT GCAGCCACGG GGACGGAGGG 120

AGCAGACTGT GGCCATCCAG GCCCTGTGGG CGGNGNGGCG CCGAAAGCCT GCAGATGACC 180

TTCCTGGGCC GGGCCGCAGT GCTGGGTCAG TTCCAGCACC CCAACATCCT GCGGCTGGAG 240

GGCGTGGTCA CCAAGAGCCG ACCCCTCATG GTGCTGACGG AGTTCATGGA GCTTNGCCCC 300

CTGGACAGCT TCCTCAGGCA GCGGGAGGGC CAGTTCAGCA GCCTGNAGCT GGTGGCCATG 360

CAGCGGGGAG TGGCTGCTGC CATGCAGTAC CTGTCCAG 398 (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 394 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

TTTTTTTTGC AAATTCCCTT CTTTCCCTTT AATGAGGAGG GGGCTGGTGG AGAGGCGGAG 60

GACCAGGGAA TGCAGCCATC GGGAGGGTTG GGTTTGGTGT GGGGCATCTC TCACAGAGGC 120

TGTTGGAGCC CGGCTCACGT CCCACATGTC CCTTCTCGGA CCAGTGTCCA GGGCTGAGTC 180

ACGGGTATCG TCATTCTCAG ACCTCCACTG AGCCCTGCTG CCTCAGGTGT TGCTGAAGGA 240

GCTGGATGTG GTGCAGCAGC TTCTTCTGGT GGCCAGCCAG GGTGATGCCC AGGGCAGGCA 300

GGTCTTCTAG GCTGAGCTGA GCCACATCAC TGAAGGTACA GAGGCCAAAC TTGGAGAAGT 360

TGTCCTGGTA GCACTCCAGT CCAATGGCTG AAAG 394 (2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 387 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

ACGAGGACCC CTGTCAGGCC ATCCGAGAAC TTGCCCGGGA AGTCGATCCT GCTTATATCA 60

AGATTGAGGA GGTCATTGGG ACAGGCTCTT TTGGAGAAGT GCGCCAGGGC CGCCTGCAGC 120

CACGGGGACG GAGGGAGCAG ACTGTGGCCA TCCAGGCCCT GTGGGCGGNG AGGCGCCGAA 180

AGCCTGCAGA TGACCTTCCT GGGCCGGGCC GCAGTGCTGG GTCAGTTCCA GCACCCCAAC 240

ATCCTGCGGC TGGAGGGCGT GGTCACCAAG AGCCGACCCC TCATGGTGCT GACGGAGTTC 300

ATGGAGCTTG GCCCCCTGGA CAGATTNCTC AGGCGGGAGG GCCAGTTCAG CAGCCTGCAG 360

CTNGTNGCCA TGCAGCGGGG GAGTTGG 387 (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 405 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GAGAACGGCC CTACTGGGAC ATGAACGAGC AGGAGGTACT AAATGCAATA GAGCAAGAGT 60

TCCGGCTGCC CCCACCTCCA GGGTGCCCCC CTGGACTACA TCTACTGATG CTAGACACTT 120

GGCAGAAGGA CCGTGCCCGT CGGCCTCATT TTGACCAGCT GGTAGCTGCA TTTGACAAGA 180

TGATTCGCAA GCCTGATACT CTCCAGGCTG AAGGGGGCTC CGGGGACAGG CCTTCCCAGG 240

CTCTTCTGAA CCCTGTGGCC TTGGACTTCC CTTGCCTGGA CTCTCCCCAG GCCTGGCTTT 300

CAGCCATTGG ACTAGAGTGC TACCAGGACA ACTTCTCCAA GTTTGGTCTT TCCACCTTCA 360

GTGATGTGGC TCAGCTCAGC CTGGAAGACC TGCCAGGCCT GGGCA 405 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 492 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

GTGCCTGCTG CTGTCCCTGT GGTGCACCAG GTGAGCCGGG CATCCAACAG CATCACGGTG 60

TCCTGGCCGC AGCCGACCAG ACCAATGGGA ACATCCTGGA CTATCAGCTC CGCTACTATG 120

ACCAGGCAGA AGACGAATCC CACTCCTTCA CCCTGACCAG CGAGACCAAC ACTGCCACCG 180

TGACACAGCT GAGCCCTGGC CACATCTATG GTTTCCAGGT GCGGGCCCGG ACTGCTGCCG 240

GCCACGGCCC TNACGGGGCA AAAGTCTATT TCCAGACACT TCCTCAAGGG GAGCTGTCTT 300

CCCAGCTTCC GGAAAGACTC TCCTTGGTGA TCGGCTCCAT CCTGGGGGGC TTTGGGCTTT 360

CCTCCTTGCT GCCATGCAGT AACCTGTTCC AAGNTTTTGN CTTTCGTTCC ATTCGGTTCG 420

CTGTTTTGNC CAAAAGCGTG CTGGGTGAAT NAAGCCAATT GGGTGTTGCA AAGGTTGGCC 480

CGTTTTGGGC AA 492 (2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 373 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

GCCACTTGGT GTGCAAGGTG GCCCGTTTTG GCCACAGTCC TCAGGGCCCA ANTTGTTTGC 60

TTCGCTGGGC AGCCCCAGAG GTCATTGCAC ATGGAAAGCA TACAACATCC AGTGATGTCT 120

GGAGCTTTGG GATACTCATG TGGGAAGTGA TGAGTTATGG AGAACGGCCT TACTGGGACA 180

TGAGTNAGCA GGAGGTACTA AATGCAATAG AGCAGGAGTT CCGGCTGCCC CCGCCTCCAG 240

GCTGTCCTCC TGGGATTACA TCTACTTATG TTGGACACTT GGCAGANAGG ACCGTGCCCG 300

GCGGCCTCAT TTTGACCAGC TGGTGGCTGC ATTTGACAAG ATGTTTCCGC AAGCCAGATT 360

ACCCTGCAGG CTG 3-73 (2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 345 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

CAAATTCCCT TCTTTCCCTT TAATGAGGAG GGGGCTGGTG GAGAGGCGGA GGACCAGGGA 60

ATGCAGCCAT CGGGAGGGTT GGGTTTGGTG TGGGGCATCT NTCACAGAGG CTNTTGGAGC 120

CCGGNTCACG TCCCACATGT CCCTTNTCGG ACCAGTNTCC AGGGNTGAGT CACGGGTATC 180

GTCATTNTCA GACCTCCACT GAGCCCTGCT GCCTCAGGTG TTGCTGAAGG AGCTGGATGT 240

GGTGCAGNAN NTTTTTCTGG TGGCCAGCCA GGGTGATGCC CAGGGCAGGC AGGTCTTNTA 300

GGCTGAGCTG AGCCACATCA CTGAAGGTAC AAGAGGGCCA AACTT 345 (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 270 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

AATCCCACTC CTTCACCCTG ACCAGCGAGA CCAACACTGC CACCGTGACA CAGCTGAGCC 60

CTGGCCACAT CTATGGTTTC CAGGTGCGGG CCCGGACTGC TGCCGGCCAC GCNCCTACGG 120

GNAAAAGTCT ATTTCCAGAC ACTTCCTCAA GGGGAGCTGT CTTCCCAGCT TCCGGAAAGA 180

CTCTCCTTGG TGATCGGCTC CATCCTGGGG GCTTTNGCCT TCCTCCTGCT GGCAGCCATC 240

ACCGTGCTGG CGGTCGTCTT CCAGCGGAAG 270 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 326 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: AGAACTTGCC CGGGAAGTCG ATCCTGCTTA TATCAAGATT GAGGAGGTCA TTGGGACAGG 60

CTCTTTTGGA GAAGTGCGCC AGGGCCGCCT GCAGCCACGG GGACGGAGGG AGCAGACTGT 120

GGCCATCCAG GCCCTGTGGG CGGNGGGCGC CGAAAGCCTG CAGATGACCT TCCTGGGCCG 180

GGCCGCAGTG CTGGGTCAGT TCCAGCACCC CAACATCCTG CGGCTGGAGG GCGTNGTCAC 240

CAAGTAGCCG ACCCCTCATG GGTGCTGGAC GGAGTTCAAT GGGAGCTTGG NCCCCTGGNA 300

CAGCTTNCTC AGTGCAGCGG TGAGGG 326 (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 241 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

GCAAATTCCC TTCTTTCCCT TTAATGAGGA GGGGGCTGGT GGAGAGGCGG AGGACCAGGG 60

ANTGCAGCCA TCGGGAGGGT TGGGTTTGGT GTGGGGCATC TCTCACAGAG GCTGTTGGAG 120

CCCGGNTCAC GTCCCACATG TCCCTTNTCG GACCAGTGTC CAGGGNTGAG TCACGGGTAT 180

CGTCATTNTC AGACCTCCAC TGAGCCCTGN TGCCTCAGGT GTTGCTGAAG GAGCTGGATG 240

T 241 (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 238 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

AATGAGGAGG GGGCTGGTGG AGAGGCGGAG GACCAGGGAA TGCAGCCATC GGGAGGGTTG 60

GGTTTGGTGT GGGGCATCTC TCACAGAGGC TGTTGGAGCC CGGCTCACGT CCCACATGTC 120

CCTTCTCGGA CCAGTGTCCA GGGCTGNGTC ACGGGTATCG TCATTCTCAG ACCTCCACTG 180

GAGCCCTGCT GCCTCAGGTN TTNCTGAAAG GAGCTGGATG TGGGTGCAGC AGCTTTCT 238 (2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 192 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

CATGTGGGAA GTGATGAGTT ATGGAGAACG GCCTTACTGG GACATGAGTG AGCAGGAGGT 60

ACTAAATGCA ATAGAGCAGG AGTTCCGGCT GCCCCCGCCT CCAGGCTGTC CTCCTGGATT 120

ACATCTACTT ATGTTGGACA CTTGGCAGAA GGACCGTGCC CGGCGGCCTC ATTTTGACCA 180

GCTGGTGGGC TN 192 (2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 203 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

TTCCTCAAGG GGAGCTGTCT TCCCAGCTTC CGGAAAGACT CTCCTTGGTG ATCGGCTCCA 60

TCCTGNGGGC TTTGGCNTTC CTCCTGCTGG CAGCCATCAC CGTGCTGGCG GTCGTCTTCC 120

AGCGGAACGG CCGTGGGACT GGCTACACGG AGCAAGCTGC AGCAATACAG CAGCCCAGGA 180

CTCGGGGTGA AGTATTACAT CGA 203 (2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 201 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CCATGCAGTA CCTGTCCAGC TTTGCCTTCG TCCATCGCTC GCTGTCTGCC CACAGCGTGC 60

TGGTGAATAG CCACTTNGTG TGCAAGGTGG CCCGTNTTGG CCACAGTCCT CAGGGCCCAA 120

GTTGTTTGCT TCGCTTGGGC AGCCCCAAGA GGTCATTNCA CANGGANAAG CATACAACAT 180

CCAGTGATGT CTGGGAGCTT T 201 (2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 173 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

CTGCCATTCG AGGACACAAC AAAGAAGAGG GCAGTCTGAA ACCTAGAAGA GAGGCTTCGC 60

AGGAACTGAC CATGCCGGCA CCCTGANCTT GGACTTCCCA GCATCTAGAC TCCAGTGTCA 120

CAATGAAGAC AAACAGGCTT GGAACCTCCT TNCCTTGTNG ATCTAGAGAT TGG 173 (2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 91 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CCTCCACTGA GCCCTGCTGC CTCAGGTGTT GCTGAAGGAG CTGGNTNTGG TGCAGCAGCT 60 TCTTCTGGTG GCCAGCCAGG GTNNTGCCCA G 91

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 88 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

CCTGTCAGCC ATCCGAGAAC TTGCCCGGGA AGTCGATCCT GCTTATATCA AGATTGAGGA 60

NGTCATTGGG ACAGGCTCTT TTGGAGAA 88 (2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3974 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

GGTACCTAAG TGAGTAGGGC GTCCGATCGA CGGACGCCTT TTTTTTGAAT TCGTAATCAT 60

GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG 120

CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG 180

CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA 240

TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA 300

CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG 360

TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC 420

AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC 480

CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 540

TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC 600

TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA 660

GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC 720

ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA 780

ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 840

CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA 900

GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG 960

GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC 1020

AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT 1080 CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCGTCGA 1140

CAATTCGCGC GCGAAGGCGA AGCGGCATGC ATTTACGTTG ACACCATCGA ATGGTGCAAA 1200

ACCTTTCGCG GTATGGCATG ATAGCGCCCG GAAGAGAGTC AATTCAGGGT GGTGAATGTG 1260

AAACCAGTAA CGTTATACGA TGTCGCAGAG TATGCCGGTG TCTCTTATCA GACCGTTTCC 1320

CGCGTGGTGA ACCAGGCCAG CCACGTTTCT GCGAAAACGC GGGAAAAAGT GGAAGCGGCG 1380

ATGGCGGAGC TGAATTACAT TCCCAACCGC GTGGCACAAC AACTGGCGGG CAAACAGTCG 1440

TTGCTGATTG GCGTTGCCAC CTCCAGTCTG GCCCTGCACG CGCCGTCGCA AATTGTCGCG 1500

GCGATTAAAT CTCGCGCCGA TCAACTGGGT GCCAGCGTGG TGGTGTCGAT GGTAGAACGA 1560

AGCGGCGTCG AAGCCTGTAA AGCGGCGGTG CACAATCTTC TCGCGCAACG CGTCAGTGGG 1620

CTGATCATTA ACTATCCGCT GGATGACCAG GATGCCATTG CTGTGGAAGC TGCCTGCACT 1680

AATGTTCCGG CGTTATTTCT TGATGTCTCT GACCAGACAC CCATCAACAG TATTATTTTC 1740

TCCCATGAAG ACGGTACGCG ACTGGGCGTG GAGCATCTGG TCGCATTGGG TCACCAGCAA 1800

ATCGCGCTGT TAGCGGGCCC ATTAAGTTCT GTCTCGGCGC GTCTGCGTCT GGCTGGCTGG 1860

CATAAATATC TCACTCGCAA TCAAATTCAG CCGATAGCGG AACGGGAAGG CGACTGGAGT 1920

GCCATGTCCG GTTTTCAACA AACCATGCAA ATGCTGAATG AGGGCATCGT TCCCACTGCG 1980

ATGCTGGTTG CCAACGATCA GATGGCGCTG GGCGCAATGC GCGCCATTAC CGAGTCCGGG 2040

CTGCGCGTTG GTGCGGATAT CTCGGTAGTG GGATACGACG ATACCGAAGA CAGCTCATGT 2100

TATATCCCGC CGTTAACCAC CATCAAACAG GATTTTCGCC TGCTGGGGCA AACCAGCGTG 2160

GACCGCTTGC TGCAACTCTC TCAGGGCCAG GCGGTGAAGG GCAATCAGCT GTTGCCCGTC 2220

TCACTGGTGA AAAGAAAAAC CACCCTGGCG CCCAATACGC AAACCGCCTC TCCCCGCGCG 2280

TTGGCCGATT CATTAATGCA GCTGGCACGA CAGGTTTCCC GACTGGAAAG CGGGCAGTGA 2340

GCGCAACGCA ATTAATGTAA GTTAGCGCGA ATTGTCGACC AAAGCGGCCA TCGTGCCTCC 2400

CCACTCCTGC AGTTCGGGGG CATGGATGCG CGGATAGCCG CTGCTGGTTT CCTGGATGCC 2460

GACGGATTTG CACTGCCGGT AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA 2520

CCAACTCGCG AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA GATCCCCGCG 2580

CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG AAGCCCAACC TTTCATAGAA 2640

GGCGGCGGTG GAATCGAAAT CTCGTGATGG CAGGTTGGGC GTCGCTTGGT CGGTCATTTC 2700

GAACCCCAGA GTCCCGCTCA GAAGAACTCG TCAAGAAGGC GATAGAAGGC GATGCGCTGC 2760 GAATCGGGAG CGGCGATACC GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC 2820

TCTTCAGCAA TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC CACACCCAGC 2880

CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA CCATGATATT CGGCAAGCAG 2940

GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA TGCGCGCCTT GAGCCTGGCG 3000

AACAGTTCGG CTGGCGCGAG CCCCTGATGC TCTTCGTCCA GATCATCCTG ATCGACAAGA 3060

CCGGCTTCCA TCCGAGTACG TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG 3120

CAGGTAGCCG GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT GGATACTTTC 3180

TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC CAATAGCAGC 3240

CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG CGCAAGGAAC GCCCGTCGTG 3300

GCCAGCCACG ATAGCCGCGC TGCCTCGTCC TGCAGTTCAT TCAGGGCACC GGACAGGTCG 3360

GTCTTGACAA AAAGAACCGG GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG 3420

CAGCCGATTG TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA AGCGGCCGGA 3480

GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC TGTCTCTTGA 3540

TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA AGAAAGCCAT CCAGTTTACT 3600

TTGCAGGGCT TCCCAACCTT ACCAGAGGGC GCCCCAGCTG GCAATTCCGG TTCGCTTGCT 3660

GTCCATAAAA CCGCCCAGTC TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT 3720

CTCTTTGCGC TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT CATCCGGGGT 3780

CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA GCCCTTGCGC 3840

CCTGAGTGCT TGCGGCAGCG TGAAGCTTAA AAAACTGCAA AAAATAGTTT GACTTGTGAG 3900

CGGATAACAA TTAAGATGTA CCCAATTGTG AGCGGATAAC AATTTCACAC ATTAAAGAGG 3960

AGAAATTACA TATG 3974 (2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 112 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: AAGCTTAAAA AACTGCAAAA AATAGTTTGA CTTGTGAGCG GATAACAATT AAGATGTACC 60 CAATTGTGAG CGGATAACAA TTTCACACAT TAAAGAGGAG AAATTACATA TG 112

79. 1

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bis)

A. The indications made below relate to the microorganism referred to in the description on page 3_ i line 5

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet D

Name of depositary institution

American Type Culture Collection

Address of depositary institution (including postal code and country)

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

Date of deposit Accession Number ATCC 97830

December 20, 1996

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet D

DNA Plasmid HKFBA76

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank, f not applicable)

The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications, e.g., "Accession Number of Deposit")

For receiving Office use only For International Bureau use only

□ This sheet was received with the international application □ This sheet was received by the International Bureau on:

Authorized officer Authorized officer 79. 2

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bis)

A. The indications made below relate to the microorganism referred to in the description on page 22 , line 8

Name of depositary institution

American Type Culture Collection

Address of depositary institution (including postal code and country)

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

Date of deposit Accession Number ATCC 209645

February 25, 1998

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet C3

DNA Plasmid pHE4a

E. SEPARATE FURNISHING OF INDICATIONS (leave blank ι) ot appl,cable)

For receiving Office use only For International Bureau use only

π This sheet was received with the international application O This sheet was received by the International Bureau on:

Authorized officer Authorized officer

Claims

-80-What Is Claimed Is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising amino acids from about -16 to about 990 in SEQ ID NO:2;

(b) a nucleotide sequence encoding a polypeptide comprising amino acids from about -14 to about 1007 in SEQ ID NO:3;

(c) a nucleotide sequence encoding a polypeptide comprising amino acids from about - 15 to about 990 in SEQ ID NO:2;

(e) a nucleotide sequence encoding a polypeptide comprising amino acids from about -13 to about 1007 in SEQ ID NO:3;

(f) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 990 in SEQ ID NO:2; (g) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 1007 in SEQ ID NO:3;

(h) a nucleotide sequence encoding a TRTK polypeptide extracellular domain;

(i) a nucleotide sequence encoding a TRTK polypeptide transmembrane domain;

(j) a nucleotide sequence encoding a TRTK polypeptide intracellular domain;

(k) a nucleotide sequence encoding a TRTK polypeptide extracellular and intracellular domains with all or part of the transmembrane domain deleted;

(1) a nucleotide sequence encoding a polypeptide having an amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No . 97830; -81-

(m) a nucleotide sequence encoding a mature TRTK polypeptide having an amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97830; and

(n) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (1), or (m).

2. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in SEQ ID NO: 1.

3. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:l encoding a TRTK polypeptide having the amino acid sequence in SEQ ID NO:2 or SEQ ID NO:3.

4. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:l encoding a mature TRTK polypeptide having the amino acid sequence in SEQ ID NO:2 or SEQ ID NO:3.

5. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence of the cDNA clone contained in ATCC Deposit No.

97830.

6. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding a TRTK polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97830.

7. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding a mature TRTK polypeptide having an amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. -82-

8. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (1), (m), or (n) of claim 1 wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

9. An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a TRTK polypeptide having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i),

G), (k), (l), or (m) of claim l .

10. The isolated nucleic acid molecule of claim 3, which encodes an epitope-bearing portion of a TRTK polypeptide selected from the group consisting of: a polypeptide comprising amino acid residues from about 19 to about 184 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 229 to about 414 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 439 to about 563 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 591 to about 684 in SEQ ID NO:2; and a polypeptide comprising amino acid residues from about 784 to about 990 in SEQ ID NO:2.

11. A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector.

12. A recombinant vector produced by the method of claim 11.

13. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 12 into a host cell. -83-

14. A recombinant host cell produced by the method of claim 13.

15. A recombinant method for producing a TRTK polypeptide, comprising culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed and recovering said polypeptide.

16. An isolated TRTK polypeptide having an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) amino acids from about -16 to about 990 in SEQ ID NO:2;

(b) amino acids from about -14 to about 1007 in SEQ ID NO:3; (c) amino acids from about -15 to about 990 in SEQ ID NO:2;

(d) amino acids from about -13 to about 1007 in SEQ ID NO:3;

(e) amino acids from about 1 to about 990 in SEQ ID NO:2;

(f) a TRTK extracellular domain; (g) a TRTK extracellular domain minus the leader sequences;

(h) a TRTK intracellular domain; (i) a TRTK transmembrane domain; (j) the amino acid sequence of TRTK polypeptide extracellular and intracellular domains with all or part of the transmembrane domain deleted; (k) the amino acid sequence of a TRTK polypeptide having a complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97830;

(1) the amino acid sequence of a mature TRTK polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97830; and

(m) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), or (1). -84-

17. A method useful during the diagnosis of cancer, comprising:

(a) assaying TRTK protein gene expression level in mammalian cells or bodily fluid; and

(b) comparing said TRTK protein gene expression level with a standard TRTK protein gene expression level, whereby an increase in said

TRTK protein gene expression level over said standard is indicative of cancer.

18. A method of treating cancer comprising introducing an effective amount of a soluble fragment of the TRTK protein into an individual to be treated in admixture with a pharmaceutically acceptable carrier.

19. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected from the group consisting of:

(a) the nucleotide sequence of a fragment of the sequence shown in SEQ ID NO: 1 , wherein said fragment comprises at least 50 contiguous nucleotides of SEQ ID NO:l, provided that said nucleotide sequence is not

HKFBA76R (SEQ ID NO:9), HTTCR36R (SEQ ID NO: 10), HPBAA66R (SEQ ID NO:l l), HJPBJ20RB (SEQ ID NO:12), HTTCR63R (SEQ ID NO:13), AA351300 (SEQ ID NO:14), T09277 (SEQ ID NO:15), AA609284 (SEQ ID NO:16), T08512 (SEQ ID NO:17), AA171336 (SEQ ID NO:18), R85150 (SEQ ID NO:19), AA297321 (SEQ ID NO:20), T08511 (SEQ ID NO:21), R61179

(SEQ ID NO:22), AA310877 (SEQ ID NO:23), T09276 (SEQ ID NO:24), T03387 (SEQ ID NO:25), H19908 (SEQ ID NO:26), AA158493 (SEQ ID NO:27), AA298297 (SEQ ID NO:28), R52792 (SEQ ID NO:29), H20300 (SEQ ID NO:30), and R87294 (SEQ ID NO:31), or any subfragment thereof; and (b) a nucleotide sequence complementary to a nucleotide sequence in (a). -85-

20. An isolated nucleic acid molecule comprising a polynucleotide encoding a TRTK polypeptide wherein, except for one to fifty conservative amino acid substitutions, said polypeptide has a sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising amino acids from about -16 to about 990 in SEQ ID NO:2;

(h) a nucleotide sequence encoding a TRTK polypeptide extracellular domain;

(i) a nucleotide sequence encoding a TRTK polypeptide transmembrane domain;

(j) a nucleotide sequence encoding a TRTK polypeptide intracellular domain;

(1) a nucleotide sequence encoding a polypeptide having an amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97830; -86-

(n) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), Q), (k), (1), or (m).

21. An isolated TRTK polypeptide wherein, except for at least one conservative amino acid substitution, said polypeptide has a sequence selected from the group consisting of:

(a) amino acids from about - 16 to about 990 in SEQ ID NO:2; (b) amino acids from about -14 to about 1007 in SEQ ID

NO:3;

(c) amino acids from about - 15 to about 990 in SEQ ID NO:2;

(d) amino acids from about -13 to about 1007 in SEQ ID NO:3; (e) amino acids from about 1 to about 990 in SEQ ID NO:2;

(f) a TRTK extracellular domain;

(g) a TRTK extracellular domain minus the leader sequences; (h) a TRTK intracellular domain;

(i) a TRTK transmembrane domain; (j ) the amino acid sequence of TRTK polypeptide extracellular and intracellular domains with all or part of the transmembrane domain deleted;

(k) the amino acid sequence of a TRTK polypeptide having a complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97830; (1) the amino acid sequence of a mature TRTK polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97830; and

(m) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), or (1).