Cloning by complementation and related processes

BACKGROUND
The present invention relates generally to novel cloning methods, to the DNA sequences obtained using these methods, the corresponding expression products of the DNA sequences and antibodies thereto, as well as to novel screening methods for compounds affecting protein activity. More specifically, the present invention provides novel complementation screening methods particularly useful in the isolation of DNAs encoding cyclic nucleotide phosphodiesterase polypeptides (PDEs) and RAS-related proteins. These DNAs, in turn, provide valuable materials useful as hybridization probes for related DNAs and useful in obtaining polypeptide expression products when used to transform suitable host cells.
Of interest to the present invention are the following discussions relating to the cyclic nucleotide phosphodiesterases and RAS related proteins.
The RAS genes were first discovered as the transforming principles of the Harvey and Kirsten murine sarcoma viruses [Ellis et al., Nature, 292:506 (1981)]. The cellular homologs of the oncogenes of Harvey and Kirsten murine sarcoma viruses (H-RAS and K-RAS) constitute two members of the RAS gene family [Shimizu et al., Proc. Natl. Acad. Sci., 80:2112 (1983)]. A third member is N-RAS [Shimizu et al., Proc. Natl. Acad. Sci., 80:2112 (1983)]. These genes are known as oncogenes since point mutations in RAS can result in genes capable of transforming non-cancerous cells into cancerous cells [Tabin et al., Nature, 300:143 (1982); Reddy et al., Nature, 300:149 (1982); Taparowsky et al., Nature, 300:762 (1982)]. Many tumor cells contain RAS genes with such mutations [Capon et al., Nature, 302:33 (1983); Capon et al., Nature, 304:507 (1983); Shimizu et al., Nature, 304:497 (1983); Taparowsky et al., Cell 34:581 (1983); Taparowsky et al., Nature, 300:762 (1982); Barbacid, Ann. Rev. Biochem., 56:779 (1987)].
Despite the importance of the RAS oncogenes to our understanding of cancer, the function of RAS genes in mammals is not known. The RAS proteins are small proteins (21,000 daltons in mammals) which bind GTP and GDP [Papageorge et al., J. Virol., 44:509 (1982)]. The RAS proteins hydrolyze GTP slowly; specific cellular proteins can accelerate this process [McGrath et al., Nature, 310:644 (1984); Trahey et al., Science, 238:542 (1987)]. RAS proteins bind to the inner surface of the plasma membrane [Willingham et al., Cell, 19:1005 (1980)] and undergo a complex covalent modification at their carboxy termini [Hancock et al., Cell, 57:1167 (1989)]. The crystal structure of H-RAS is known [De Vos et al., Science, 239:888 (1988)].
The yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, that have structural and functional homology with mammalian RAS oncogenes [Powers et al., Cell, 36:607 (1984); Kataoka et al., Cell, 40:19 (1985); Defeo-Jones et al., Science, 228:179 (1985); Dhar et al., Nucl. Acids Res., 12:3611 (1984)]. Both RAS1 and RAS2 have been cloned from yeast plasmid libraries and the complete nucleotide sequence of their coding regions has been determined [Powers et al., Cell, 36:607 (1984); DeFeo-Jones et al., Nature, 306:707 (1983)]. The two genes encode proteins with nearly 90% identity to the first 80 amino acid positions of the mammalian RAS proteins, and nearly 50% identity to the next 80 amino acid positions. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% identity for the first 180 positions. After this, at nearly the same position that the mammalian RAS proteins begin to diverge from each other, the two yeast RAS proteins diverge radically. The yeast RAS proteins, like proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid, and X is the terminal amino acid [Barbacid, Ann Rev. Biochem., 56:779 (1987)]. Monoclonal antibody directed against mammalian RAS proteins immumoprecipitates RAS proteins in yeast cells [Powers et al., Cell, 47:413 (1986)]. Thus, the yeast RAS proteins have the same overall structure and interrelationship as is found in the family of mammalian RAS proteins.
RAS genes have been detected in a wide variety of eukaryotic species, including Schizosaccharomyces pombe, Dictyostelium discoidiem and Drosophila melanogaster [Fukui et al., EMBO, 4:687 (1985); Reymond et al., Cell, 39:141 (1984); Shilo et al., Proc. Natl. Acad. Sci. (U.S.A.), 78:6789 (1981); Neuman-Silberberg, Cell, 37:1027 (1984)]. The widespread distribution of RAS genes in evolution indicates that studies of RAS in simple eukaryotic organisms may elucidate the normal cellular functions of RAS in mammals.
Extensive genetic analyses of the RAS1 and RAS2 of S. cerevisiae have been performed. By constructing in vitro RAS genes disrupted by selectable biochemical markers and introducing these by gene replacement into the RAS chromosomal loci, it has been determined that neither RAS1 nor RAS2 is, by itself, an essential gene. However, doubly RAS deficient (ras1.sup.- ras2.sup.-) spores of doubly heterozygous diploids are incapable of resuming vegetative growth. At least some RAS function is therefore required for viability of S. cerevisiae [Kataoka et al., Cell, 37:437 (1984)]. It has also been determined that RAS1 is located on chromosome XV, 7 cM from ADE2 and 63 cM from HIS3; and that RAS2 is located on chromosome XIV, 2 cM from MET4 [Kataoka et al., Cell, 37:437 (1984)].
Mammalian RAS expressed in yeast can function to correct the phenotypic defects that otherwise would result from the loss of both RAS1 and RAS2 [Kataoka et al., Cell, 40:19 (1985)]. Conversely, yeast RASI is capable of functioning in vertebrate cells [De Feo-Jones et al., Science, 228:179 (1985)]. Thus, there has been sufficient conservation of structure between yeast and human RAS proteins to allow each to function in heterologous host cells.
The missense mutant, RAS2.sup.va119 which encodes valine in place of glycine at the nineteenth amino acid position, has the same sort of mutation that is found in some oncogenic mutants of mammalian RAS genes [Tabin et al., Nature, 300:143 (1982); Reddy et al., Nature, 300:149 (1982); Taparowsk et al., Nature, 300:762 (1982)]. Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type RAS alleles [Kataoka et al., Cell, 37:437 (1984)]. When an activated form of the RAS2 gene (e.g., RAS2.sup.va119) is present in haploid cells, yeast cells fail to synthesize glycogen, are unable to arrest in G1, die rapidly upon nutrient starvation, and are acutely sensitive to heat shock [Toda et al., Cell, 40:27 (1985); Sass et al., Proc. Natl. Acad. Sci., 83:9303 (1986)].
S. cerevisiae strains containing RAS2.sup.va119 have growth and biochemical properties strikingly similar to yeast carrying the IAC or bcy1 mutations, which activate the cAMP pathway in yeast [Uno et al., J. Biol. Chem., 257:14110 (1981)]. Yeast strains carrying the IAC mutation have elevated levels of adenylylate cyclase activity. bcy1.sup.- cells lack the regulatory component of the cAMP dependent protein kinase [Uno et al., J. Biol. Chem., 257:14110 (1982); Toda et al., Mol. Cell. Biol., 7:1371 (1987)]. Yeast strains deficient in RAS function exhibit properties similar to adenylyl cyclase-deficient yeast [Toda et al., Cell, 40:27 (1985)]. The bcy1.sup.- mutation suppresses lethality in ras1.sup.- ras2.sup.- yeast. These results suggest that in the yeast S. cerevisiae, RAS proteins function in the cAMP signalling pathway.
Adenylyl cyclase has been shown to be controlled by RAS proteins [Toda et al., Cell, 40:27 (1985)]. RAS proteins, either from yeast or mutans, can stimulate adenylyl cyclase up to fifty fold in in vitro biochemical assays. RAS proteins will stimulate adenylyl cyclase only when bound with GTP [Field et al., Mol. Cell. Biol., 8:2159 (1988)].
The phenotypes resulting from the activation of RAS, including sensitivity to heat shock and starvation, are primarily the result of overexpression or uncontrolled activation of the cAMP effector pathway via adenylyl cyclase [Kataoka et al., Cell, 37:437 (1984); Kataoka et al., Cell, 43:493 (1985); Toda et al., Cell, 40:27 (1985); Field et al., Mol. Cell. Biol., 8:2159 (1988)].
Two S. cerevisiae yeast genes, PDE1 and PDE2, which encode the low and high affinity cAMP phosphodiesterases, respectively, have been isolated [Sass et al., Proc. Natl. Acad. Sci., 83:9303 (1986); Nikawa et al., Mol. Cell. Biol., 7:3629 (1987)]. These genes were cloned from yeast genomic libraries by their ability to suppress the heat shock sensitivity in yeast cells harboring an activated RAS2.sup.va119 gene. Cells lacking the PDE genes (i.e., pde1.sup.- pde2.sup.- yeast) are heat shock sensitive, are deficient in glycogen accumulation, fail to grow on an acetate carbon source, and in general have defects due to activation of the cAMP signaling pathway [Nikawa et al., Mol. Cell. Biol., 7:3629 (1987)].
Genetic analysis clearly indicates that RAS proteins have other functions in S. cerevisiae in addition to stimulating adenylyl cyclase [Toda et al., Japan Sci Soc. Press., Tokyo/VNU Sci. Press., pp. 253 (1987); Wigler et al,, Cold Spring Harbor Symposium, LIII:649 (1988); Michaeli et al., EMBO, 8:3039 (1989)]. The precise biochemical nature of these functions is unknown. Experiments with other systems, such as S. pombe and Xenopus laevis oocytes, indicate that RAS stimulation of adenylyl cyclase is not. widespread in evolution [Birchmeier et al., Cell, 43:615 (1985)]. It is unlikely that RAS stimulates adenylyl cyclase in mammals (Beckner et al., Nature, 317:1 (1985)).
Phosphodiesterases (PDEs) are the enzymes responsible for the degradation of cyclic AMP (cAMP) to AMP and cGMP to GMP. Cyclic AMP is a "second messenger" that mediates the response of cells to a variety of hormones and neurotransmitters including calcitonin, chorionic gonadotropin, corticotropin, epinephrine, follicle-stimulating hormone, glucagon, leutenizing hormone, lipotropin, melanocyte-stimulating hormone, norepinephrine, parathyroid hormone, thyroid-stimulating hormone, and vasopressin.
Cellular concentrations of cyclic adenosine monophosphate (cAMP) are controlled not only the rate of cAMP production by adenylyl cyclase, but also by the rate of cAMP degradation by phosphodiesterases. In humans, a number of important physiological responses are controlled by cAMP levels, including mental function, smooth muscle relaxation, strength of cardiac contractility, release of histamine and other immunoreactive molecules, lymphocyte proliferation, and platelet aggregation [Robison et al., Cyclic AMP, Academic Press, New York and London (1971)]. Thus, the range of diseases which can potentially be affected by agents or pharmaceutical compounds which alter cAMP levels include inflammatory processes (e.g., arthritis and asthma), heart failure, smooth muscle cramps, high blood pressure, blood clotting, thrombosis, and mental disorders.
Given the importance of cAMP in the regulation of a variety of metabolic processes, considerable effort has been directed toward developing and evaluating cAMP analogues, as well as inhibitors of phosphodiesterases. One way to modulate cAMP levels in cells is through the modulation of cAMP phosphodiesterase activity. Certain drugs useful in treating heart failure, asthma, depression, and thrombosis, appear to work by inhibiting cAMP phosphodiesterases. The pharmaceutical industry has not been notably successful in finding suitably specific drugs, in part because effective drug screens have not been available. Most tissues contain so many different isoforms of phosphodiesterases that drug screening based on traditional methods involving inhibition of crude tissue extracts is unlikely to yield anything other than a broadly acting inhibitor of phosphodiesterases. Broadly acting inhibitors of cAMP phosphodiesterases, such as theophylline, have many deleterious side effects.
As noted above, PDE inhibitor research has as its goal the development of highly specific PDE inhibitors. This lack of PDE inhibitor specificity is in part attributable to the existence of several distinct molecular forms of PDE present within a single tissue type, indeed, present among the various cell-types comprising a particular tissue type. These various forms can be distinguished according to substrate specificity (cAMP vs. cGMP), intracellular location (soluble vs. membrane bound), response to calmodulin, and can, in certain instances, be selectively inhibited by various therapeutic agents. Developing agents that will selectively act upon PDEs is directed toward reproducing the desirable effects of cyclic nucleotides, e.g., bronchodilation, increased myocardial contractility, anti-inflammation, yet without causing the undesirable effects, e.g., increased heart rate or enhanced lipolysis.
One approach to screening agents for their potential utility as PDE inhibitors, e.g. drug screening, requires "kinetically pure" preparations of PDE enzymes. That is, the use of whole tissue homogenates or extracts is unlikely to identify inhibitors selective for an individual PDE isozyme because most tissues are heterogeneous with respect to cell type and even many cell types contain multiple PDE isozymes.
At least five different families of PDEs have been described based on characteristics such as substrate specificity, kinetic properties, cellular regulatory control, size, and in some instances, modulation by selective inhibitors. [Beavo, Adv. in. Second Mess. and Prot. Phosph. Res. 22.:1-38 (1988)]. The five families include:
______________________________________I Ca.sup.2+ /calmodulin-stimulatedII cGMP-stimulatedIII cGMP-inhibitedIV cAMP-specificV cGMP-specific______________________________________
Within each family there are multiple forms of closely related PDEs. See Beavo, "Multiple Phosphodiesterase Isozymes Background, Nomenclature and Implications", pp. 3-15 In: Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action, Beavo, J. and Houslay, M. D., Eds.; John Wiley & Sons, New York (1990). See, also, Beavo, TIPS, 11:150 (1990).
Of the many distinct PDE enzymes now recognized, for only certain of the cGMP specific PDEs is complete cDNA sequence information available. With the acquisition of complete structural information for all PDEs, it may be possible to identify and localize (cellular and subcellular distribution) each PDE isozyme and thereby design isozyme-selective PDE inhibitors as therapeutic agents for specific diseases allowing avoidance of untoward side-effects. However, the heterogeneity, instability, and relatively low abundance of some of the PDE isozymes have presented major obstacles in purifying and characterizing these enzymes.
Several methods are presently available for cloning mammalian genes. A standard approach to cloning mammalian genes requires obtaining purified protein, determining a partial amino acid sequence of the purified protein, using the partial amino acid sequence to produce degenerate oligonucleotide probes, and screening cDNA libraries with these probes to obtain cDNA encoding the protein. This method is time consuming and, because of the degeneracy of the probes used, may identify sequences other than those encoding the protein(s) of interest. Many mammalian genes have been cloned this way including, for example, the gene encoding the cGMP phosphodiesterase expressed in retina Ovchinnikov et al., FEBS, 223:169 (1987)].
A second approach to cloning genes encoding a protein of interest is to use a known gene as a probe to find homologs. This approach is particularly useful when members of a gene family or families are sufficiently homologous. The Drosophila melanogaster dunce phosphodiesterase gene was used, for example to clone rat homologs. Davis et al., Proc. Natl. Acad. Sci. (U.S.A.), 86:3604 (1989); and Swinnen et al., Proc. Natl. Acad. Sci. (U.S.A.), 86:5325 (1989). Although additional members of one family of phosphodiesterase genes might be cloned once a first member of that family has been cloned, it is never known in advance whether the nucleotide sequences of genes belonging to different phosphodiesterase gene families will exhibit sufficient homology to use probes derived from one family to identify members of another family.
Yet another approach to cloning genes is known as complementation, A number of researchers have reported the isolation of yeast genes by their ability to complement a mutation/defect in the corresponding gene in another yeast. See, for example: McKnight et al., EMBO J., 4:2093 (1985)--Aspergillus nidulans gene encoding alcohol dehydrogenase isolated by its ability to complement an adh1 mutation in S. cerevisiae; Sass et al., PNAS (U.S.A.), 83:9303 (1986)--S. cerevisiae PDE2 gene isolated by its ability to complement a RAS2.sup.va119 allele in S. cerevisiae strain TK161-R2V; Nikawa et al., Mol. Cell. Biol., 7:3629 (1987)--S. cerevisiae PDE1 gene isolated by transforming S. cerevisiae strain TK161-R2V; and Wilson, Molec. Cell. Biol., 8:505 (1988)--S. cerevisiae SRA5 gene isolated by virtue of its ability to rescue a RAS.sup.+ sra5-5 S. cerevisiae strain RW60- 12C.
Yeast have also been used to isolate non-yeast genes. For example, Henikoff et al., Nature, 289:33 (1981), reported the isolation of a D. melanogaster gene by complementation of yeast mutants and Lee et al., Nature, 327:31 (1987), reported the isolation of human gene by its ability to complement a mutation in the cdc2 gene in S. pombe. The expression vector employed included a viral (SV40) promoter.
More recently, complementation screening has been used by the applicants herein to detect and isolate mammalian cDNA clones encoding certain types of phosphodiesterases (PDEs). Colicelli et al., PNAS (U.S.A.), 86:3599 (1989) reports the construction of a rat brain cDNA library in a Saccharomyces cerevisiae expression vector and the isolation therefrom of genes having the capacity to function in yeast to suppress the phenotypic effects of RAS2.sup.va119, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human H-RAS gene. A rat species cDNA so cloned and designated DPD (dunce-like phosphodiesterase) has the capacity to complement the loss of growth control associated with an activated RAS2.sup.va119 gene harbored in yeast strains TK161-R2V. The gene encodes a high-affinity cAMP specific phosphodiesterase that is highly homologous to the cAMP phosphodiesterase encoded by the dunce locus of D. melanogaster.
Relatively few PDE genes have been cloned to date. Of those cloned, most belong to the cAMP-specific family of phosphodiesterases (cAMP-PDEs). See Davis, "Molecular Genetics of the Cyclic Nucleotide Phosphodiesterases", pp. 227-241 in Cyclic Nucleotide Phosphodiesterases: Structure, Regulation, and Drug Action, Beavo, J. and Houslay, M. D., Eds.; John Wiley & Sons, New York; 1990. See also, e.g., Faure et al., PNAS (U.S.A.), 85:8076 (1988)--D. discoideum; Sass et al., supra--S. cerevisiae, PDE class IV, designated PDE2; Nikawa et al., supra--S. cerevisiae, designated PDE1; Wilson et al., supra--S. cerevisiae, designated SRA5; Chen et al., PNAS (U.S.A.), 83:9313 (1986)--D. melanogaster, designated dnc.sup.+ ; Ovchinnikov, et al., supra--bovine retina, designated GMP PDE; Davis et al., supra--rat liver, designated Sat dnc-1; Colicelli, et al., supra--rat brain, designated DPD; Swinnen, et al., PNAS (U.S.A.), 86:5325 (1989)--rat testis, rat PDE1, PDE2, PDE3 and PDE4; and Livi, et al., Mol. Cell. Biol., 10:2678 (1990)--human monocyte, designated hPDE1. See also, LeTrong et al., Biochemistry, 29:10280 (1990) reporting cloning of a DNA encoding a fragment of a bovine adrenal cGMP stimulated PDE and Thompson et al., FASEBJ., 5(6):A1592 (Abstract No. 7092, 1991) reporting the cloning of a "Type II PDE" from rat pheochromocytoma cells.
Thus, there continues to exist a need in the art for improved cloning procedures effective for isolating genes, both of known and unknown function, for expression products sufficiently kinetically pure so as to be suitable for use in drug improved immunological specificity, and for drug screening methods that do not require kinetically pure protein preparations.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to methods for cloning mammalian genes encoding proteins which can function in microorganisms, particularly yeast, and can modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the microorganism. Provided by the invention are mammalian genes cloned according to the method, as well as products encoded by such genes, and antibodies immunologically reactive with the encoded proteins.
More specifically, the present invention relates to a method of detecting mammalian genes that encode products that modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates, or in a biochemical pathway which is controlled, directly or indirectly, by a RAS protein (i.e., "RAS-related Protein); to the genes so cloned; to products (nucleic acids, proteins) encoded by the mammalian genes cloned including novel mammalian genes that encode, for example, cAMP phosphodiesterases, proteins that interact with RAS proteins, and other proteins affecting cell growth and maintenance.
The present method can be used to detect a mammalian gene of interest that functions in a microorganism that is genetically altered or defective in a defined manner (an altered microorganism) to correct the genetic alteration or defect and, as a result, modifies an identifiable phenotypic alteration or characteristic associated with the genetic alteration or defect (produces a phenotype more like that off normal or unaltered microorganism). Altered microorganisms illustrating those useful in practice of methods of the invention include S. cerevisiae strains TK161-R2V, 10DAB and SKN37 and S. pombe strain SP65.
The present invention thus provides novel methods for detecting, in a genetically altered microorganism (such as a mutant yeast or mammalian host cell), a mammalian gene that is capable of modifying a phenotypic alteration associated with a genetic alteration. The steps of the novel methods include: (a) providing mammalian cDNA in an expression vector capable of expressing the mammalian cDNA in the genetically altered microorganism (preferred vectors including an endogenous host cell promoter DNA sequence operatively associated with the cDNA); (b) introducing the expression vector into the genetically altered microorganism; (c) maintaining the genetically altered microorganisms containing the expression vector under conditions appropriate for growth; and (d) identifying genetically altered microorganisms in which the phenotypic alteration associated with the genetic alteration in the microorganism is modified. Optionally included is the step of isolating the cDNA inserted in microorganisms identified in step (d).
Although use of the present method to clone mammalian genes is described in detail in respect to cAMP phosphodiesterases and proteins that interact with RAS proteins, it can be used to clone and identify other mammalian genes that function in an appropriately-selected altered microorganism to correct, complement or supplement the genetic alteration and, as a result, correct the associated phenotypic alteration. Phenotypic alterations of yeast cells which illustrate the invention include heat shock sensitivity, nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on acetate and failure to sporulate.
In presently preferred forms, the novel DNA sequences comprise cDNA sequences; however, alternate DNA forms such as genomic DNA, and DNA prepared by partial or total chemical synthesis from nucleotides, as well as DNA with deletions or mutations, is also within the contemplation of the invention.
Association of DNA sequences provided by the invention with homologous or heterologous species expression control DNA sequences, such as promoters, operators, regulators and the like, allows for in vivo and in vitro transcription to form messenger RNA which, in turn, is susceptible to translation to provide the invention proteins, and related poly- and oligo-peptides in large quantities. Presently preferred vectors for use in practice of the invention include plasmids pADNS, pADANS, pAAUN and pAAUN-ATG.
Specifically provided by the invention are mammalian DNA sequences encoding cyclic nucleotide phosphodiesterases and fragments thereof as well as RAS protein-related DNA sequences which are present as mammalian DNA inserts in bacterial plasmids which are the subject of deposits made Apr. 15, 1991 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 in accordance with U.S. Patent and Trademark Office and Budapest Treaty requirements. Mammalian PDE DNAs made subject of the deposits include:
1. Plasmid pRATDPD in E. coli (A.T.C.C. accession No. 68586) containing a rat brain cDNA insert encoding a dunce-like PDE;
2. Plasmid pJC44x in E. coli (A.T.C.C. accession No. 68603) containing a human glioblastoma cell cDNA insert encoding a cAMP specific PDE;
3. Plasmid pTM3 in E. coli (A.T.C.C. accession No. 68600) containing a human glioblastoma cell cDNA insert encoding a cAMP specific PDE;
4. Plasmid pTM72 in E. coli (A.T.C.C. accession No. 68602) containing a human glioblastoma cell cDNA insert encoding a cAMP specific PDE;
5. Plasmid pPDE21 In E. coli (A.T.C.C. accession No. 68595) containing a human temporal cortical cell cDNA insert encoding a cAMP specific PDE;
6. Plasmid pGB18ARR in E. coli (A.T.C.C. accession No. 68596) containing a human temporal cortical cell cDNA insert encoding a cAMP specific PDE;
7. Plasmid pGB25 In E. coli (A.T.C.C. accession No. 68594) containing a human temporal cortical cell cDNA insert encoding a cAMP specific PDE; and,
8. Plasmid pTM22 In E. coli (A.T.C.C. accession No. 68601) containing a human glioblastoma cell cDNA insert encoding a PDE of unclassifiable family designation.
Mammalian RAS-related DNAs made the subject of deposit include:
9. Plasmid pJC99 in E. coli (A.T.C.C. accession No. 68599) containing a human glioblastoma cell cDNA insert encoding a RAS-related polypeptide;
10. Plasmid pJC265 in E. coli (A.T.C.C. accession No. 68598) containing a human glioblastoma cell cDNA insert encoding a BAS-related polypeptide;
11. Plasm pJC310 in E. coli (A.T.C.C. accession No. 68597) containing a human glioblastoma cell cDNA insert encoding a RAS-related polypeptide;
12. Plasmid pML5 in E. coli (A.T.C.C. accession No. 68593) containing a human glioblastoma cell cDNA insert encoding a RAS-related polypeptide;
13. Plasmid pATG16 in E. coli (A.T.C.C. accession No. 68592) containing a human glioblastoma cell cDNA insert encoding a RAS-related polypeptide; and,
14. Plasmid pATG29 in E. coli (A.T.C.C. accession No. 68591) containing a human glioblastoma cell cDNA insert encoding a RAS-related polypeptide.
Yeast expression plasmids deposited in connection with the present invention include:
15. Plasmid pAAUN in E. coli (A.T.C.C. accession No. 68590);
16. Plasmid pAAUN-ATG in E. coli (A.T.C.C. accession No. 68589);
17. Plasmid pADANS in E. coli (A.T.C.C. accession No. 68587); and,
18. Plasmid pADNS in E. coli (A.T.C.C. accession No. 68588).
Yeast host cells made the subject of deposit in connection with the present invention include:
19. S. pombe SP565 (A.T.C.C. accession No. 74047);
20. S. cerevisiae SKN37 (A.T.C.C. accession No. 74048);
21. S. cerevisiae 10DAB (A.T.C.C. accession No. 74049); and,
22. S. cerevisiae TK161-R2V (A.T.C.C. accession No. 74050).
Novel protein products of the invention include polypeptides having the primary structural conformation (i.e., amino acid sequence) of phosphodiesterase proteins as well as those having the primary structural conformation of non-phosphodiesterase proteins, including peptide fragments thereof and synthetic peptides assembled to be duplicative of amino acid sequences thereof. Proteins, protein fragments, and synthetic peptides of the invention are projected to have numerous uses including therapeutic, diagnostic and prognostic uses and will provide the basis for preparation of monoclonal and polyclonal antibodies specifically immunoreactive with these proteins. Preferred protein fragments and synthetic peptides include those duplicating regions of the proteins which are not involved in substrate binding functions and the most preferred are those which share at least one antigenic epitope with the proteins of the invention.
Use of mammalian host cells for expression of DNAs of the invention is expected to provide for such post-translational modifications (e.g., truncation, lipidation, glycosylation, and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.
Also provided by the present invention are antibody substances (including polyclonal and monoclonal antibodies, chimeric antibodies and single chain antibodies) characterized by their ability to bind with high immunospecificity to the proteins and to their fragments and peptides, recognizing unique epitopes which are not common to other proteins, especially phosphodiesterases.
Also provided by the present invention are novel procedures for the detection and/or quantification of normal, abnormal, or mutated forms of the proteins as well as nucleic acids (e.g., DNA and mRNA) associated therewith. Illustratively, antibodies of the invention may be employed in known immunological procedures for quantitative detection of the proteins in fluid and tissue samples, of DNA sequences of the invention that may be suitably labelled and employed for quantitative detection of mRNA encoding these proteins.
Among the multiple aspects of the present invention, therefore, is the provision of (a) novel nucleic acid sequences encoding cyclic nucleic acid phosphodiesterase polypeptides and RAS-related proteins as hereinafter described, and (b) DNA sequences which hybridize thereto under hybridization conditions of the stringency equal to or greater than the conditions described herein and employed in the initial isolation of certain cDNAs of the invention, as well as (c) DNA sequences encoding the same, or allelic variant, or analog polypeptides through use of, at least in part, degenerate codons. Correspondingly provided are viral vectors or circular plasmid DNA vectors incorporating such DNA sequences and procaryotic and eucaryotic host cells transformed or transfected with such DNA sequences and vectors as well as novel methods for the recombinant production of proteins encoded by the DNA sequences through cultured growth of such hosts and isolation of these proteins from the hosts or their culture media.
The present invention further relates to a method of identifying agents that modify or alter (i.e., reduce or stimulate) the activity of the protein products of such mammalian genes expressed in microorganisms, such as yeast. Identification of such agents can be carried out using two types of screening procedures: one based on biochemical assays of mammalian proteins of known enzymatic function and one based on phenotypic assays for proteins of determined or as yet undetermined function. In the former case, if the encoded proteins are phosphodiesterases, for example, pharmacological screens include assays for chemical agents that alter (i.e., reduce or stimulate) phosphodiesterase activity. In the latter case, if the encoded proteins interact with RAS proteins, for example, pharmacological screens include the assay for agents that reduce or stimulate interactions with RAS proteins. These screening methods can be used with either whole cell preparations or cell extracts and do not require enzyme purification.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof which includes numerous illustrative examples of the practice of the invention, reference being made to the drawing wherein:

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 [FIG. 1(A), 1(B), 1(C) and 1(D), 1(E), 1(F), 1(G), 1(H), 1(I), and 1(J)] is a comparative alignment of the nucleotide sequences of the human cDNA inserts of plasmids pJC44X, pTM3, pGB14 and pGB18ARR, wherein smaller letters designate lack of homology and gaps indicate absence of corresponding base positions;
FIG. 2 [FIG. 2(A), 2(B), 2(C) and 2(D), 2(E), 2(F), 2(G), 2(H), 2(I), and 2(J)] is a comparative alignment of the nucleotide sequences of the human cDNA inserts of plasmids pPDE2RR, pTM72, pPDE7 and pPDE 10x-INV, with smaller letters designating lack of homology and gaps indicating the absence of corresponding base positions;
FIGS. 3(A) and 3(B) is a comparative alignment of the nucleotide sequences of the human cDNA inserts of plasmids pPDE18 and pGB25, with smaller letters designating lack of homology and gaps indicating the absence of corresponding base positions; and
FIG. 4 [FIG. 4(A) 4(B), 4(C) and 4(D)] is a comparative alignment of deduced amino acid sequences of plasmids pTM72 (TM72), pRATDPD, pJC44X, pPDE18 and pPDE21, wherein smaller letters designate non-homologous residues and gaps indicate lack of any residue at the aligned position.

DETAILED DESCRIPTION
The following examples illustrate practice of the invention. Example 1 relates to cloning and identification of mammalian genes by complementation in yeast. Example 2 relates to cloning and identification of mammalian genes by hybridization with mammalian genes cloned by complementation. Example 3 relates to characterization of cloned genes by complementation capacity. Example 4 relates to further characterization of cloned genes by nucleotide sequence analysis. Example 5 relates to screening and identification of agents which alter phosphodiesterase enzymatic activity.
EXAMPLE 1
Cloning of Mammalian Genes by Complementation in Yeast
In its most general form, the methods of the present invention are as follows. ##STR1##
First, a cDNA library of mammalian mRNAs is produced using known techniques. This library can be made by cloning double stranded cDNA into an expression vector. The cDNA can be prepared from a pre-existing cDNA library, or it can be prepared by the reverse transcription of mRNA purified from a tissue or cell line of choice, using standard procedures. Watson et al., In: DNA Cloning, a Practical Approach, IRL Press Oxford (1984)).
The cDNA so obtained is cloned into an expression vector capable of expressing mammalian. cDNA inserts as mRNA which in turn can be translated into protein in a host cell of choice, e.g., altered yeast such as S. pombe SP565 (ras1::Leu2/ras1::Leu2) (A.T.C.C. 74047), S. cerevisiae SKN37 (cap::H1S3) (A.T.C.C. 74048), S. cerevisiae 10DAB (pde1.sup.-, pde2.sup.-) (A.T.C.C. 74049); and S. cerevisiae TK161-R2V (RAS2.sup.va119) (A.T.C.C. 74050). Expression vectors which have been used for this purpose are described in the examples which follow and include pAAUN (A.T.C.C. 68590), pAAUNATG (A.T.C.C. 68589), pADNS (A.T.C.C. 68587), and pADANS (A.T.C.C. 68588).
Preferred expression vectors contain a transcriptional promoter specific for the host cell into which the vector is introduced, e.g., promoters specific for expression in S. cerevisiae. The transcribed mRNA may utilize the ATG of the cDNA insert as the "start" codon or may express the cDNA product as a fusion protein.
The cDNA library (present as cDNA inserts in a selected expression vector) is introduced into a suitable host cell. This host cell contains genetic alterations which cause the host cell to have an identifiable phenotypic alteration or abnormality associated with the genetic alteration. The host cell may be a eukaryotic microorganism, such as the yeast S. cerevisiae or a mammalian cell.
Known methods, such as lithium acetate-induced transformation, are used to introduce the cDNA-containing expression vector. In the examples that follow, transformation of yeast cells was performed with lithium acetate. Yeast cells were grown in either rich medium (YPD) or synthetic medium with appropriate auxotrophic supplements (SC). Mortimer et al., In: The Yeast, 1:385 (1969). Ito et al., J. Bacteriol., 153:163 (1983).
The genetic alterations of the selected host cell, may for example, lead to defects in the metabolic pathways controlled by the RAS proteins and the associated readily discernible phenotype may be sensitivity to heat shock or nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on certain carbon sources, failure to sporulate, failure to mate, or other properties associated with defects in the pathways controlled by or controlling RAS proteins. For example, the genetic alteration can be the presence of the RAS2.sup.va119 gene Yeast containing such an alteration-exhibit heat shock sensitivity, which can be overcome by expression of mammalian genes. In the examples that follow, heat shock experiments were performed by replica plating onto preheated SC plates which were maintained at 55.degree. C. for 10 minutes, allowed to cool, and incubated at 30.degree. C. for 24-48 hrs.
Other host cells with genetic alterations can be chosen, such as disruptions of the PDE1 and PDE2 genes in S. cerevisiae or disruptions of, or the presence of an activated allele of ras1 in S. pombe. Other genetic alterations in a host cell may be correctable by different subsets of mammalian cDNA genes.
After introduction of the cDNA insert-containing expression vector, host cells are maintained under conditions appropriate for host cell growth. Those host cells which have been corrected for their phenotypic alteration are selected or otherwise identified and the mammalian gene which they express can be recovered e.g., by transformation of E. coli with DNA isolated from the host cell. Segregation analysis in the examples that follow was performed by growing yeast transformants in YPD for 2-3 days, plating onto YPD plates, and replica plating onto YPD, SC-leucine (plasmid selection), and YPD heat shock plates. E. coli strain HB101 was used for plasmid propagation and isolation, and strain SCS1 (Stratagene) was used for transformation and maintenance of the cDNA library. Mandel et al., Mol. Biol., 53:159 (1970); Hanahan J. Mol. Biol., 166:557 (1983).
If desired, the mammalian gene can be isolated and sequenced; alternatively, the protein encoded by the gene can be identified and expressed in cultured cells for use in further processes.
Parts A, B, and C below describe the isolation of mammalian genes by complementation in yeast and their subsequent biochemical characterization.
A. Isolation and Biochemical Characterization of a Rat Brain cDNA Encoding a Phosphodiesterase
A rat brain cDNA library was produced and cloned into the yeast expression vector, pADNS. RNA was purified from Sprague-Dawley rat brains by published procedures. Chirgwin et al., Biochem., 18:5294 (1979); Lizardi, Methods Enzymol., 96:24 (1983); Watson et al., In: DNA cloning, a practical approach, IRL, Press Oxford (1984). pADNS consists of a 2.2 kbp BglII to HpaI fragment containing the S. cerevisiae LEU2 gene from YEp213 [Sherman et al., Laboratory Manual for Methods in Yeast Genetics, Sherman, F., Fink, G. R. and Hicks, J. B., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1986)], a 1.6 kbp HpaI to HindIII fragment of the S. cerevisiae 2.mu. plasmid containing the origin of replication, and a 2.1 kbp SspI to EcoRI fragment containing the ampicillin resistance gene from the plasmid pUC18. It also contains a 1.5 kbp BamHI to HindIII fragment of the modified S. cerevisiae alcohol dehydrogenase (ADH1) promoter [Bennetzen et al., J. Biol. Chem., 257:3018 (1982); Ammerer, Meth. Enzymol., 101:192 (1983)] and a 0.6 kbp HindIII to BamHI fragment containing the ADH1 terminator sequences. The promoter and terminator sequences are separated by a polylinker that contains the restriction endonuclease sites NotI, SacII, and SfiI between the existing HindIII and SacI sites.
Double stranded cDNAs were prepared and ligated to NotI linkers, cleaved with NotI restriction enzyme, and cloned into pADNS at the NotI site situated between the alcohol dehydrogenase promoter and termination sequences of the vector. The use of the rare cutting NotI obviated the need for restriction site methylases commonly used in cDNA cloning. cDNAs were ligated to the NotI linker oligonucleotides:
SEQ ID NO: 1
5'-AAGCGGCCGC, and
SEQ ID NO: 2
5'-GCGGCCGCTT.
Approximately 1.5.times.10.sup.5 independent cDNA inserts were contained in the library, with an average insert size of 1.5 kbp. DNA prepared from the cDNA expression library was used to transform the RAS2.sup.va119 yeast strain TK161- R2V. The 50,000 Leu.sup.+ transformants obtained were subsequently tested for heat shock sensitivity. Only one transformant displayed heat shock resistance which was conditional upon retention of the expression plasmid. The plasmid, designated pRATDPD, was isolated from this transformant and the 2.17 kb NotI insert was analyzed by restriction site mapping and nucleotide sequencing. SEQ ID NO: 3 and SEQ ID NO: 4 provide the nucleotide sequence of the insert and the corresponding deduced amino acid sequence. Sequencing was performed using the dideoxy chain termination method. Sanger et al., Proc. Natl. Acad. Sci. (U.S.A.), 74:5463 (1977); Biggin, et al., Proc. Natl. Acad. Sci. (U.S.A.), 80:3963 (1983)). Genalign was used to align the DPD and dunce sequences (GENALIGN is a copyrighted software product of IntelliGenetics, Inc.; developed by Dr. Hugo Martinez).
A large open reading frame of 562 codons was found. The first ATG appears at codon 46 and a protein which initiates at this codon would have a predicted molecular weight of approximately 60 kDa. This rat gene is designated RATDPD. A search for similar sequences was performed by computer analysis of sequence data banks, and the Drosophila melanogaster dunce gene was found. The two genes would encode proteins with an 80% amino acid identity, without the introduction or gaps, over a 252 amino acid region located in the center of the rat DPD cDNA. The dunce gene has been shown to encode a high affinity cAMP phosphodiesterase. Chen et al., Proc. Natl. Acad. Sci. (U.S.A.), 83:9313 (1986); Davis et al., J. Cell Biol., 90:101 (1981); Walter et al., Neurosci., 4:494 (1984)).
To demonstrate that the sequences upstream and downstream of the large sequence identity region were in fact contiguous with that region in the mRNA, rather than artifacts of the method for cDNA cloning, the structure of the cloned cDNA was compared with the structure of DPD cDNAs contained in an independently prepared, first strand cDNA population obtained by reverse transcribing total rat brain poly (A).sup.+ RNA With an oligo dT primer. Oligonucleotide primers complementary to sequences located within the identity region, and to sequences near the 5' or 3' ends of the coding strand, were made. Using either the cloned pRATDPD DNA or the total first strand cDNA material as template, polymerase chain reactions (PCR) were carried out using four different primer sets and the reaction products were analysed by polyacrylamide gel electrophoresis.
Polymerase chain reactions (PCRs) were carried out in thermocycler (Perkin Elmer, Cetus) using a modification of published procedures. Saiki et al., Science, 239:487 (1988). Reaction mixtures contained template DNA (1 ng of cloned DNA, or 1 .mu.g of total first strand cDNA), 25 pmoles of oligonucleotide primers, 200 .mu.M deoxyribonucleotide triphosphates, 10 mM Tris HCl (pH 8.4), 50 mM KCl, 3 mM MgCl.sub.2, and 0.01% (w/v) gelatin. The oligonucleotide primers used were:
SEQ ID NO: 5
A, 5'-CACCCTGCTGACAAACCT.sup.44 ;
SEQ ID NO: 6
B, 5'-ATGGAGACGCTGGAGGAA.sup.153 ;
SEQ ID NO: 7
C, 5'-ATACGCCACATCAGAATG.sup.676 ;
SEQ ID NO: 8
D, 5'-TACCAGAGTATGATTCCC.sup.1449 ;
SEQ ID NO: 9
E, 5'-GTGTCGATCAGAGACTTG.sup.1668 ; and
SEQ ID NO: 10
F, 5'-GCACACAGGTTGGCAGAC.sup.2048.
The superscript numbers indicate position coordinates in pRATDPD SEQ ID NO: 3. Primers C, E and F are non-coding strand sequences. Thirty cycles (1.5 min at 94.degree. C., 3 min at 55.degree. C., and 7 min at 72.degree. C.) were performed and the reaction products were analyzed by polyacrylamide gel electrophoresis.
In each case, a fragment of the predicted length was obtained using either of the template DNAs. The band assignments were confirmed by cleavage with restriction endonucleases having recognition sites within the amplified DNA product. Again, in each case, the primary PCR product obtained using either source of template yielded cleavage products of the predicted sizes. The results indicate that the sequence arrangement in the cloned cDNA faithfully reflects the structure of the rat mRNA.
To analyse the biochemical properties of the pRATDPD gene product, crude cell extracts were prepared from one liter cultures of 10DAB yeast cells which had been transformed with either pADNS or pRATDPD. Yeast strain 10DAB cells are pde1.sup.- and pde2.sup.- and do not have a measureable level of endogenous cyclic nucleotide phosphodiesterase activity. Phosphodiesterase activity assays were performed using cAMP as substrate as follows. Yeast cells were grown at 30.degree. C. for 36.hours in one liter cultures of synthetic media (SC-leucine). Cells were harvested and washed with buffer C (20 mM MES (pH 6.2), 0.1 mM MgCl.sub.2, 0.1 mM EGTA, 1 mM .beta.-mercaptoethanol), were resuspended in 30 ml buffer C with 50 .mu.l 1M PMSF, and were disrupted with a French press. The extracts Were centrifuged at 1,600 g for 10 min and the supernatants were spun at 18,000 g for 90 min (4.degree. C.). The supernatant was assayed for phosphodiesterase activity as in Collicelli et al., supra. All the reactions contained Tris-HCl (pH7.5) (100 mM), cell extract (50 .mu.g protein/ml), 5'-nucleotidase (Sigma, 20 ng/ml) and 10 mM Mg.sup.2+ (unless otherwise stated) and the indicated cyclic nucleotide concentrations. Assays for the cGMP hydrolysis used 1.5 .mu.M cGMP. Inhibition studies employed 5 .mu.M cAMP in the presence of varying amounts of cGMP up to 500 .mu.M. [.sup.3 H]cAMP and [.sup.3 H]cGMP were obtained from NEN (New England Nuclear). Reactions were incubated for 10 min at 30.degree. C. and stopped with 5.times. stop solution (250 mM EDTA, 25 mM AMP, 100 mM cAMP).
Control extracts (10DAB with pADNS) showed no cAMP phosphodiesterase activity. Results with the controls were unchanged when performed at 0.degree. C. or in the absence of Mg.sup.2+ and were comparable to results obtained when no extract was added. These results indicate that there is no detectable background phosphodiesterase activity in the non-transformed control strain 10DAB.
In contrast, considerable cAMP phosphodiesterase activity was seen in the 10AB yeast strain transformed with pRATDPD. The rate of cAMP hydrolysis in the resulting transformants was measured as a function of cAMP concentration. The deduced K.sub.m for cAMP is 3.5 .mu.M and the calculated V.sub.max is 1.1 nmol/min/mg.
The assay conditions were varied to ascertain the cation preferences of the enzyme and to determine the ability of calcium and calmodulin to stimulate its activity. In these assays, Mn.sup.2+ can be utilized as well as Mg.sup.2+, and either cation in 1 mM final concentration was sufficient. Calcium/calmodulin was unable to stimulate the measured phosphodiesterase activity in the extract. A parallel assay using beef heart phosphodiesterase (Boeringer Mannheim) yielded a 6.5 fold stimulation with the addition of calcium/calmodulin. Finally, no cGMP phosphodiesterase activity was detected in these assays. Beef heart phosphodiesterase was again used as a positive control. In addition, cGMP present in amounts 100 fold over substrate concentrations was unable to inhibit cAMP phosphodiesterase activity.
Biochemical characterization of the pRATDPD cDNA product expressed in yeast indicates that it is a high affinity cAMP specific phosphodiesterase, as is dunce. Davis et al., J. Cell. Biol., 90:101 (1981); Walter et al., J. Neurosci., 4 (1984). In addition, the phosphodiesterase activity is not stimulated by the presence of calcium/calmodulin. This property is shared with dunce and is distinct from some other phosphodiesterases. Beavo, In Advances in second messenger and phosphoprotein research Greengard et al., eds., Raven Press (1988). The two proteins, pRATDPD and dunce, thus appear to have similar biochemical characteristics. However, it should also be noted that pRATDPD encodes a protein product which shows much less significant homology (35%) to dunce beyond the previously described highly conserved core region. These non-conserved sequences could result in an altered or refined function for this mammalian dunce homolog.
The pRATDPD nucleotide sequence as set forth in SEQ ID NO: 3 encodes a methionine codon at position 46 and the established reading frame remains open through to position 563, resulting in a protein with a predicted molecular weight of 60 kDa. The same reading frame, however, is open beyond the 5' end of the coding strand. At present, it is not known if the methionine codon at position 46 is the initiating condon for the DPD protein. The coding sequence is interrupted by three closely spaced terminator codons. However, the established reading frame then remains open for an additional 116 codons, followed by more terminator codons, a polyadenylylation consensus signal and a polyadenine stretch. This 3' open reading frame could be incorporated into another dunce-like phosphodiesterase through alternate splicing.
B. Cloning of Human Glioblastoma Cell cDNAs by Complementation
A cDNA library was constructed in .lambda.ZAP using. NotI linkers. In this example, the cDNA derived from mRNA was purified from the human glioblastoma cell line U118MG. Inserts from the .lambda. vector were transferred into two yeast expression vectors pADNS and pADANS. Plasmid pADANS differs from pADNS in that the mRNA transcribed will direct the synthesis of a fusion protein including an N terminal portion derived from the alcohol dehydrogenase protein and the remainder from the mammalian cDNA insert.
The two mammalian cDNA expression libraries so constructed were screened, as in the previous example, for cDNAs capable of correcting the heat shock sensitivity of the S. cerevisiae host TK161-R2V. Several cDNAs were isolated and analysed by sequencing. Four different cDNAs, contained as inserts in plasmids pJC44x, pJC99, pJC265, and pJC310, were thereby discovered, and their DNA and deduced amino acid sequences are provided in SEQ ID NOs: 11 and 112; 13 and 14; 15 and 16; 17 and 118 respectively.
The insert of pJC44x was shown by computer analysis to be homologous to the rat pRATDPD gene and biochemical analysis of cellular lysates demonstrated that it encodes a cAMP phosphodiesterase. The inserts in pJC99, pJC265, and pJC310, show no significant homology to previously isolated genes.
C. Cloning of Human Glioblastoma Cell Phosphodiesterase cDNAs by Complementation
The human gliobastoma cDNA expression library previously described was screened for cDNAs capable of correcting the heat shock sensitivity of the phosphodiesterase deficient yeast strain 10DAB. Several cDNAs were so isolated and analyzed by nucleotide and restriction endonuclease sequencing mapping. The cDNA insert in pTM22 encodes a novel human gene. .Its nucleotide sequence and deduced amino acid sequence are shown in SEQ ID NOs: 19 and 20.
From a computer analysis of the nucleotide sequence of the pTM22 insert putatively encodes a protein homologous to various cAMP phosphodiesterases, such as the bovine Ca.sup.2+ /calmodulin dependent cAMP phosphodiesterase and the rat DPD phosphodiesterase described in Example 1A. Biochemical analysis has proven that the isolated DNA encodes a novel cAMP phosphodiesterase.
Sequences related to the pTM22 insert were found to be expressed in the human heart as well, and splicing variants of TM22 were isolated from a human heart cDNA library using pTM22 insert sequences as a nucleic acid hybridization probe.
Plasmid pTM22 was unable to correct the heat shock sensitivity of RAS2.sup.va119 yeast strains, i.e., of TK161-R2V. It thus appears that the pde1.sup.- pde2.sup.- yeast strain 10DAB is more sensitive to phenotypic reversion by mammalian cAMP phosphodiesterase clones than is the RAS2.sup.va119 yeast strain.
Several other human glioblastoma cDNAs, isolated as inserts in the plasmids designated pTM3 and pTM72, were similarly characterized. These two different cAMP phosphodiesterase cDNAs were found to be very closely related to, but distinct from, the pRATDPD cDNA insert and the pJC99 cDNA insert. Their nucleotide sequences and deduced amino acid sequences are shown in SEQ ID NOs: 21 and 22 (pTM3) 23 and 24 (pTM72), respectively.
Biochemical analysis of cell lysates has established that the cDNAs of pTM3 and pTM72, pJC44x and pRATDPD encode rolipram sensitive cAMP phosphodiesterases.
D. Kinetic Analysis of pPDE cDNA Expression Products
Samples containing approximately 10.sup.10 transformed S. cerevisiae 10DAB cells expressing the human cDNAs inserted in pJC44x, pTM3, a pTM22-like plasmid (designated L22 Met and including a 1.7 kb fragment insert derived from pTM22 and encoding the PDE activity) and pAD72 (a TM72-like clone) were resuspended in 2.5 ml PBS and disrupted by vortexing in the presence of glass beads at 4.degree. C. The supernatant fraction following. Centrifugation for 5 min at 12,000.times.g was the source for enzyme in these studies.
Phosphodiesterase activity was determined as described, with minor modifications, in Davis et al., J. Cyc. Nuc. Res., 5:65-74 (1979). Incubation mixtures contained 40 mM Tris pH 8.0, 1 mM EGTA, 5 mM MgCl.sub.2, 0.1 mg/ml BSA, diluted yeast extract, [.sup.3 H]cAMP, and varying amounts of unlabeled cyclic nucleotides to a final volume of 0.25 ml. Reactions were terminated by the addition of 0.25 ml stop buffer containing 0.5 carbonate pH 9.3, 0.5M.multidot.NaCl and 0.1% SDS. Nucleotide products and unreacted substrates were separated on boronate columns (8.times.33 mm). The products were eluted from the boronate columns with sorbitol into scintillation vials for tritium analysis. All kinetic data represent measurements of initial rates, determined by incubations for multiple time intervals at suitable dilutions of enzyme. Analysis of kinetic data by the Lineweaver-Burk transformation of the Michaelis-Menten kinetic model demonstrates a linear double reciprocal plot indicative of a simple kinetic model for each enzyme tested. Cyclic nucleotide concentrations varied from 3.times.10.sup.-8 to 1.times.10.sup. -4 M [cAMP]. The results obtained are shown in Table 1, below.
TABLE 1______________________________________Preliminary Kinetic Analysis of Human CyclicNucleotide Phosphodiesterases Derivedby Yeast ComplementationClone Name K.sub.m.sup.1 V.sub.max.sup.2______________________________________pJC44x 3 .mu.M 830pAD72 1.3 .mu.M 670pTM3 4.5 .mu.M 16pL22Met 0.1 .mu.M 240______________________________________ .sup.1 expressed as .mu.M cAMP .sup.2 expressed as nmol/min/10.sup.12 cells
E. Cloning of Human Glioblastoma Cell RAS-related cDNAs by Complementation in Yeast
In this example, four human glioblastoma cell cDNAs were isolated which do not encode PDEs. They were obtained by complementation of two genetically altered S. cerevisiae and S. Pombe yeast strains.
Clone S46 was selected by complementation in S. cerevisiae strain RS60.15B. This strain contains a mutant allele of RAS2, RAS2.sup.val119ala15, which renders cells unable to grow at 36.degree. C. [Powers et al., Mol. Cell Biol., 9:390-395 (1989)], because such cells are defective in RAS function at elevated temperatures. Human cDNAs from a human glioblastoma cell library were selected that could complement this defect. One cDNA found this way was designated S46. Its nucleotide and deduced amino acid sequences are provided in SEQ ID NOs: 25 and 26. The deduced amino acid sequence is homologous to a Xenopus laevis gene that encodes a known protein kinase, the S6 protein kinase.
Plasmid pML5 was selected by complementation in another S. cerevisiae strain, SKN37. This particular strain contains a disrupted allele of CAP, cap::HIS3. CAP encodes an adenylyl cyclase associated protein of undetermined function. [Field et al., Cell, 61:319-327 (1990)]. As a consequence of this gene disruption, SKN37 fails to grow in medium rich in amino acids [Gerst et al., Mol. Cell Biol., 11:1248-1257 (1991)]. Human cDNAs were selected that could complement this defect. One cDNA insert found this way is present in pML5. Its nucleotide and deduced amino acid sequences are provided in SEQ ID NOs: 27 and 28. Its coding capacity is not yet certain.
Plasmids pATG16 and pATG29 were selected by complementation in the S. pombe diploid strain SP565. This strain is homozygous for disruptions of ras1 (ras1::LEU2). As a consequence, this strain fails to sporulate [Fukui et al., Cell, 44:329-336 (1986)] and human cDNAs were selected that could complement this defect. DNA sequence information for the inserts of pATG16 and pATG29 is set forth in SEQ ID NOs: 29 and 30; and 31 and 32, respectively. These genes have unknown function The vector used for screening in S. pombe differs from the vector used for screening in S. cerevisiae. This vector, pAAUN-ATG, utilizes an S. pombe specific promoter, the adh promoter, and was constructed as follows. The cloning vector pAAUN was derived from plasmid pART1 (McLeod et al., EMBO J., 6:729-736 (1987) by replacing the S. cerevisiae LEU2 gene with a 1.8 kbp HindIII ura4 fragment from S. pombe and adding NotI linkers at the SmaI site of the polylinker (PL) derived from Viera et al., Methods in Enzymology, 153:3-11 (1987). pAAUN contains the S. pombe adh promter for gene expression and an ARS region for DNA replidation. Plasmid pAAUN-ATG, was derived from plasmid pART8, obtained from David Beach, at Cold Spring Harbor Laboratory, and from pAAUN. The fragment of BamHI-EcoRV in pAAUN was replaced with the fragment of BamHI and EcoRV in pART8 which had a ATG start codon supplied by NdeI site in the polylinker.
EXAMPLE 2
Cloning and Identification of Mammalian Genes by Hybridization with Mammalian Genes Cloned by Complementation
This example relates to the cloning and identification of additional mammalian genes by hybridization to probes having sequences derived from the genes described in Example 1, i.e., those genes cloned via complementation in yeast.
Low and high stringency hybridizations were done under the same conditions for 12 to 16 hours at 65.degree. C. in an aqueous solution consisting of 6 times the normal concentration of sodium citrate (SSC), 5 times the normal concentration of Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 0.05 mg/ml of denatured salmon sperm DNA and probe. After hybridization, nitrocellulose filters are incubated for five minutes in 2.times.SSC, 0.5% SDS, at room temperature, and for twenty minutes in fresh 2.times.SSC, 0.5% SDS, at 60.degree. C.
For high stringency hybridizations only, a third wash is performed for twenty minutes at 60.degree. C. in 0.1.times.SSC, 0.1% SDS. The normal concentration of SSC is 0.15M sodium citrate and 0.15M sodium chloride, and the normal concentration of Denhardt's solution is 0.2 g/l Ficoll, 0.2 g/l polyvinyl/pyrrolidone, and 0.2 g/l bovine serum albumin.
Plasmids pPDET, pPDE10X inv, and pPDE2RR were isolated by low stringency hybridization screens of a human temporal lobe cDNA library using the pRATDPD insert as probe. Nucleotide sequence (SEQ ID NOs: 33, 34 and 35, respectively) comparisons indicate that the inserts are representatives of the same genetic locus as the insert in pTM72. SEQ ID NO: 36 sets out the deduced amino acid sequence of the insert of pPDE2RR.
Plasmids pGB14 and pGB18ARR were obtained in the same manner. DNA sequence analysis (SEQ ID NOs: 37 and 39, respectively) revealed that they are representatives of the same genetic locus as the inserts in pTM3 and pJC44x. The deduced amino acid sequences of the inserts are set out in SEQ ID NOs: 38 and 40, respectively.
Plasmid pGB25 was also obtained by low stringency hybridization using the pRATDPD insert as a probe. Judged by its nucleotide and deduced amino acid sequence as set out in SEQ ID NOS: 41 and 42 it represents a novel member of PDE family IV.
The cDNA insert of pGB25 was used as a probe to obtain pPDE18 and pPDE21. The cDNA of pPDE18 (SEQ ID NO: 43) represents the same locus as that of pGB25 (SEQ ID NO: 41) and contains more sequence information than the pGB25 cDNA. The pPDE21 insert represents a fourth member of PDE family IV. Its DNA and deduced amino acid sequences are set out in SEQ ID NOs: 45 and 46.
No biochemical data on expression products of these clones has yet been obtained. Their assignment to class IV is made solely based on sequence relationships.
EXAMPLE 3
Characterization of Cloned Genes by Complementation Capacity
This example relates to the further characterization of the genes cloned in Example 1 by their capacity to complement yeast strains other than the yeast strain originally used to clone the gene.
For example, 10DAB cells (pde1.sup.- pde2.sup.-) were transformed with the DPD expression plasmid, pRATDPD, and assayed for heat shock sensitivity. Expression of the rat DPD gene indeed rendered this host resistant to heat shock. Similarly, pJC44x was able to correct the phenotypic defects of this pde1.sup.- pde2.sup.- yeast strain.
In contrast, pJC99, pJC265, and pJC310 were unable to do so. This suggests that the cDNAs of the latter inserts do not encode cAMP phosphodiesterases. Rather, these genes encode proteins of undetermined function which appear to be able to correct phenotypic defects in yeast with activated RAS proteins as reflected by their capacity to complement yeast strain TK161-R2V.
The procedures described below operate to establish that cDNAs need not be cloned by complementation (or by hybridization to DNAs cloned by complementation) in order to be functional in a genetically altered host. Put another way, the following procedures demonstrate that chemical agent screening methodologies according to the present invention need not involve initial direct or indirect cloning of pertinent DNAs by means of complementation.
A. Yeast Phenotype Complementation by Expression of a cDNA Encoding Bovine Brain CaM-PDE
Plasmid pCAM-40 (in E. coli, A.T.C.C. accession No. 68576) includes a bovine brain cDNA insert encoding a 61 kDa Ca.sup.2+ /calmodulin stimulated cyclic nucleotide phosphodiesterase.
A 2.2 kb cDNA fragment, adapted for insertion. into yeast expression plasmids pADNS and pADANS was derived from the plasmid pCAM-40 by polymerase chain reaction. Briefly, the following PCR amplification was employed to alter the pCAM-40 DNA insert to align it appropriately with the ADH1 promoter in the-vectors.
One oligonucleotide primer (Oligo A) used in the PCR reaction
SEQ ID NO: 47
5'-TACGAAGCTTTGATGGGGTCTACTGCTAC-3'
anneals to the pCaM-40 cDNA clone at base pair positions 100-116 and includes a HinDIII site before the initial methionine codon. A second oligonucleotide primer (Oligo B)
SEQ ID NO: 48
5'-TACGAAGCTTTGATGGTTGGCTTGGCATATC-3'
was designed to anneal at positions 520-538 and also includes a HinDIII site two bases before a methionine codon. The third oligonucleotide
SEQ ID NO: 49
5'-ATTACCCCTCATAAAG-3'
annealed to a position in the plasmid that was 3' of the insert. For one reaction, Oligo A and Oligo C were used as primers with pCAM-40 as the template. The nucleic acid product of this reaction included the entire open reading frame. A second reaction used Oligo B and Oligo C as primers on the template pCAM-40 and yielded a nucleic acid product that lacked the portion of the cDNA sequence encoding the calmodulin binding domain. These amplified products were digested with HinDIII and NotI and ligated to HinDIII/NotI-digested yeast expression vectors pADNS and pADANS. Plasmid clones containing inserts were selected and transformed into S. cerevisiae strain 10DAB by lithium acetate transformation.
Transformed yeast were streaked in patches on agar plates containing synthetic medium lacking the amino acid-leucine (SC-leucine agar) and grown for 3 days at 30.degree. C. Replicas of this agar plate were made with three types of agar plates: one replica on SC-leucine agar, one replica on room temperature YPD agar, and three replicas on YPD agar plates that had been warmed to 56.degree. C. The three warmed plates were maintained at 56.degree. C. for 10, 20, or 30 minutes. These replicas were than allowed to cool to room temperature and then all of the plates were placed at 30.degree. C. Yeast transformed with plasmids constructed to express the CaM-PDE were resistant to the thermal pulse. More specifically, both the construct designed too express the complete open reading frame and that designed to express the truncated protein (including the catalytic region but not the calmodulin binding domain), in either pADNS or pADANS, complemented the heat shock sensitivity phenotype of the 10DAB host cells, i.e., rendered them resistant to the 56.degree. C. temperature pulse.
B. Biochemical Assay of Expression Products
The CaM-PDE expression product was evaluated by preparing cell-free extracts from the yeast and measuring the extracts' biochemical phosphodiesterase activity. For this purpose, 200 ml cultures of transformed yeast Were grown in liquid SC-leucine to a density of about 6 million cells per ml. The cells were collected by centrifugation and the cell pellets were frozen. Extracts were prepared by thawing the frozen cells on ice, mixing the cells with 1 ml of PBS and an equal volume of glass beads, vortexing them to disrupt the yeast cells, and centrifuging the disrupted cells at approximately 12,000.times.g for 5 min to remove insoluble debris. The supernatant was assayed for phosphodiesterase activity.
Extracts of yeast cells, up to 50 .mu.l, were assayed for phosphodiesterase activity in 50 mM Tris (pH 8.0), 1.0 mM EGTA, 0.01 mg/ml BSA (bovine serum albumin), [.sup.3 H]-cyclic nucleotide (4-10,000 cpm/pmol), and 5 mM MgCl.sub.2 in a final volume of 250 .mu.l at 30.degree. C. in 10.times.75 mm glass test tubes. The incubations were terminated by adding 250 .mu.l of 0.5M sodium carbonate (pH 9.3), 1M NaCl, and 0.1% SDS. The products of the phosphodiesterase reaction were separated from the cyclic nucleotide by chromatography on 8.times.33 mm columns of BioRad Affi-Gel 601 boronic acid gel. The columns were equilibrated with 0.25M sodium bicarbonate (pH 9.3) and 0.5M NaCl. The reactions were applied to the columns. The assay tubes were rinsed with 0.25M sodium bicarbonate (pH 9.3) and 0.5M NaCl and this rinse was applied to the columns. The boronate columns were washed twice with 3.75 ml of 0.25M sodium bicarbonate (pH 9.3) and 0.5M NaCl followed by 0.5 ml of 50 mM sodium acetate (pH 4.5). The product was eluted with 2.5 ml of 50 mM sodium acetate (pH 4.5) containing 0.1M sorbitol and collected in scintillation vials. The eluate was mixed with 4.5 ml Ecolite Scintillation Cocktail and the radioactivity measured by liquid scintillation spectrometry.
Both the construct designed to express the complete open reading frame and that designed to express a truncated protein, in either pADNS or pADANS, expressed active protein as determined by biochemical phosphodiesterase assay of cell extracts using cAMP substrate.
C. Yeast Phenotype Complementation by Expression of a cDNA Encoding a Bovine Adrenal cGS-PDE
The plasmid p3CGS-5 (A.T.C.C. 68579) which contains a 4.2-kb DNA fragment encoding the bovine cGMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE), was adapted for cloning into pADNS and pADANS by replacing the first 147 bases of the cDNA with a restriction site suitable for use in the insertion into the plasmids. The oligonucleotide BS1, having the sequence
SEQ ID NO: 50
5'-TACGAAGCTTTGATGCGCCGACAGCCTGC-3',
encodes a HinDIII site and anneals to positions 148-165 of the cDNA insert. An oligonucleotide designated BS3
SEQ ID NO: 51
5'-GGTCTCCTGTTGCAGATATTG-3',
anneals to positions 835-855 just 3' of a unique NsiI site The resulting. PCR-generated fragment following digestion with HinDIII and NsiI was then ligated to HinDIII- and NsiI-digested p3CGS-5 thereby replacing the original 5' end of the bovine cDNA. A plasmid derived from this ligation was digested with HinDIII and NotI to release the modified cDNA insert. The insert was cloned into pADNS and pADANS at their HinDIII and NotI sites. These plasmids were then transformed into the yeast strain 10DAB by the lithium acetate method and the transformed cells were grown and subjected to elevated temperatures as in Section A, above. Both transformations resulted in complementation of the heat shock sensitivity phenotype of the 10DAB host cells.
D. Biochemical Assay of Expression Product
The expression of the cGS-PDE was also evaluated by preparing cell-free extracts from the yeast and measuring the extracts' biochemical phosphodiesterase activity. For this purpose, 50 ml cultures of transformed yeast were grown in liquid SC-leucine to a density of about 10 million cells per ml. Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1986). The cells were collected by centrifugation, the cell pellets were washed once with water, and the final cell pellets were frozen. To prepare an extract, the frozen cells were thawed on ice, mixed with 1 ml of PBS and an equal volume of glass beads, vortexed to disrupt the yeast cells, and centrifuged to remove debris. The supernatant was then assayed for phosphodiesterase activity as in Section B, above.
Constructs in either pADNS or pADANS expressed active protein as determined by biochemical phosphodiesterase assay of cell extracts using cGMP.
EXAMPLE 4
Further Characterization of Cloned Genes by Nucleotide Sequence Analysis
This example describes the family-relatedness of the various human PDE clones described in the preceeding examples. These clones include both those obtained by complementation and those obtained by hybridization.
______________________________________COMPLEMENTATION HYBRIDIZATION______________________________________pJC44x pPDE7pTM22 pPDE10 .times. invpTM3 pPE2RRpTM72 pGB14 pGB18ARR pGB25 pPDE21 pPDE18______________________________________
The uniqueness of its DNA sequence indicates that the pPDE21 cDNA derives from a locus herein designated PDE Class IV1. Plasmid pTM3, pJC44x, pGB18ARR and pGB14 cDNA all derive from the same genetic locus, herein designated PDE Class IV2. Evidence for this relation is shown in FIGS. 1(A)-1(J) demonstrating virtual sequence identity.
Likewise pTM72, pPDE7, pPDE10Xinv, and pPDE2RR cDNAs all derive from a genetic locus, herein designated PDE Class IV4. Evidence for this relation is shown in FIGS. 2(A)-2(J) demonstrating virtual sequence identity.
The cDNAs of pGB25 and pPDE18 derive from yet another genetic locus, herein designated PDE class IV3. Evidence of this relation is shown in FIGS. 3(A)-(B) which demonstrates virtual sequence identity.
The sequences derived from any given locus are not precisely identical. These sequence deviations can derive from a number of different sources including, sequencing errors, true polymorphisms in human populations, cloning artifacts, and differences in splicing patterns. Differences in splicing patterns perhaps account for the major differences in the pTM3 and pJC44x inserts. The pJC44x insert cDNA also may contain some cloning artifacts. Sequence errors, not only for the clones described above, but also for published PDE sequences may have occurred. Naturally occuring sequence variations, or polymorphisms, may also account for the observed results. This introduces some uncertainty into the deduced amino acid sequence of the product of a given locus. Accordingly, it is to be appreciated that the nucleotide sequences claimed encompass not only the specific sequences claimed but also DNA sequences which are substantially the same as those provided herein for cloned cDNAs of interest.
The PDE family IV classes 1-4 comprise a gene family that is related to the rat DPD. The evidence for this is based on the similarity of the encoded amino acid sequences of representatives of this family.
Ostensibly, there are just four members of PDE family IV. In the description that follows, the term "human dunce PDEs" refers to all members of family IV, i.e., the genes that show nucleotide sequence homology to the Drosophila dunce PDE.
Only a subset of the members of a gene family may be expressed in any given tissue. Attempts to quantitate a gene family by studying cDNAs cloned from one, or only a few, tissues may therefore underestimate the total number of members of the family. However, analysis of genomic DNA avoids this problem. Human genomic DNA was used as a substrate in PCR reactions performed in parallel, each containing one of a number of different pairs of oligonucleotides corresponding to various regions of the family IV PDEs. The regions chosen were those strongly conserved in evolution and/or present in all the known members of this human gene family. The oligonucleotides were comprised of mixtures representing the full degeneracy of codons specifying the desired amino acid sequence. The vast majority of the oligonucleotide pairs tested produced several different PCR products, which were heterogenous in length but always equal to or longer than those produced from the corresponding cDNA. However, two pairs produced only products identical in length to the cDNA. The longer, heterogenous populations of products resulted from the priming of oligonucleotide pairs located on two separate exons. The two oligonucleotide pairs that produced identical length products primed off the same exon.
To confirm that the heterogenous fragment populations truly represented priming from separate exons, human family. IV PDE genomic DNA clones were used as substrates in control PCR reactions. In these experiments, each of these clones produced a single PCR product, which was always equal in length to one of the heterogenous products obtained from genomic DNA.
The products from one of the reactions using oligonucleotides pairs that primed from one exon were cloned and sequenced. The oligonucleotides used were
SEQ ID NO: 52
5'TTYAARTCTNYTNCARGRNGA, and
SEQ ID NO: 53
5'ACNATRTCTRATNACCATYTT
wherein: N is any of the four-nucleotides; Y is C or T; and R is G or A. This corresponds to the fully degenerate codons specifying four potential amino acid sequences
FKLLQ(E/G)EN
represented by SEQ ID NOs: 54 and 55, and
DMVID(M/I)V
represented by SEQ ID NOs: 56 and 57
respectively, the two conserved domains boxed in FIG. 4. Using these primers, four different PCR clones were obtained, each corresponding in nucleotide sequence to one of the members of the known human family IV PDEs. The numbers of clones falling into each category were as follows:
______________________________________TYPE TOTALS______________________________________TM72 type (Class IV4): 16JC44 type (Class IV2): 29PDE18 type (Class IV3): 25PDE21 type (Class IV1): 9Total: 79______________________________________
Assuming that the human genes each exist as single copies (which is consistent with this analysis of the available genomic clones), the four PCR products should be obtained ideally at equal frequency. The slightly skewed distribution obtained here probably reflects differing efficiencies in the production of these products in a PCR reaction due to mismatches with the PCR oligonucleotides. However, all four previously known genes were represented in the final PCR product, and no new sequences were identified. Therefore, the human PDE family IV most likely consists of a total of four members. Had this method identified a novel member of the family, the PCR clone could have been used as a probe to isolate cDNA clones. It is possible, however, that this family IV family has other members which have diverged at the codons specifying the amino acids sequences boxed in FIGS. 4(A)-(D).
The cDNA insert pTM22 represents a genetic locus that is not a member of family IV. The evidence for this is that while the deduced amino acid sequence of the pTM22 insert has the general features expected of a cAMP phosphodiesterase, this sequence is not particularly closely related to the sequences of members of the family IV or of the family I, the Ca.sup.2+ /calmodulin sensitive PDEs, or of the other known PDE families.
EXAMPLE 5
Screening and Identification of Agents Which Alter Enzymatic Activity
In their most general form, the pharmacological screening methods of the invention permit screening for agents that reduce or stimulate the activity of any mammalian protein whose presence or expression in an altered microbial host cell in which a genetic alteration is associated with an identifiable phenotypic alteration results in correction of the phenotypic alteration. Two general types of screens are possible. Both methods are applicable to either living cells, or cell preparations, or cell extracts.
A. Identification of Agents that Affect Proteins of Known Activity
The first type of pharmacological screen is applicable when the mammalian gene encodes a protein of known and assayable biochemical function. The mammalian gene is first expressed in a microbial host by utilizing an appropriate host expression vector of the type already described. Either whole cells or extracts of host cells can be used. Extracts are prepared, using known techniques, i.e., the cells are disrupted and their cellular constituents released. Crude cellular extract of purified mammalian protein is assayed for the known biochemical function in the presence of agents, the effects of which on the protein are to be assessed. In this manner, agents which inhibit or stimulate the activity of the mammalian protein can be identified.
This type of procedure can be carried out to analyze the effects of selected agents on mammalian cAMP phosphodiesterases. For example, a yeast strain lacking both endogenous PDE1 and PDE2 genes can be used as the host cell, into which cDNA encoding mammalian cAMP phosphodiesterase is introduced in an appropriate expression vector and expressed. Such a host cell is particularly useful because there is no endogenous (background) cAMP phosphodiesterase activity. [Colicelli et al., Proc. Natl. Acad. Sci. (U.S.A.), 86:3599 (1989)]. Hence, activity of the mammalian enzyme can be cleanly assayed even in crude cell extracts. This procedure is illustrated below, in which it is demonstrated that the enzymatic activity of the rat DPD gene product is readily inhibited by the pharmacological agents Rolipram and R020 1724, but not as readily by the pharmacological agent theophylline.
The genes and cells described in the preceeding examples can be used to identify chemical compounds which inhibit the activity of a known enzyme, the rat DPD phosphodiesterase. To test the efficiency of known inhibitory compounds, cell free extracts were made. Yeast cells deficient in endogenous phosphodiesterase (10DAB), and expressing the rat DPD or yeast PDE2 genes from the described expression vector, were used. One liter cultures were harvested, washed in buffer C (20 mM MES(pH 6.2)/0.1 mM MgCl.sub.2 /0.1 mM EGTA/1 mM 2-mercaptoethanol), resuspended in buffer C containing 1.5 mM phenylmethylsulfonyl fluoride, and disrupted in a French press at 4.degree. C. Cell extracts were clarified at 100 g for 10 minutes and at 18000 g for 90 minutes. PDE activities were assayed as published (Charbonneau et al., Proc. Natl. Acad. Sci. (U.S.A.), 83:9308-9312 (1986); Tempel et al., Proc. Natl. Acad. Sci. (U.S.A.), 80:1482-1486 (1983)) in a reaction mix containing 50 .mu.g of cell protein/ml, 100 mM Tris (pH 7.5), 10 mM Mg.sup.++, 5 .mu.M cAMP, 5'-nucleotidase and [.sup.3 H]cAMP. AMP was separated from cAMP using AG1-X8 resin from Bio Rad. About 10.sup.4 cpm were obtained for 10 min reactions and backgrounds (phosphodiesterase deficient-yeast or no extract) were about 300 cpm. The cytosolic fraction was assayed in the presence or absence of inhibitory compounds. These assays measure the amount of adenosine 5' monophosphate (AMP) produced by phosphodiesterase-catalysed hydrolysis of adenosine 3', 5'-cyclic adenosine monophosphate (cAMP). For each extract the percent inhibition for various concentrations of known inhibitors is given in Table 2. The percent inhibition represents the decrease in phosphodiesterase activity relative to measurements made in the absence of inhibitors. Rolipram, and the related compound R020 1724, were the most effective inhibitors of DPD activity.
TABLE 2______________________________________Inhibition of Phosphodiesterases by ChemicalsPhospho- Concentrationdiesterase Agent (.mu.M) Inhibition (%)______________________________________PDE2 Theophylline 250 0.0 IBMX 250 0.0 R020 1724 100 3.0 Rolipram 100 0.0rat DPD Theophylline 250 42. IBMX 250 87. R020 1724 0.1 35. 1.0 52. 10.0 79. 100.0 92. Rolipram 0.1 50. 1.0 72. 10.0 92. 100.0 95.______________________________________
This analysis can, of course, be extended to test new or related chemical compounds for their ability to inhibit PDE activity, or the activity of another phosphodiesterase expressed in this system. Clearly, this form of analysis can also be extended to other genes cloned and expressed in a similar manner for which there is an assayable enzymatic activity.
Phosphodiesterase activity was determined as described in the previous table using 0.04 and 1.0 .mu.M cAMP for pL22 Met and pJC44x, respectively. These concentrations of cAMP were specifically chosen to be below the K.sub.m for their respective enzymes. Thus, the EC.sub.50 closely approximates the inhibitor constant or K.sub.i of each enzyme. All kinetic data represent initial velocities of enzyme catalysis.
TABLE 3______________________________________Inhibitor Sensitivities of Human CyclicAMP Phosphodiesterases Derived byYeast Complementation EC.sub.50.sup.1Agent pJC44x pL22 Met______________________________________cAMP 3 0.2cGMP >300 >300Rolipram 0.4 >300RO 20-1724 3 >300Milrinone 30 30Theophylline 300 >300______________________________________ .sup.1 EC.sub.50 = Inhibitor concentration at 50% enzyme velocity, concentration expressed in .mu.M
The following procedure was applied to the screening of whole transformed host cells. The yeast strain 10DAB was transformed with the expression vector pAD72, which expresses a human family IV phosphodiesterase, i.e., a cAMP specific PDE. This transformed strain was grown in SC-leucine medium for three days at 30.degree. C. These cultures achieved a cell density of about 50 million cells per ml. Aliquots of this culture (300 .mu.l) were taken and mixed with 4.8 .mu.l 10% DMSO or 10% DMSO containing an appropriate concentration of phosphodiesterase inhibitor. The treated cultures were then incubated for two hours at 30.degree. C., after which two 3 .mu.l aliquots were removed and transferred to an SC-leucine agar plate. Then, a 100 .mu.l aliquot was removed from the treated cultures and transferred to a glass 12.times.75 mm test tube and the test tubes were incubated at 50.degree. C. in a mineral oil-containing hot block for 30 min. The test tubes were removed from the hot block and placed at room temperature. Two 3 .mu.l aliquots were removed and transferred to an SC-leucine plate. The agar plates were then incubated at 30.degree. C. and examined at various times to evaluate growth.
Yeast treated with 10% DMSO alone showed a slight decrease in the number of viable cells following the 50.degree. C. heat treatment. Treatment of cells with Rolipram reduced the number of viable cells, such that at 100 .mu.M Rolipram, less than 10 out of approximately 150,000 cells remained viable. Milrinone up, to 100 .mu.M, had no observable effect on the culture.
B. Identification of Agents Which Affect Proteins of Unspecified Function
This example illustrates the use of the genes and methods described above for use in identifying chemical compounds which affect the function of the encoded mammalian proteins expressed in yeast, even when the function of that protein has not yet been determined.
10DAB cells, which are phosphodiesterase deficient, are sensitive to heat shock. As already discussed, when these cells acquire the capacity to express the cDNA of pRATDPD, they become resistant to heat shock. 10DAB cells expressing the cDNA of pRATDPD were maintained in rich medium (YPD) for three days at stationary phase. These cultures were then treated with Rolipram, a known phosphodiesterase inhibitor, for 40 minutes at a final concentration of 100 .mu.M. Control cultures were not treated with any inhibitor. These cultures were then heat shocked in glass tubes at 50.degree. C. for 30 minutes. One microliter of each culture was plated. Cultures treated with Rolipram were much more sensitive to heat shock, reflecting an inhibition of enzymatic function.
The second type of pharmacological screen is applicable even when the mammalian gene encodes a protein of undetermined function, and, thus, cannot be assayed by a biochemical activity. In this method, agents to be tested are applied or introduced directly to the genetically altered microbial host expressing the mammalian protein. Agents capable of inhibiting the mammalian gene or gene product are identified by their ability to reverse the phenotype originally corrected by expression of the mammalian protein in the altered host.
This procedure has been used for mammalian cDNAs encoding cyclic nucleotide phosphodiesterases and a yeast containing RAS2.sup.va119 as the host strain. When the rat DPD gene is introduced into the heat shock sensitive host and expressed, the host strain becomes heat shock resistant. When the now resistant cells are incubated in Rolipram, they become heat shock sensitive again, indicating that Rolipram inhibits the activity of the rat DPD gene product. This pharmacological screen does not require that the function of the DPD gene product be known. This same approach can be applied to assess other genes.
In addition, and other phenotype that is dependent on DPD phosphodiesterase activity should be affected by the presence of the inhibitory drug. The effect of a drug or agent can be assessed as described. Finally, in the most generalized case, inhibitory chemicals for proteins of unknown function, expressed from mammalian cDNAs in yeast can be discovered in a similar way. This approach depends only on the phenotype consequent to expression of the protein and not on knowledge of its function.
For example, tyrosine kinases comprise a very large and diverse superfamily of proteins. They are important in regulation of cell growth. Certain tyrosine kinases are expressed ubiquitously in cells. Other tyrosine kinases display tissue specific distribution. Truly specific inhibitors of such tyrosine kinases could thus be expected to have specific and desirable therapeutic effects without unwanted side effects. For example, specific inhibitors of the PDGF receptor-tyrosine kinase could be expected to retard the growth of atherosclerotic plaques or retard scar formation; specific inhibitors of the lck tyrosine kinase, which mediates signals from the CD4 and CD8 T-cell receptors, could be expected to be anti-inflammatory without being cytotoxic.
It is probable that yeast can be used to screen pharmacological agents for inhibition of specific tyrosine kinases. Brugge et al., Mol. Cell. Biol., 7:2180-2187 (1987) demonstrated that expression of the avian v-src gene in the yeast S. cerevisiae inhibits growth. This vital gene encodes a tyrosine specific protein kinase that closely resembles the cellular src genes that are expressed ubiquitously in mammalian and avian cells. If this is a general property of active mammalian tyrosine kinases expressed in yeast, then the following design for a pharmacological screen would be expected to be effective.
A specific mammalian tyrosine kinase cDNA gene can thus be inserted in a yeast shuttle vector such that it is under the control of an inducible yeast promoter, such as the GAL10 promoter which is inducible in the presence of galactose and in the absence of glucose. Introduction of this vector into a yeast cell can be anticipated to render that cell unable to grow in induction medium (containing galatose in the absence of glucose), since under such conditions the mammalian tyrosine kinase would be expressed to the detriment of the cell. In the presence of an inhibitor of the tyrosine kinase, such cells would thrive on induction medium. This provides a simple screen for pharmacological agents that inhibit mammalian tyrosine kinases. False positives would include agents that blocked induction of the expression of kinase. Such false positives could be distinguished by the failure of the masalian kinase to be induced, which can be determined by quantitation with specific antibodies.
While the present invention has been described in terms of specific illustrative methods and materials, it is understood that modifications and variations thereof will occur to those skilled in the art upon consideration of the above detailed description. Consequently only such limitations as appear in the appended claims should be placed thereon.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 57(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AAGCGGCCGC 10(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GCGGCCGCTT 10(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2158 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A ) NAME/KEY: CDS(B) LOCATION: 1..1688(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:AGCTTGCGAATCGTAAGAAACAATTTCACCCTGCTGACAAACCTTCAC48SerLeuArgIleValArgAsnAsnPheThrLeuLeuThrAsnLeuHis1 51015GGAGCACCGAACAAGAGGTCGCCAGCGGCTAGTCAGGCTCCAGTCACC96GlyAlaProAsnLysArgSerProAlaAlaSerGlnAlaProValThr 202530AGAGTCAGCCTGCAAGAAGAATCATATCAGAAACTAGCAATGGAGACG144ArgValSerLeuGlnGluGluSerTyrGlnLysLeuAlaMetGluThr35 4045CTGGAGGAACTAGACTGGTGCCTAGACCAGCTAGAGACCATCCAGACC192LeuGluGluLeuAspTrpCysLeuAspGlnLeuGluThrIleGlnThr50 5560TACCGCTCTGTCAGCGAGATGGCTTCAAACAAGTTCAAAAGGATGCTG240TyrArgSerValSerGluMetAlaSerAsnLysPheLysArgMetLeu6570 7580AACCGGGAGCTGACACACCTCTCAGAGATGAGCAGATCAGGGAACCAA288AsnArgGluLeuThrHisLeuSerGluMetSerArgSerGlyAsnGln 859095GTGTCTGAATACATTTCGAACACGTTCTTAGACAAGCAGAACGATGTG336ValSerGluTyrIleSerAsnThrPheLeuAspLysGlnAsnAspVal1 00105110GAAATCCCATCTCCCACCCAGAAGGACAGGGAGAAGAAGAAGAAGCAG384GluIleProSerProThrGlnLysAspArgGluLysLysLysLysGln115 120125CAGCTCATGACCCAGATAAGTGGAGTGAAGAAACTGATGCACAGCTCA432GlnLeuMetThrGlnIleSerGlyValLysLysLeuMetHisSerSer130 135140AGCCTGAACAACACAAGCATCTCACGCTTTGGAGTCAACACGGAAAAT480SerLeuAsnAsnThrSerIleSerArgPheGlyValAsnThrGluAsn145150 155160GAGGATCATCTAGCCAAGGAGCTGGAAGACCTGAACAAATGGGGCCTT528GluAspHisLeuAlaLysGluLeuGluAspLeuAsnLysTrpGlyLeu1 65170175AACATCTTCAACGTGGCTGGGTACTCCCATAATCGGCCCCTCACATGC576AsnIlePheAsnValAlaGlyTyrSerHisAsnArgProLeuThrCys1 80185190ATCATGTACGCCATTTTCCAGGAAAGAGACCTTCTAAAGACGTTTAAA624IleMetTyrAlaIlePheGlnGluArgAspLeuLeuLysThrPheLys195 200205ATCTCCTCCGACACCTTCGTAACCTACATGATGACTTTAGAAGACCAT672IleSerSerAspThrPheValThrTyrMetMetThrLeuGluAspHis210 215220TACCATTCTGATGTGGCGTATCACAACAGCCTGCACGCTGCTGACGTG720TyrHisSerAspValAlaTyrHisAsnSerLeuHisAlaAlaAspVal225230 235240GCCCAGTCAACGCACGTTCTCCTCTCTACGCCAGCACTGGATGCTGTC768AlaGlnSerThrHisValLeuLeuSerThrProAlaLeuAspAlaVal2 45250255TTCACAGACCTGGAAATCCTGGCTGCCATTTTTGCAGCTGCCATCCAT816PheThrAspLeuGluIleLeuAlaAlaIlePheAlaAlaAlaIleHis2 60265270GATGTTGATCATCCTGGAGTCTCCAATCAGTTTCTCATCAATACAAAT864AspValAspHisProGlyValSerAsnGlnPheLeuIleAsnThrAsn275 280285TCCGAACTTGCTTTGATGTATAATGACGAATCTGTGCTGGAAAACCAT912SerGluLeuAlaLeuMetTyrAsnAspGluSerValLeuGluAsnHis290 295300CACCTCGCTGTGGGATTCAAGCTCCTTCAAGAGGAACATTGCGACATC960HisLeuAlaValGlyPheLysLeuLeuGlnGluGluHisCysAspIle305310 315320TTTCAGAATCTTACCAAGAAGCAACGCCAGACACTCAGGAAAATGGTG1008PheGlnAsnLeuThrLysLysGlnArgGlnThrLeuArgLysMetVal3 25330335ATTGACATGGTGTTAGCAACTGATATGTCCAAGCACATGAGCCTCCTG1056IleAspMetValLeuAlaThrAspMetSerLysHisMetSerLeuLeu3 40345350GCTGACCTTAAAACGATGGTAGAAACCAAAAAGGTGACGAGCTCCGGT1104AlaAspLeuLysThrMetValGluThrLysLysValThrSerSerGly355 360365GTTCTCCTCCTGGACAACTATACTGACCGGATACAGGTTCTTCGCAAC1152ValLeuLeuLeuAspAsnTyrThrAspArgIleGlnValLeuArgAsn370 375380ATGGTACATTGTGCAGACCTGAGCAACCCTACCAAGTCCTTGGAGTTG1200MetValHisCysAlaAspLeuSerAsnProThrLysSerLeuGluLeu385390 395400TATCGGCAATGGACTGATCGCATCATGGAGGAGTTTTTCCAACAGGGA1248TyrArgGlnTrpThrAspArgIleMetGluGluPhePheGlnGlnGly4 05410415GACAAAGAACGGGAGAGGGGAATGGAGATTAGCCCAATGTGTGATAAA1296AspLysGluArgGluArgGlyMetGluIleSerProMetCysAspLys4 20425430CACACAGCTTCTGTGGAAAAGTCCCAGGTTGGTTTCATTGACTACATT1344HisThrAlaSerValGluLysSerGlnValGlyPheIleAspTyrIle435 440445GTCCATCCATTGTGGGAGACCTGGGCAGACCTGGTTCAGCCTGATGCT1392ValHisProLeuTrpGluThrTrpAlaAspLeuValGlnProAspAla450 455460CAAGACATTTTGGACACACTAGAAGATAACAGGAACTGGTACCAGAGT1440GlnAspIleLeuAspThrLeuGluAspAsnArgAsnTrpTyrGlnSer465470 475480ATGATTCCCCAGAGCCCCTCTCCACCACTGGACGAGAGGAGCAGGGAC1488MetIleProGlnSerProSerProProLeuAspGluArgSerArgAsp4 85490495TGCCAAGGCCTTATGGAGAAGTTTCAGTTCGAACTGACCCTTGAAGAA1536CysGlnGlyLeuMetGluLysPheGlnPheGluLeuThrLeuGluGlu5 00505510GAGGATTCTGAAGGACCGGAAAAGGAGGGAGAAGGCCCCAACTATTTC1584GluAspSerGluGlyProGluLysGluGlyGluGlyProAsnTyrPhe515 520525AGCAGCACAAAGACACTTTGTGTGATCGATCCAGAGAACAGGGATTCT1632SerSerThrLysThrLeuCysValIleAspProGluAsnArgAspSer530 535540CTGGAAGAGACTGACATAGACATTGCCACAGAAGACAAGTCTCTGATC1680LeuGluGluThrAspIleAspIleAlaThrGluAspLysSerLeuIle545550 555560GACACATAATCTCCCTCTGTGTGGAGGTGAACATTCTATCCTTGACGAGCATGCCAGC1738AspThrTGAGTGGTAGGGCCCACCTACCAGAGCCAAGGCCTGCACAAAACAAAGGCCACCTGGCTT1798TGCAGTTACTTGAGTTTGGAGCCAGAATGCAAGGCCGTGAAGCAAATAGCAGTTCCGTGC1858TGCCTTGCCTTGCCGGCGAGCTTGGCGAGACCCGCAGCTGTAGTAGAAGCCAGTTCCCAG1918CACAGCTAAATGGCTTGAAAACAGAGGACAGAAAGCTGAGAG ATTGCTCTGCAATAGGTG1978TTGAGGGGCTGTCCCGACAGGTGACTGAACTCACTAACAACTTCATCTATAAATCTCACC2038CATCCTGTTGTCTGCCAACCTGTGTGCCTTTTTTGTAAAATGTTTTCGTGTCTTTGAAAT2098GCCTGTTGAATATCTAG AGTTTAGTACCTCCTTCTACAAACTTTTTTGAGTCTTTCTGGG2158(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 562 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:SerLeuArgIleValArgAs nAsnPheThrLeuLeuThrAsnLeuHis151015GlyAlaProAsnLysArgSerProAlaAlaSerGlnAlaProValThr20 2530ArgValSerLeuGlnGluGluSerTyrGlnLysLeuAlaMetGluThr354045LeuGluGluLeuAspTrpCysLeuAspGlnLeuGluThr IleGlnThr505560TyrArgSerValSerGluMetAlaSerAsnLysPheLysArgMetLeu65707580A snArgGluLeuThrHisLeuSerGluMetSerArgSerGlyAsnGln859095ValSerGluTyrIleSerAsnThrPheLeuAspLysGlnAsnAspVal 100105110GluIleProSerProThrGlnLysAspArgGluLysLysLysLysGln115120125GlnLeuMetThrGlnIleSe rGlyValLysLysLeuMetHisSerSer130135140SerLeuAsnAsnThrSerIleSerArgPheGlyValAsnThrGluAsn145150155 160GluAspHisLeuAlaLysGluLeuGluAspLeuAsnLysTrpGlyLeu165170175AsnIlePheAsnValAlaGlyTyrSerHisAsnArg ProLeuThrCys180185190IleMetTyrAlaIlePheGlnGluArgAspLeuLeuLysThrPheLys195200205I leSerSerAspThrPheValThrTyrMetMetThrLeuGluAspHis210215220TyrHisSerAspValAlaTyrHisAsnSerLeuHisAlaAlaAspVal225 230235240AlaGlnSerThrHisValLeuLeuSerThrProAlaLeuAspAlaVal245250255PheThrAspLeuGluIl eLeuAlaAlaIlePheAlaAlaAlaIleHis260265270AspValAspHisProGlyValSerAsnGlnPheLeuIleAsnThrAsn275280 285SerGluLeuAlaLeuMetTyrAsnAspGluSerValLeuGluAsnHis290295300HisLeuAlaValGlyPheLysLeuLeuGlnGluGluHisCysAsp Ile305310315320PheGlnAsnLeuThrLysLysGlnArgGlnThrLeuArgLysMetVal325330335IleAspMetValLeuAlaThrAspMetSerLysHisMetSerLeuLeu340345350AlaAspLeuLysThrMetValGluThrLysLysValThrSerSerGly 355360365ValLeuLeuLeuAspAsnTyrThrAspArgIleGlnValLeuArgAsn370375380MetValHisCysAlaAspLeuSerAs nProThrLysSerLeuGluLeu385390395400TyrArgGlnTrpThrAspArgIleMetGluGluPhePheGlnGlnGly405 410415AspLysGluArgGluArgGlyMetGluIleSerProMetCysAspLys420425430HisThrAlaSerValGluLysSerGlnValGlyPhe IleAspTyrIle435440445ValHisProLeuTrpGluThrTrpAlaAspLeuValGlnProAspAla450455460GlnAspI leLeuAspThrLeuGluAspAsnArgAsnTrpTyrGlnSer465470475480MetIleProGlnSerProSerProProLeuAspGluArgSerArgAsp 485490495CysGlnGlyLeuMetGluLysPheGlnPheGluLeuThrLeuGluGlu500505510GluAspSerGluGlyPr oGluLysGluGlyGluGlyProAsnTyrPhe515520525SerSerThrLysThrLeuCysValIleAspProGluAsnArgAspSer530535 540LeuGluGluThrAspIleAspIleAlaThrGluAspLysSerLeuIle545550555560AspThr(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:CACCCTGCTGACAAACCT18(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:ATGGAGACGCTGGAGGAA18(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(iv) ANTI-SENSE: YES(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:ATACGCCACATCAGAATG18(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:TACCAGAGTATGATTCCC18(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(iv) ANTI-SENSE: YES(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:GTGTCGATCAGAGACTTG18(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:( A) LENGTH: 18 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(iv) ANTI-SENSE: YES(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:GCACACAGGTTGGCAGAC18(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2702 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 2..2701(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:AAGCTTGCGGCCGCGCGGCCTAGGCCGCATCCCG GAGCTGCAACTG46SerLeuArgProArgGlyLeuGlyArgIleProGluLeuGlnLeu151015GTGGCCTTCCCGGTGGCGGTGGCGGCTGAGG ACGAGGCGTTCCTGCCC94ValAlaPheProValAlaValAlaAlaGluAspGluAlaPheLeuPro202530GAGCCCCTGGCCCCGCGCGCGCCCCGCC GCCGCGTTCGCCGCCCTCCT142GluProLeuAlaProArgAlaProArgArgArgValArgArgProPro354045CGCCCGTCTTCTTCGCCAGCCCGTCCCC AACTTTCCGCAGACGCCTTC190ArgProSerSerSerProAlaArgProGlnLeuSerAlaAspAlaPhe505560GGCTTCTCCGCAGCTGCCAGGATTTGGGCCG CCAGGCTTGGGCTGGGG238GlyPheSerAlaAlaAlaArgIleTrpAlaAlaArgLeuGlyLeuGly657075CTGGCTTCGAGGCAGAGAATGGGCCGACACCATCTCC TGGCCGCAGCC286LeuAlaSerArgGlnArgMetGlyArgHisHisLeuLeuAlaAlaAla80859095CCTGGACTGCAGGCGAGCCCAGGACTCGTGC TGCACGCCGGGGCGGCC334ProGlyLeuGlnAlaSerProGlyLeuValLeuHisAlaGlyAlaAla100105110ACCAGCCAGCGCCGGGAGTCCTTCCTGT ACCGCTCAGACAGCGACTAT382ThrSerGlnArgArgGluSerPheLeuTyrArgSerAspSerAspTyr115120125GACATGTCACCCAAGACCATGTCCCGGA ACTCATCGGTCACCAGCGAG430AspMetSerProLysThrMetSerArgAsnSerSerValThrSerGlu130135140GCGCACGCTGAAGACCTCATCGTAACACCAT TTGCTCAGGTGCTGGCC478AlaHisAlaGluAspLeuIleValThrProPheAlaGlnValLeuAla145150155AGCCTCCGGAGCGTCCGTAGCAACTTCTCACTCCTGA CCAATGTGCCC526SerLeuArgSerValArgSerAsnPheSerLeuLeuThrAsnValPro160165170175GTTCCCAGTAACAAGCGGTCCCCGCTGGGCG GCCCCACCCCTGTCTGC574ValProSerAsnLysArgSerProLeuGlyGlyProThrProValCys180185190AAGGCCACGCTGTCAGAAGAAACGTGTC AGCAGTTGGCCCGGGAGACT622LysAlaThrLeuSerGluGluThrCysGlnGlnLeuAlaArgGluThr195200205CTGGAGGAGCTGGACTGGTGTCTGGAGC AGCTGGAGACCATGCAGACC670LeuGluGluLeuAspTrpCysLeuGluGlnLeuGluThrMetGlnThr210215220TATCGCTCTGTCAGCGAGATGGCCTCGCACA AGTTCAAAAGGATGTTG718TyrArgSerValSerGluMetAlaSerHisLysPheLysArgMetLeu225230235AACCGTGAGCTCACACACCTGTCAGAAATGAGCAGGT CCGGAAACCAG766AsnArgGluLeuThrHisLeuSerGluMetSerArgSerGlyAsnGln240245250255GTCTCAGAGTACATTTCCACAACATTCCTGG ACAAACAGAATGAAGTG814ValSerGluTyrIleSerThrThrPheLeuAspLysGlnAsnGluVal260265270GAGATCCCATCACCCACGATGAAGGAAC GAGAAAAACAGCAAGCGCCG862GluIleProSerProThrMetLysGluArgGluLysGlnGlnAlaPro275280285CGACCAAGACCCTCCCAGCCGCCCCCGC CCCCTGTACCACACTTACAG910ArgProArgProSerGlnProProProProProValProHisLeuGln290295300CCCATGTCCCAAATCACAGGGTTGAAAAAGT TGATGCATAGTAACAGC958ProMetSerGlnIleThrGlyLeuLysLysLeuMetHisSerAsnSer305310315CTGAACAACTCTAACATTCCCCGATTTGGGGTGAAGA CCGATCAAGAA1006LeuAsnAsnSerAsnIleProArgPheGlyValLysThrAspGlnGlu320325330335GAGCTCCTGGCCCAAGAACTGGAGAACCTGA ACAAGTGGGGCCTGAAC1054GluLeuLeuAlaGlnGluLeuGluAsnLeuAsnLysTrpGlyLeuAsn340345350ATCTTTTGCGTGTCGGATTACGCTGGAG GCCGCTCACTCACCTGCATC1102IlePheCysValSerAspTyrAlaGlyGlyArgSerLeuThrCysIle355360365ATGTACATGATATTCCAGGAGCGGGACC TGCTGAAGAAATTCCGCATC1150MetTyrMetIlePheGlnGluArgAspLeuLeuLysLysPheArgIle370375380CCTGTGGACACGATGGTGACATACATGCTGA CGCTGGAGGATCACTAC1198ProValAspThrMetValThrTyrMetLeuThrLeuGluAspHisTyr385390395CACGCTGACGTGGCCTACCATAACAGCCTGCACGCAG CTGACGTGCTG1246HisAlaAspValAlaTyrHisAsnSerLeuHisAlaAlaAspValLeu400405410415CAGTCCACCCACGTACTGCTGGCCACGCCTT GGCCAACCTTAAGGAAT1294GlnSerThrHisValLeuLeuAlaThrProTrpProThrLeuArgAsn420425430GCAGTGTTCACGGACCTGGAGATTCTCG CCGCCCTCTTCGCGGCTGCC1342AlaValPheThrAspLeuGluIleLeuAlaAlaLeuPheAlaAlaAla435440445ATCCACGATGTGGATCACCCTGGGGTCT CCAACCAGTTCCTCATCAAC1390IleHisAspValAspHisProGlyValSerAsnGlnPheLeuIleAsn450455460ACCAATTCGGAGCTGGCGCTCATGTACAACG ATGAGTCGGTGCTCGAG1438ThrAsnSerGluLeuAlaLeuMetTyrAsnAspGluSerValLeuGlu465470475AATCACCACCTGGCCGTGGGCTTCAAGCTGCTGCAGG AGGACAACTGC1486AsnHisHisLeuAlaValGlyPheLysLeuLeuGlnGluAspAsnCys480485490495GACATCTTCCAGAACCTCAGCAAGCGCCAGC GGCAGAGCCTACGCAAG1534AspIlePheGlnAsnLeuSerLysArgGlnArgGlnSerLeuArgLys500505510ATGGTCATCGACATGGTGCTGGCCACGG ACATGTCCAAGCACATGACC1582MetValIleAspMetValLeuAlaThrAspMetSerLysHisMetThr515520525CTCCTGGCTGACCTGAAGACCATGGTGG AGACCAAGAAAGTGACCAGC1630LeuLeuAlaAspLeuLysThrMetValGluThrLysLysValThrSer530535540TCAGGGGTCCTCCTGCTAGATAACTACTCCG ACCGCATCCAGGTCCTC1678SerGlyValLeuLeuLeuAspAsnTyrSerAspArgIleGlnValLeu545550555CGGAACATGGTGCACTGTGCCGACCTCAGCAACCCCA CCAAGCCGCTG1726ArgAsnMetValHisCysAlaAspLeuSerAsnProThrLysProLeu560565570575GAGCTGTACCGCCAGTGGACAGACCGCATCA TGGCCGAGTTCTTCCAG1774GluLeuTyrArgGlnTrpThrAspArgIleMetAlaGluPhePheGln580585590CAGGGTGACCGAGAGCGCGAGCGTGGCA TGGAAATCAGCCCCATGTGT1822GlnGlyAspArgGluArgGluArgGlyMetGluIleSerProMetCys595600605GACAAGCACACTGCCTCCGTGGAGAAGT CTCAGGTGGGTTTTATTGAC1870AspLysHisThrAlaSerValGluLysSerGlnValGlyPheIleAsp610615620TACATTGTGCACCCATTGTGGGAGACCTGGG CGGACCTTGTCCACCCA1918TyrIleValHisProLeuTrpGluThrTrpAlaAspLeuValHisPro625630635GATGCCCAGGAGATCTTGGACACTTTGGAGGACAACC GGGACTGGTAC1966AspAlaGlnGluIleLeuAspThrLeuGluAspAsnArgAspTrpTyr640645650655TACAGCGCCATCCGGCAGAGCCCATCTCCGC CACCCGAGGAGGAGTCA2014TyrSerAlaIleArgGlnSerProSerProProProGluGluGluSer660665670AGGGGGCCAGGCCACCCACCCCTGCCTG ACAAGTTCCAGTTTGAGCTG2062ArgGlyProGlyHisProProLeuProAspLysPheGlnPheGluLeu675680685ACGCTGGAGGAGGAAGAGGAGGAAGAAAT ATCAATGGCCCAGATACCG2110ThrLeuGluGluGluGluGluGluGluIleSerMetAlaGlnIlePro690695700TGCACAGCCCAAGAGGCATTGACTGAGCAGGG ATTGTCAGGAGTCGAG2158CysThrAlaGlnGluAlaLeuThrGluGlnGlyLeuSerGlyValGlu705710715GAAGCTCTGGATGCAACCATAGCCTGGGAGGCATCCCC GGCCCAGGAG2206GluAlaLeuAspAlaThrIleAlaTrpGluAlaSerProAlaGlnGlu720725730735TCGTTGGAAGTTATGGCACAGGAAGCATCCCT GGAGGCCGAGCTGGAG2254SerLeuGluValMetAlaGlnGluAlaSerLeuGluAlaGluLeuGlu740745750GCAGTGTATTTGACACAGCAGGCACAGTC CACAGGCAGTGCACCTGTG2302AlaValTyrLeuThrGlnGlnAlaGlnSerThrGlySerAlaProVal755760765GCTCCGGATGAGTTCTCGTCCCGGGAGGA ATTCGTGGTTGCTGTAAGC2350AlaProAspGluPheSerSerArgGluGluPheValValAlaValSer770775780CACAGCAGCCCCTCTGCCCTGGCTCTTCAAAG CCCCCTTCTCCCTGCT2398HisSerSerProSerAlaLeuAlaLeuGlnSerProLeuLeuProAla785790795TGGAGGACCCTGTCTGTTTCAGAGCATGCCCGGCCTCC CGGGCCTCCC2446TrpArgThrLeuSerValSerGluHisAlaArgProProGlyProPro800805810815CTCCACGGCGGCCGAGGTGGAGGCCCAACGAG AGCACCAGGCTGCCAA2494LeuHisGlyGlyArgGlyGlyGlyProThrArgAlaProGlyCysGln820825830GAGGGCTTGCAGTGCCTGCGCAGGGACAT TTGGGGAGGACACATCCGC2542GluGlyLeuGlnCysLeuArgArgAspIleTrpGlyGlyHisIleArg835840845ACTCCCAGCTCCTGGTGGCGGGGGGTCAG GTGGAGACCCTACCTGATC2590ThrProSerSerTrpTrpArgGlyValArgTrpArgProTyrLeuIle850855860CCCAGACCTCTGTCCCTGTTCCCCTCCACTCC TCCCCTCACTCCCCTG2638ProArgProLeuSerLeuPheProSerThrProProLeuThrProLeu865870875CTCCCCCGACCACCTCCTCCTCTGCCTCAAAGACTCTT GTCCTCTTGT2686LeuProArgProProProProLeuProGlnArgLeuLeuSerSerCys880885890895CCGCGGCCGCAAGCTT 2702ProArgProGlnAla900(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 900 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:SerLe uArgProArgGlyLeuGlyArgIleProGluLeuGlnLeuVal151015AlaPheProValAlaValAlaAlaGluAspGluAlaPheLeuProGlu 202530ProLeuAlaProArgAlaProArgArgArgValArgArgProProArg354045ProSerSerSerProAlaArgPro GlnLeuSerAlaAspAlaPheGly505560PheSerAlaAlaAlaArgIleTrpAlaAlaArgLeuGlyLeuGlyLeu657075 80AlaSerArgGlnArgMetGlyArgHisHisLeuLeuAlaAlaAlaPro859095GlyLeuGlnAlaSerProGlyLeuValLeuHisAlaGly AlaAlaThr100105110SerGlnArgArgGluSerPheLeuTyrArgSerAspSerAspTyrAsp115120125MetSe rProLysThrMetSerArgAsnSerSerValThrSerGluAla130135140HisAlaGluAspLeuIleValThrProPheAlaGlnValLeuAlaSer145150 155160LeuArgSerValArgSerAsnPheSerLeuLeuThrAsnValProVal165170175ProSerAsnLysArgSerPro LeuGlyGlyProThrProValCysLys180185190AlaThrLeuSerGluGluThrCysGlnGlnLeuAlaArgGluThrLeu195200 205GluGluLeuAspTrpCysLeuGluGlnLeuGluThrMetGlnThrTyr210215220ArgSerValSerGluMetAlaSerHisLysPheLysArgMetLeuAsn225230235240ArgGluLeuThrHisLeuSerGluMetSerArgSerGlyAsnGlnVal245250255Se rGluTyrIleSerThrThrPheLeuAspLysGlnAsnGluValGlu260265270IleProSerProThrMetLysGluArgGluLysGlnGlnAlaProArg275 280285ProArgProSerGlnProProProProProValProHisLeuGlnPro290295300MetSerGlnIleThrGlyLeuLysLysLeu MetHisSerAsnSerLeu305310315320AsnAsnSerAsnIleProArgPheGlyValLysThrAspGlnGluGlu325330 335LeuLeuAlaGlnGluLeuGluAsnLeuAsnLysTrpGlyLeuAsnIle340345350PheCysValSerAspTyrAlaGlyGlyArgSerLeuThr CysIleMet355360365TyrMetIlePheGlnGluArgAspLeuLeuLysLysPheArgIlePro370375380ValAspThrMe tValThrTyrMetLeuThrLeuGluAspHisTyrHis385390395400AlaAspValAlaTyrHisAsnSerLeuHisAlaAlaAspValLeuGln 405410415SerThrHisValLeuLeuAlaThrProTrpProThrLeuArgAsnAla420425430ValPheThrAspLeuGluIle LeuAlaAlaLeuPheAlaAlaAlaIle435440445HisAspValAspHisProGlyValSerAsnGlnPheLeuIleAsnThr450455 460AsnSerGluLeuAlaLeuMetTyrAsnAspGluSerValLeuGluAsn465470475480HisHisLeuAlaValGlyPheLysLeuLeuGlnGluAspAsn CysAsp485490495IlePheGlnAsnLeuSerLysArgGlnArgGlnSerLeuArgLysMet500505510Va lIleAspMetValLeuAlaThrAspMetSerLysHisMetThrLeu515520525LeuAlaAspLeuLysThrMetValGluThrLysLysValThrSerSer530 535540GlyValLeuLeuLeuAspAsnTyrSerAspArgIleGlnValLeuArg545550555560AsnMetValHisCysAlaAspLeu SerAsnProThrLysProLeuGlu565570575LeuTyrArgGlnTrpThrAspArgIleMetAlaGluPhePheGlnGln580585 590GlyAspArgGluArgGluArgGlyMetGluIleSerProMetCysAsp595600605LysHisThrAlaSerValGluLysSerGlnValGlyPheIle AspTyr610615620IleValHisProLeuTrpGluThrTrpAlaAspLeuValHisProAsp625630635640AlaGl nGluIleLeuAspThrLeuGluAspAsnArgAspTrpTyrTyr645650655SerAlaIleArgGlnSerProSerProProProGluGluGluSerArg 660665670GlyProGlyHisProProLeuProAspLysPheGlnPheGluLeuThr675680685LeuGluGluGluGluGluGluGlu IleSerMetAlaGlnIleProCys690695700ThrAlaGlnGluAlaLeuThrGluGlnGlyLeuSerGlyValGluGlu705710715 720AlaLeuAspAlaThrIleAlaTrpGluAlaSerProAlaGlnGluSer725730735LeuGluValMetAlaGlnGluAlaSerLeuGluAlaGlu LeuGluAla740745750ValTyrLeuThrGlnGlnAlaGlnSerThrGlySerAlaProValAla755760765ProAs pGluPheSerSerArgGluGluPheValValAlaValSerHis770775780SerSerProSerAlaLeuAlaLeuGlnSerProLeuLeuProAlaTrp785790 795800ArgThrLeuSerValSerGluHisAlaArgProProGlyProProLeu805810815HisGlyGlyArgGlyGlyGly ProThrArgAlaProGlyCysGlnGlu820825830GlyLeuGlnCysLeuArgArgAspIleTrpGlyGlyHisIleArgThr835840 845ProSerSerTrpTrpArgGlyValArgTrpArgProTyrLeuIlePro850855860ArgProLeuSerLeuPheProSerThrProProLeuThrProLeuLeu865870875880ProArgProProProProLeuProGlnArgLeuLeuSerSerCysPro885890895Ar gProGlnAla900(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1721 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 60..1274 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:AAGCTTGCGGCCGCATTGGGTACCGCGTGCCAGCAGGCAGTGGCCCTAGCCTTCCGCCT59ATGCCCTCCCTCCAAGAGGTGGACTGCGGCTCCCCCAGCAGCTCCGAG107MetProSerLeuGlnGluValAsp CysGlySerProSerSerSerGlu151015GAGGAGGGGGTGCCAGGGTCCCGGGGGAGCCCAGCGACCTCACCCCAC155GluGluGlyValProGlySer ArgGlySerProAlaThrSerProHis202530CTGGGCCGCCGACGACCTCTGCTTCGGTCCATGAGCGCCGCCTTCTGC203LeuGlyArgArgArgProLeu LeuArgSerMetSerAlaAlaPheCys354045TCCCTACTGGCACCGGAGCGGCAGGTGGGCCGGGCTGCGGCAGCACTG251SerLeuLeuAlaProGluArgGln ValGlyArgAlaAlaAlaAlaLeu505560ATGCAGGACCGACACACAGCCGCGGGCCAGCTGGTGCAGGACCTACTG299MetGlnAspArgHisThrAlaAlaGlyGln LeuValGlnAspLeuLeu65707580ACCCAGGTGCGGGATGGGCAGAGGCCCCAGGAGCTCGAGGGCATCCGT347ThrGlnValArgAspGlyGlnArg ProGlnGluLeuGluGlyIleArg859095CAGGCGCTGAGCCGGGCCCGGGCCATGCTGAGTGCGGAGCTGGGCCCT395GlnAlaLeuSerArgAlaArg AlaMetLeuSerAlaGluLeuGlyPro100105110GAGAAGCTCGTGTCGCCTAAGAGGCTGGAACATGTCCTGGAGAAGTCA443GluLysLeuValSerProLys ArgLeuGluHisValLeuGluLysSer115120125TTGCATTGCTCTGTGCTCAAGCCTCTCCGGCCCATCCTGGCAGCCCGC491LeuHisCysSerValLeuLysPro LeuArgProIleLeuAlaAlaArg130135140CTGCGGCGCCGGCTTGCCGCAGACGGCTCCCTGGGCCGCCTAGCTGAG539LeuArgArgArgLeuAlaAlaAspGlySer LeuGlyArgLeuAlaGlu145150155160GGCCTCCGCCTGGCCCGGGCCCAGGGCCCCGGAGCCTTCGGGTCCCAC587GlyLeuArgLeuAlaArgAlaGln GlyProGlyAlaPheGlySerHis165170175CTGAGCCTGCCCTCCCCAGTAGAGTTGGAGCAAGTGCGCCAGAAGCTG635LeuSerLeuProSerProVal GluLeuGluGlnValArgGlnLysLeu180185190CTGCAGCTCGTCCGCACCTACTCACCCAGCGCCCAGGTCAAGCGGCTC683LeuGlnLeuValArgThrTyr SerProSerAlaGlnValLysArgLeu195200205CTGCAGGCCTGCAAGCTGCTCTACATGGCCCTGAGGACCCAGGAAGGG731LeuGlnAlaCysLysLeuLeuTyr MetAlaLeuArgThrGlnGluGly210215220GAGGGCTCGGGTGCCGACGGGTTCCTGCCTCTGCTGAGCCTCGTCTTG779GluGlySerGlyAlaAspGlyPheLeuPro LeuLeuSerLeuValLeu225230235240GCCCACTGTGACCTTCCTGAGCTGCTGCTGGAGGCCGAGTACATGTCG827AlaHisCysAspLeuProGluLeu LeuLeuGluAlaGluTyrMetSer245250255GAGCTGCTGGAGCCCAGCCTGCTTACTGGAGAGGGTGGCTACTACCTG875GluLeuLeuGluProSerLeu LeuThrGlyGluGlyGlyTyrTyrLeu260265270ACCAGCCTCTCTGCCAGCCTGGCCCTGCTGAGTGGCCTGGGTCAGGCC923ThrSerLeuSerAlaSerLeu AlaLeuLeuSerGlyLeuGlyGlnAla275280285CACACCCTCCCACTGAGCCCCGTGCAGGAGCTACGGCGCTCCCTCAGC971HisThrLeuProLeuSerProVal GlnGluLeuArgArgSerLeuSer290295300CTCTGGGAGCAGCGCCGCCTGCCTGCCACCCACTGCTTCCAGCACCTC1019LeuTrpGluGlnArgArgLeuProAlaThr HisCysPheGlnHisLeu305310315320CTCCGAGTAGCCTATCAGGATCCCAGCAGTGGCTGCACCTCCAAGACC1067LeuArgValAlaTyrGlnAspPro SerSerGlyCysThrSerLysThr325330335CTGGCCGTGCCCCCAGAGGCCTCGATTGCCACCCTGAACCAGCTCTGT1115LeuAlaValProProGluAla SerIleAlaThrLeuAsnGlnLeuCys340345350GCCACCAAGTTCCGAGTGACCCAGCCCAACACTTTTGGCCTCTTCCTG1163AlaThrLysPheArgValThr GlnProAsnThrPheGlyLeuPheLeu355360365TACAAGGAGCAGGGCTACCACCGCCTGCCCCCTGGGCCCTGGCCCACA1211TyrLysGluGlnGlyTyrHisArg LeuProProGlyProTrpProThr370375380GGCTGCCCACCACTGGCTACCTCGTCTACCGCCGGGCAGAGTGGCCTG1259GlyCysProProLeuAlaThrSerSerThr AlaGlyGlnSerGlyLeu385390395400AGACCCAGGGGGCTGTGACAGAGGAGGAGGGCAGTGGGCAGTCAGAGGCAAGAAG1314ArgProArgGlyLeu 405CAGAGGGGAGGAGCAAGGGTGCCAGGGAGATGGGGATGCTGGGGTCAAAGCCAGCCCCAG1374GGACATTCGGGAACAGTCTGAGACAACTGCTGAAGGGGGCCAGGGTCAAGCCCAGGAAGG1434CCCTGCTCAGCCAGGGGAACCAGAGGCAGAGG GAAGCCGGGCAGCAGAGGAGTAGCTTGA1494AGTGGCCAGAAGGGTCATTCGGGGCGGGAGACCCTGAGCCTGCTGAGAAATCCTTTTAGC1554GCCAGCAAGCCCCACCCAGGGCCCTGTCCTGTGTCTGCCACCACCTTTGTCTGATACTTG1614TTTCCAG GGAAGCTGGGGGAACTGCCACATCTGAGGAACTGGAATAAAGATGAGGGGCCT1674TCGGGGGCCAATGCGGCCGCCGCGGCCTTTTTGGCCAGCTCGAATTC1721(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 405 amino acids (B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:MetProSerLeuGlnGluValAspCysGlySerProSerSerSerGlu151015GluGluGl yValProGlySerArgGlySerProAlaThrSerProHis202530LeuGlyArgArgArgProLeuLeuArgSerMetSerAlaAlaPheCys35 4045SerLeuLeuAlaProGluArgGlnValGlyArgAlaAlaAlaAlaLeu505560MetGlnAspArgHisThrAlaAlaGlyGlnLeuVal GlnAspLeuLeu65707580ThrGlnValArgAspGlyGlnArgProGlnGluLeuGluGlyIleArg8590 95GlnAlaLeuSerArgAlaArgAlaMetLeuSerAlaGluLeuGlyPro100105110GluLysLeuValSerProLysArgLeuGluHisValLeuGluLys Ser115120125LeuHisCysSerValLeuLysProLeuArgProIleLeuAlaAlaArg130135140LeuArgArgArgLeuAl aAlaAspGlySerLeuGlyArgLeuAlaGlu145150155160GlyLeuArgLeuAlaArgAlaGlnGlyProGlyAlaPheGlySerHis165 170175LeuSerLeuProSerProValGluLeuGluGlnValArgGlnLysLeu180185190LeuGlnLeuValArgThrTyrSerPro SerAlaGlnValLysArgLeu195200205LeuGlnAlaCysLysLeuLeuTyrMetAlaLeuArgThrGlnGluGly210215220GluGlySerGlyAlaAspGlyPheLeuProLeuLeuSerLeuValLeu225230235240AlaHisCysAspLeuProGluLeuLeuLeuGluAlaGluTyrMetSer245250255GluLeuLeuGluProSerLeuLeuThrGlyGluGlyGlyTyrTyrLeu260265270ThrSerLe uSerAlaSerLeuAlaLeuLeuSerGlyLeuGlyGlnAla275280285HisThrLeuProLeuSerProValGlnGluLeuArgArgSerLeuSer290 295300LeuTrpGluGlnArgArgLeuProAlaThrHisCysPheGlnHisLeu305310315320LeuArgValAlaTyrGlnAspProSerSer GlyCysThrSerLysThr325330335LeuAlaValProProGluAlaSerIleAlaThrLeuAsnGlnLeuCys340345 350AlaThrLysPheArgValThrGlnProAsnThrPheGlyLeuPheLeu355360365TyrLysGluGlnGlyTyrHisArgLeuProProGlyProTrpProThr370375380GlyCysProProLeuAlaThrSerSerThrAlaGlyGlnSerGlyLeu385390395400ArgProArgGl yLeu405(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1829 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 30..1421 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:GCGGCCGCGGCCGGCAGCGGCTGAGCGACATGAGCATTTCTACTTCCTCCTCC53MetSerIleSerThrSerSerSer1 5GACTCGCTGGAGTTCGACCGGAGCATGCCTCTGTTTGGCTACGAGGCG101AspSerLeuGluPheAspArgSerMetProLeuPheGlyTyrGluAla101520GACACCAACAGCAGCCTGGAGGACTACGAGGGGGAAAGTGACCAAGAG149AspThrAsnSerSerLeuGluAspTyrGluGlyGluSerAspGlnGlu253035 40ACCATGGCGCCCCCCATCAAGTCCAAAAAGAAAAGGAGCAGCTCCTTC197ThrMetAlaProProIleLysSerLysLysLysArgSerSerSerPhe4550 55GTGCTGCCCAAGCTCGTCAAGTCCCAGCTGCAGAAGGTGAGCGGGGTG245ValLeuProLysLeuValLysSerGlnLeuGlnLysValSerGlyVal6065 70TTCAGCTCCTTCATGACCCCGGAGAAGCGGATGGTCCGCAGGATCGCC293PheSerSerPheMetThrProGluLysArgMetValArgArgIleAla7580 85GAGCTTTCCCGGGACAAATGCACCTACTTCGGGTGCTTAGTGCAGGAC341GluLeuSerArgAspLysCysThrTyrPheGlyCysLeuValGlnAsp9095100TACGTGAGCTTCCTGCAGGAGAACAAGGAGTGCCACGTGTCCAGCACC389TyrValSerPheLeuGlnGluAsnLysGluCysHisValSerSerThr105110115 120GACATGCTGCAGACCATCCGGCAGTTCATGACCCAGGTCAAGAACTAT437AspMetLeuGlnThrIleArgGlnPheMetThrGlnValLysAsnTyr125130 135TTGTCTCAGAGCTCGGAGCTGGACCCCCCCATCGAGTCGCTGATCCCT485LeuSerGlnSerSerGluLeuAspProProIleGluSerLeuIlePro140145 150GAAGACCAAATAGATGTGGTGCTGGAAAAAGCCATGCACAAGTGCATC533GluAspGlnIleAspValValLeuGluLysAlaMetHisLysCysIle155160 165TTGAAGCCCCTCAAGGGGCACGTGGAGGCCATGCTGAAGGACTTTCAC581LeuLysProLeuLysGlyHisValGluAlaMetLeuLysAspPheHis170175180ATGGCCGATGGCTCATGGAAGCAACTCAAGGAGAACCTGCAGCTTGTG629MetAlaAspGlySerTrpLysGlnLeuLysGluAsnLeuGlnLeuVal185190195 200CGGCAGAGGAATCCGCAGGAGCTGGGGGTCTTCGCCCCGACCCCTGAT677ArgGlnArgAsnProGlnGluLeuGlyValPheAlaProThrProAsp205210 215TTTGTGGATGTGGAGAAAATCAAAGTCAAGTTCATGACCATGCAGAAG725PheValAspValGluLysIleLysValLysPheMetThrMetGlnLys220225 230ATGTATTCGCCGGAAAAGAAGGTCATGCTGCTGCTGCGGGTCTGCAAG773MetTyrSerProGluLysLysValMetLeuLeuLeuArgValCysLys235240 245CTCATTTACACGGTCATGGAGAACAACTCAGGGAGGATGTATGGCGCT821LeuIleTyrThrValMetGluAsnAsnSerGlyArgMetTyrGlyAla250255260GATGACTTCTTGCCAGTCCTGACCTATGTCATAGCCCAGTGTGACATG869AspAspPheLeuProValLeuThrTyrValIleAlaGlnCysAspMet265270275 280CTTGAATTGGACACTGAAATCGAGTACATGATGGAGCTCCTAGACCCA917LeuGluLeuAspThrGluIleGluTyrMetMetGluLeuLeuAspPro285290 295TCGCTGTTACATGGAGAAGGAGGCTATTACTTGACAAGCGCATATGGA965SerLeuLeuHisGlyGluGlyGlyTyrTyrLeuThrSerAlaTyrGly300305 310GCACTTTCTCTGATAAAGAATTTCCAAGAAGAACAAGCAGCGCGACTG1013AlaLeuSerLeuIleLysAsnPheGlnGluGluGlnAlaAlaArgLeu315320 325CTCAGCTCAGAAACCAGAGACACCCTGAGGCAGTGGCACAAACGGAGA1061LeuSerSerGluThrArgAspThrLeuArgGlnTrpHisLysArgArg330335340ACCACCAACCGGACCATCCCCTCTGTGGACGACTTCCAGAATTACCTC1109ThrThrAsnArgThrIleProSerValAspAspPheGlnAsnTyrLeu345350355 360CGAGTTGCATTTCAGGAGGTCAACAGTGGTTGCACAGGAAAGACCCTC1157ArgValAlaPheGlnGluValAsnSerGlyCysThrGlyLysThrLeu365370 375CTTGTGAGACCTTACATCACCACTGAGGATGTGTGTCAGATCTGCGCT1205LeuValArgProTyrIleThrThrGluAspValCysGlnIleCysAla380385 390GAGAAGTTCAAGGTGGGGGACCCTGAGGAGTACAGCCTCTTTCTCTTC1253GluLysPheLysValGlyAspProGluGluTyrSerLeuPheLeuPhe395400 405GTTGACGAGACATGGCAGCAGCTGGCAGAGGACACTTACCCTCAAAAA1301ValAspGluThrTrpGlnGlnLeuAlaGluAspThrTyrProGlnLys410415420 ATCAAGGCGGAGCTGCACAGCCGACCACAGCCCCACATCTTCCACTTT1349IleLysAlaGluLeuHisSerArgProGlnProHisIlePheHisPhe425430435 440GTCTACAAACGCATCAAGAACGATCCTTATGGCATCATTTTCCAGAAC1397ValTyrLysArgIleLysAsnAspProTyrGlyIleIlePheGlnAsn445450 455GGGGAAGAAGACCTCACCACCTCCTAGAAGACAGGCGGGACTTCCCAGTGGTGC1451GlyGluGluAspLeuThrThrSer460ATCCAAAGGGGAGCTGGAAGCCTTGCCTTCCCGCTTCTACATGCTTGAGCTTGA AAAGCA1511GTCACCTCCTCGGGGACCCCTCAGTGTAGTGACTAAGCCATCCACAGGCCAACTCGGCCA1571AGGGCAACTTTAGCCACGCAAGGTAGCTGAGGTTTGTGAAACAGTAGGATTCTCTTTTGG1631CAATGGAGAATTGCATCTGATGGTTCAAG TGTCCTGAGATTGTTTGCTACCTACCCCCAG1691TCAGGTTCTAGGTTGGCTTACAGGTATGTATATGTGCAGAAGAAACACTTAAGATACAAG1751TTCTTTTGAATTCAACAGCAGATGCTTGCGATGCAGTGCGTCAGGTGATTCTCACTCCTG1811TGG ATGGCTTCATCCCTG1829(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 464 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:MetSerIle SerThrSerSerSerAspSerLeuGluPheAspArgSer151015MetProLeuPheGlyTyrGluAlaAspThrAsnSerSerLeuGluAsp20 2530TyrGluGlyGluSerAspGlnGluThrMetAlaProProIleLysSer354045LysLysLysArgSerSerSerPheVal LeuProLysLeuValLysSer505560GlnLeuGlnLysValSerGlyValPheSerSerPheMetThrProGlu657075 80LysArgMetValArgArgIleAlaGluLeuSerArgAspLysCysThr859095TyrPheGlyCysLeuValGlnAspTyrValSerPheLeuGlnGl uAsn100105110LysGluCysHisValSerSerThrAspMetLeuGlnThrIleArgGln115120125PheMetThr GlnValLysAsnTyrLeuSerGlnSerSerGluLeuAsp130135140ProProIleGluSerLeuIleProGluAspGlnIleAspValValLeu145150 155160GluLysAlaMetHisLysCysIleLeuLysProLeuLysGlyHisVal165170175GluAlaMetLeuLysAspPheHisM etAlaAspGlySerTrpLysGln180185190LeuLysGluAsnLeuGlnLeuValArgGlnArgAsnProGlnGluLeu195200 205GlyValPheAlaProThrProAspPheValAspValGluLysIleLys210215220ValLysPheMetThrMetGlnLysMetTyrSerProGluLysLysVal225 230235240MetLeuLeuLeuArgValCysLysLeuIleTyrThrValMetGluAsn245250255AsnSer GlyArgMetTyrGlyAlaAspAspPheLeuProValLeuThr260265270TyrValIleAlaGlnCysAspMetLeuGluLeuAspThrGluIleGlu275 280285TyrMetMetGluLeuLeuAspProSerLeuLeuHisGlyGluGlyGly290295300TyrTyrLeuThrSerAlaTyrGlyAlaLeuSerL euIleLysAsnPhe305310315320GlnGluGluGlnAlaAlaArgLeuLeuSerSerGluThrArgAspThr325330 335LeuArgGlnTrpHisLysArgArgThrThrAsnArgThrIleProSer340345350ValAspAspPheGlnAsnTyrLeuArgValAlaPheGlnGluVa lAsn355360365SerGlyCysThrGlyLysThrLeuLeuValArgProTyrIleThrThr370375380GluAspValCysGlnI leCysAlaGluLysPheLysValGlyAspPro385390395400GluGluTyrSerLeuPheLeuPheValAspGluThrTrpGlnGlnLeu405 410415AlaGluAspThrTyrProGlnLysIleLysAlaGluLeuHisSerArg420425430ProGlnProHisIlePheHisPheVa lTyrLysArgIleLysAsnAsp435440445ProTyrGlyIleIlePheGlnAsnGlyGluGluAspLeuThrThrSer450455460(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1299 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..1299(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GGCCGCATTG CCGACCCGGCCCGTAGTGTGGAAGCAGCTTCAGCTCAA48GlyArgIleAlaAspProAlaArgSerValGluAlaAlaSerAlaGln151015AGATTAG AACGACTCCGAAAAGAGAGACAAAACCAGATCAAATGCAAA96ArgLeuGluArgLeuArgLysGluArgGlnAsnGlnIleLysCysLys202530AATATTC AGTGGAAAGAAAGAAATTCTAAGCAATCAGCCCAGGAGTTA144AsnIleGlnTrpLysGluArgAsnSerLysGlnSerAlaGlnGluLeu354045AAGTCACTGT TTGAAAAAAAATCTCTCAAAGAGAAGCCTCCAATTTCT192LysSerLeuPheGluLysLysSerLeuLysGluLysProProIleSer505560GGGAAGCAGTCGATATT ATCTGTACGCCTAGAACAGTGCCCTCTGCAG240GlyLysGlnSerIleLeuSerValArgLeuGluGlnCysProLeuGln65707580CTGAATAACCC TTTTAACGAGTATTCCAAATTTGATGGCAAGGGTCAT288LeuAsnAsnProPheAsnGluTyrSerLysPheAspGlyLysGlyHis859095GTAGGTAC AACAGCAACCAAGAAGATCGATGTCTACCTCCCTCTGCAC336ValGlyThrThrAlaThrLysLysIleAspValTyrLeuProLeuHis100105110TCGAGCCA GGACAGACTGCTGCCAATGACCGTGGTGACAATGGCCAGC384SerSerGlnAspArgLeuLeuProMetThrValValThrMetAlaSer115120125GCCAGGGTGCA GGACCTGATCGGGCTCATCTGCTGGCAGTATACAAGC432AlaArgValGlnAspLeuIleGlyLeuIleCysTrpGlnTyrThrSer130135140GAAGGACGGGAGCCGAA GCTCAATGACAATGTCAGTGCCTACTGCCTG480GluGlyArgGluProLysLeuAsnAspAsnValSerAlaTyrCysLeu145150155160CATATTGCTGA GGATGATGGGGAGGTGGACACCGATTTCCCCCCGCTG528HisIleAlaGluAspAspGlyGluValAspThrAspPheProProLeu165170175GATTCCAA TGAGCCCATTCATAAGTTTGGCTTCAGTACTTTGGCCCTG576AspSerAsnGluProIleHisLysPheGlyPheSerThrLeuAlaLeu180185190GTTGAAAA GTACTCATCTCCTGGTCTGACATCCAAAGAGTCACTCTTT624ValGluLysTyrSerSerProGlyLeuThrSerLysGluSerLeuPhe195200205GTTCGAATAAA TGCTGCTCATGGATTCTCCCTTATTCAGGTGGACAAC672ValArgIleAsnAlaAlaHisGlyPheSerLeuIleGlnValAspAsn210215220ACAAAGGTTACCATGAA GGAAATCTTACTGAAGGCAGTGAAGCGAAGA720ThrLysValThrMetLysGluIleLeuLeuLysAlaValLysArgArg225230235240AAAGGATCCCA GAAAGTTTCAGGCCCTCAGTACCGCCTGGAGAAGCAG768LysGlySerGlnLysValSerGlyProGlnTyrArgLeuGluLysGln245250255AGCGAGCC CAATGTCGCCGTTGACCTGGACAGCACTTTGGAGAGCCAG816SerGluProAsnValAlaValAspLeuAspSerThrLeuGluSerGln260265270AGCGCATG GGAGTTCTGCCTGGTCCGCGAGAACAGTTCAAGGGCAGAC864SerAlaTrpGluPheCysLeuValArgGluAsnSerSerArgAlaAsp275280285GGGGTTTTTGA GGAGGATTCGCAAATTGACATAGCCACAGTACAGGAT912GlyValPheGluGluAspSerGlnIleAspIleAlaThrValGlnAsp290295300ATGCTTAGCAGCCACCA TTACAAGTCATTCAAAGTCAGCATGATCCAC960MetLeuSerSerHisHisTyrLysSerPheLysValSerMetIleHis305310315320AGACTGCGATT CACAACCGACGTACAGCTAGGTATCTCTGGAGACAAA1008ArgLeuArgPheThrThrAspValGlnLeuGlyIleSerGlyAspLys325330335GTAGAGAT AGACCCTGTTACGAATCAGAAAGCCAGCACTAAGTTTTGG1056ValGluIleAspProValThrAsnGlnLysAlaSerThrLysPheTrp340345350ATTAAGCA GAAACCCATCTCAATCGATTCCGACCTGCTCTGTGCCTGT1104IleLysGlnLysProIleSerIleAspSerAspLeuLeuCysAlaCys355360365GACCTTGCTGA AGAGAAAAGCCCCAGTCACGCAATATTTAAACTCACG1152AspLeuAlaGluGluLysSerProSerHisAlaIlePheLysLeuThr370375380TATCTAAGCAATCACGA CTATAAACACCTCTACTTTGAATCGGACGCT1200TyrLeuSerAsnHisAspTyrLysHisLeuTyrPheGluSerAspAla385390395400GCTACCGTCAA TGAAATTGTGCTCAAGGTTAACTACATCCTGGAATCG1248AlaThrValAsnGluIleValLeuLysValAsnTyrIleLeuGluSer405410415CGAGCTAG CACTGCCCGGGCTGACTACTTTGCTCAAAAAAAAAGCGGC1296ArgAlaSerThrAlaArgAlaAspTyrPheAlaGlnLysLysSerGly420425430CGC 1299Arg(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 433 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GlyArgIle AlaAspProAlaArgSerValGluAlaAlaSerAlaGln151015ArgLeuGluArgLeuArgLysGluArgGlnAsnGlnIleLysCysLys20 2530AsnIleGlnTrpLysGluArgAsnSerLysGlnSerAlaGlnGluLeu354045LysSerLeuPheGluLysLysSerLeuL ysGluLysProProIleSer505560GlyLysGlnSerIleLeuSerValArgLeuGluGlnCysProLeuGln657075 80LeuAsnAsnProPheAsnGluTyrSerLysPheAspGlyLysGlyHis859095ValGlyThrThrAlaThrLysLysIleAspValTyrLeuProLe uHis100105110SerSerGlnAspArgLeuLeuProMetThrValValThrMetAlaSer115120125AlaArgVal GlnAspLeuIleGlyLeuIleCysTrpGlnTyrThrSer130135140GluGlyArgGluProLysLeuAsnAspAsnValSerAlaTyrCysLeu145150 155160HisIleAlaGluAspAspGlyGluValAspThrAspPheProProLeu165170175AspSerAsnGluProIleHisLysP heGlyPheSerThrLeuAlaLeu180185190ValGluLysTyrSerSerProGlyLeuThrSerLysGluSerLeuPhe195200 205ValArgIleAsnAlaAlaHisGlyPheSerLeuIleGlnValAspAsn210215220ThrLysValThrMetLysGluIleLeuLeuLysAlaValLysArgArg225 230235240LysGlySerGlnLysValSerGlyProGlnTyrArgLeuGluLysGln245250255SerGlu ProAsnValAlaValAspLeuAspSerThrLeuGluSerGln260265270SerAlaTrpGluPheCysLeuValArgGluAsnSerSerArgAlaAsp275 280285GlyValPheGluGluAspSerGlnIleAspIleAlaThrValGlnAsp290295300MetLeuSerSerHisHisTyrLysSerPheLysV alSerMetIleHis305310315320ArgLeuArgPheThrThrAspValGlnLeuGlyIleSerGlyAspLys325330 335ValGluIleAspProValThrAsnGlnLysAlaSerThrLysPheTrp340345350IleLysGlnLysProIleSerIleAspSerAspLeuLeuCysAl aCys355360365AspLeuAlaGluGluLysSerProSerHisAlaIlePheLysLeuThr370375380TyrLeuSerAsnHis AspTyrLysHisLeuTyrPheGluSerAspAla385390395400AlaThrValAsnGluIleValLeuLysValAsnTyrIleLeuGluSer405 410415ArgAlaSerThrAlaArgAlaAspTyrPheAlaGlnLysLysSerGly420425430Arg(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3987 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 3..1498(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GGCGGCCGCGGCAGGGCGGGCGCCGCGCGGAGGCAGGGCG GGCGTATTCAATGGAAGTGT60GTTACCAGCTGCCGGTACTGCCCCTGGACAGGCCGGTCCCCCAGCACGTCCTCAGCCGCC120GAGGAGCCATCAGCTTCAGCTCCAGCTCCGCTCTCTTCGGCTGCCCCAATCCCCGGCAGC180TCTCTCAGAGGCGT GGAGCTATTTCCTATGACAGTTCTGATCAGACTGCATTATACATTC240GTATGCTAGGAGATGTACGTGTAAGGAGCCGAGCAGGATTTGAATCAGAAAGAAGAGGTT300CTCACCCATATATTGATTTTCGTATTTTCCACTCTCAATCTGAAATTGAAGTGTCTGT CT360CTGCAAGGAATATCAGAAGGCTACTAAGTTTCCAGCGATATCTTAGATCTTCACGCTTTT420TTCGTGGTACTGCGGTTTCAAATTCCCTAAACATTTTAGATGATGATTATAATGGACAAG480CCAAGTGTATGCTGGAAAAAGTTGGAAATTGG AATTTTGATATCTTTCTATTTGATAGAC540TAACAAATGGAAATAGTCTAGTAAGCTTAACCTTTCATTTATTTAGTCTTCATGGATTAA600TTGAGTACTTCCATTTAGATATGATGAAACTTCGTAGATTTTTAGTTATGATTCAAGAAG660ATTACCA CAGTCAAAATCCTTACCATAACGCAGTCCACGCTGCGGATGTTACTCAGGCCA720TGCACTGTTACTTAAAGGAACCTAAGCTTGCCAATTCTGTAACTCCTTGGGATATCTTGC780TGAGCTTAATTGCAGCTGCCACTCATGATCTGGATCATCCAGGTGTTAAT CAACCTTTCC840TTATTAAAACTAACCATTACTTGGCAACTTTATACAAGAATACCTCAGTACTGGAAAATC900ACCACTGGAGATCTGCAGTGGGCTTATTGAGAGAATCAGGCTTATTCTCACATCTGCCAT960TAGAAAGCAGGCAACAAATGGAGAC ACAGATAGGTGCTCTGATACTAGCCACAGACATCA1020GTCGCCAGAATGAGTATCTGTCTTTGTTTAGGTCCCATTTGGATAGAGGTGATTTATGCC1080TAGAAGACACCAGACACAGACATTTGGTTTTACAGATGGCTTTGAAATGTGCTGATATTT1140 GTAACCCATGTCGGACGTGGGAATTAAGCAAGCAGTGGAGTGAAAAAGTAACGGAGGAAT1200TCTTCCATCAAGGAGATATAGAAAAAAAATATCATTTGGGTGTGAGTCCACTTTGCGATC1260GTCACACTGAATCTATTGCCAACATCCAGATTGGTTTTATGAC TTACCTAGTGGAGCCTT1320TATTTACAGAATGGGCCAGGTTTTCCAATACAAGGCTATCCCAGACAATGCTTGGACACG1380TGGGGCTGAATAAAGCCAGCTGGAAGGGACTGCAGAGAGAACAGTCGAGCAGTGAGGACA1440CTGATGCTGCATTTGAGT TGAACTCACAGTTATTACCTCAGGAAAATCGGTTATCATAAC1500CCCCAGAACCAGTGGGACAAACTGCCTCCTGGAGGTTTTTAGAAATGTGAAATGGGGTCT1560TGAGGTGAGAGAACTTAACTCTTGACTGCCAAGGTTTCCAAGTGAGTGATGCCAGCCAGC 1620ATTATTTATTTCCAAGATTTCCTCTGTTGGATCATTTGAACCCACTTGTTAATTGCAAGA1680CCCGAACATACAGCAATATGAATTTGGCTTTCATGTGAAACCTTGAATATNNAAAGCCCA1740GCAGGAGAGAATCCGAAAGGAGTAACAAAGGAAGTT TTGATATGTGCCACGACTTTTTCA1800AAGCATCTAATCTTCAAAACGTCAAACTTGAATTGTTCAGCAACAATCTCTTGGAATTTA1860ACCAGTCTGATGCAACAATGTGTATCTTGTACCTTCCACTAAGTTCTCTCTGAGAAAATG1920GAAATGTGAA GTGCCCAGCCTCTGCNTGCCTCTGGCAAGACAATGTTTACAAATCAACTC1980TGAAAATATTGGTTCTAAATTGCCTTGGAGCATGATTGTGAAGGAACCACTCAAACAAAT2040TTAAAGATCAAACTTTAGACTGCAGCTCTTTCCCCCTGGTTTGCCTTTTTCTTC TTTGGA2100TGCCACCAAAGCCTCCCATTTGCTATAGTTTTATTTCATGCACTGGAAACTGAGCATTTA2160TCGTAGAGTACCGCCAAGCTTTCACTCCAGTGCCGTTTGGCAATGCAATTTTTTTTAGCA2220ATTAGTTTTTAATTTGGGGTGGGAGGGGA AGAACACCAATGTCCTAGCTGTATTATGATT2280CTGCACTCAAGACATTGCATGTTGTTTTCACTACTGTACACTTGACCTGCACATGCGAGA2340AAAAGGTGGAATGTTTAAAACACCATAATCAGCTCAGNGTATTTGCCAATCTGAAATAAA2400AGT GGGATGGGAGAGCGTGTCCTTCAGATCAAGGGTACTAAAGTCCCTTTCGCTGCAGTG2460AGTGAGAGGTATGTTGTGTGTGAATGTACGGATGTGTGTTTGNGTGNATGTTTGTGCATG2520TGTGACNGTGCATGTTATGTTTCTCCATGTGGGCAAAGATTTGAAAN GTAAGCTTTTATT2580TATTATTTTAGAATGTGACATAATGAGCAGCCACACTCGGGGGAGGGGAAGGTTGGTAGG2640TAAGCTGTAACAGATTGCTCCAGTTGCCTTAAACTATGCACATAGCTAAGTGACCAAACT2700TCTTGTTTTGATTTGAAAAAA GTGCATTGTTTTCTTGTCCCTCCCTTTGATGAAACGTTA2760CCCTTTGACGGGCCTTTTGATGTGAACAGATGTTTTCTAGGACAAACTATAAGGACTAAT2820TTTAAACTTCAAACATTCCACTTTTGTAATTTGTTTTAAATTGTTTTATGTATAGTAAGC288 0ACAACTGTAATCTAGTTTTAAGAGAAACCGGTGCTTTCTTTTAGTTCATTTGTATTTCCC2940TTGTTACTGTAAAAGACTGTTTATTAATTGTTTACAGTTTGTTGCAACAGCCATTTTCTT3000GGGAGAAAGCTTGAGTGTAAAGCCATTTGTAAAAGGCTTT GCCATACTCATTTTAATATG3060TGCCTGTTGCTGTTAACTTTTGATGAATAAAAACCTATCTTTTCATGAAACTTCTCTCTA3120TACAAATTGAAATACATAATGCTTTCTGGTTCTTCTTCAAACCAAAACTTGTCAAATTCA3180TAGACAAGATAACA GTAAAACTGATGAAAGTGTTCCATTGTTGGTATACCAGGAACAAGG3240TTATAGAGATGAAACTTCAAAGCTTCACTCTTCAGTAAGCTATAAGCCATCTCTGTAAGA3300TTGATTCCAACTATTGCATAAGAATACCCTAATTTTGGATGATTTGAACGGGAAAGAA TC3360TGATGAGCTTCACTAGTGTAATTTTCACTGAAATACACAAGATTGATTAACCCAAGTATG3420CCCATGCCTCTGAAGTCTGTCTTGGGATCATCACCCTGAAAACCAATTTCAGCCCACTGC3480TTGGAGATTCTAGCGTTTAACTTCTTCGTGGG CATTAGAAGATTCCAAAGCTTCATGAGT3540AGCTCTTCATGCTGTAGGTTATCAGAATCATATGGCCTTTTCCTCACACTTTCTACATCC3600AAATACAGCTGTTTATAACCAGTTATCTGCAGTAAGCACATCTTCATGCATATTTTAAAA3660CTGGCAT CCTTCTCAGGGTTAATATTCTTTTCCTTCATAATATCATCTACATATTTGTCC3720ACTTCACTCTGAACAACATGTGTCGCCTTCTGTAAAACCTTATTCTTGGAGTATGTCAAG3780GAATTTTCTATCCTGTGTGTCCTTTGTGCACCTACATAGGTATCAAATAT TCGCTGCAAT3840TCACACTTCCCAGTCATCTGTCGTAATAGCCATTTCATCCAAAATCGAAAAAAGTGCCCA3900TAGAAGAACTCCCACAAAGAAATAAACATTTTTTTTTCCTCACAGGAGCGGAAGAACTAG3960GGGGAGCAGGAGCTGCAATGCGGCC GC3987(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 498 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:ArgProArgGlnGlyGlyArgArg AlaGluAlaGlyArgAlaTyrSer151015MetGluValCysTyrGlnLeuProValLeuProLeuAspArgProVal2025 30ProGlnHisValLeuSerArgArgGlyAlaIleSerPheSerSerSer354045SerAlaLeuPheGlyCysProAsnProArgGlnLeuSerGlnArg Arg505560GlyAlaIleSerTyrAspSerSerAspGlnThrAlaLeuTyrIleArg65707580MetLeuGly AspValArgValArgSerArgAlaGlyPheGluSerGlu859095ArgArgGlySerHisProTyrIleAspPheArgIlePheHisSerGln100 105110SerGluIleGluValSerValSerAlaArgAsnIleArgArgLeuLeu115120125SerPheGlnArgTyrLeuArgSerSerAr gPhePheArgGlyThrAla130135140ValSerAsnSerLeuAsnIleLeuAspAspAspTyrAsnGlyGlnAla145150155 160LysCysMetLeuGluLysValGlyAsnTrpAsnPheAspIlePheLeu165170175PheAspArgLeuThrAsnGlyAsnSerLeuValSerLeuThrPhe His180185190LeuPheSerLeuHisGlyLeuIleGluTyrPheHisLeuAspMetMet195200205LysLeuArgA rgPheLeuValMetIleGlnGluAspTyrHisSerGln210215220AsnProTyrHisAsnAlaValHisAlaAlaAspValThrGlnAlaMet225230 235240HisCysTyrLeuLysGluProLysLeuAlaAsnSerValThrProTrp245250255AspIleLeuLeuSerLeuIleAlaAl aAlaThrHisAspLeuAspHis260265270ProGlyValAsnGlnProPheLeuIleLysThrAsnHisTyrLeuAla275280 285ThrLeuTyrLysAsnThrSerValLeuGluAsnHisHisTrpArgSer290295300AlaValGlyLeuLeuArgGluSerGlyLeuPheSerHisLeuProLeu305 310315320GluSerArgGlnGlnMetGluThrGlnIleGlyAlaLeuIleLeuAla325330335ThrAspI leSerArgGlnAsnGluTyrLeuSerLeuPheArgSerHis340345350LeuAspArgGlyAspLeuCysLeuGluAspThrArgHisArgHisLeu355 360365ValLeuGlnMetAlaLeuLysCysAlaAspIleCysAsnProCysArg370375380ThrTrpGluLeuSerLysGlnTrpSerGluLysVa lThrGluGluPhe385390395400PheHisGlnGlyAspIleGluLysLysTyrHisLeuGlyValSerPro405410 415LeuCysAspArgHisThrGluSerIleAlaAsnIleGlnIleGlyPhe420425430MetThrTyrLeuValGluProLeuPheThrGluTrpAlaArgPhe Ser435440445AsnThrArgLeuSerGlnThrMetLeuGlyHisValGlyLeuAsnLys450455460AlaSerTrpLysGlyL euGlnArgGluGlnSerSerSerGluAspThr465470475480AspAlaAlaPheGluLeuAsnSerGlnLeuLeuProGlnGluAsnArg485 490495LeuSer(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3131 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: 1652(D) OTHER INFORMATION: /note="A shift in reading framemay occur at this residue."(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: join(743..1648, 1651..2661)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:GCGGCCGCGCGGCCTAGGCCGCATCCCGGAGCTGCAACTGGTGGCCTTCCCGGTGGCG GT60GGCGGCTGAGGACGAGGCGTTCCTGCCCGAGCCCCTGGCCCCGCGCGCGCCCCGCCGCCC120GCGTTCGCCGCCCTCCTCGCCCGTCTTCTTCGCCAGCCCGTCCCCAACTTTCCGCAGACG180CCTTCGGCTTCTCCGCAGCTGCCAGGATTTGG GCCGCCAGGCTTGGGCTGGGGCTGGCTT240CGAGGCAGAGAATGGGCCGACACCATCTCCTGGCCGCAGCCCCCTGGACTCGCAGGCGAG300CCCAGGACTCGTGCTGCACGCCGGGGCGCCACCAGCCAGCGCCGGGAGTCCTTCCTGTAC360CGCTCAG ACAGCGACTATGACATGTCACCCAAGACCATGTCCCGGAACTCATCGGTCACC420AGCGAGGCACAGTTGCTTCTCTGCGGACCCCTGACCTGCCTCTGTCCTCAATCACAGGCA480CGCTGAAGACCTCATCGTAACACCATTTGCTCAGGTGCTGGCCAGCCTCC GGAGCGTCCG540TAGCAACTTCTCACTCCTGACCAATGTGCCCGTTCCCAGTAACAAGCGGTCCCGCTGGGC600GGCCCCACCCCTGTCTGCAAGGCCACGCTGTCAGACCTTCTCAGTCACTACCCTGGCTGC660CCCTTCCTTAGAAGAAACGTGTCAG CAGTTGGCCCGGGAGACTCTGGAGGAGCTGGACTG720GTGTCTGGAGCAGCTGGAGACCATGCAGACCTATCGCTCTGTCAGCGAGATG772MetGlnThrTyrArgSerValSerGluMet 1510GCCTCGCACAAGTTCAAAAGGATGTTGAACCGTGAGCTCACACACCTG820AlaSerHisLysPheLysArgMetLeuAsnArgGluLeuThrHisLeu 152025TCAGAAATGAGCAGGTCCGGAAACCAGGTCTCAGAGTACATTTCCACA868SerGluMetSerArgSerGlyAsnGlnValSerGluTyrIleSerTh r303540ACATTCCTGGACAAACAGAATGAAGTGGAGATCCCATCACCCACGATG916ThrPheLeuAspLysGlnAsnGluValGluIleProSerProThrMe t455055AAGGAACGAGAAAAACAGCAAGCGCCGCGACCAAGACCCTCCCAGCCG964LysGluArgGluLysGlnGlnAlaProArgProArgProSerGlnPro 606570CCCCCGCCCCCTGTACCACACTTACAGCCCATGTCCCAAATCACAGGG1012ProProProProValProHisLeuGlnProMetSerGlnIleThrGly75 808590TTGAAAAAGTTGATGCATAGTAACAGCCTGAACAACTCTAACATTCCC1060LeuLysLysLeuMetHisSerAsnSerLeuAsnAsnSerAsnIlePro 95100105CGATTTGGGGTGAAGACCGATCAAGAAGAGCTCCTGGCCCAAGAACTG1108ArgPheGlyValLysThrAspGlnGluGluLeuLeuAlaGlnGluLe u110115120GAGAACCTGAACAAGTGGGGCCTGAACATCTTTTGCGTGTCGGATTAC1156GluAsnLeuAsnLysTrpGlyLeuAsnIlePheCysValSerAspTy r125130135GCTGGAGGCCGCTCACTCACCTGCATCATGTACATGATATTCCAGGAG1204AlaGlyGlyArgSerLeuThrCysIleMetTyrMetIlePheGlnGlu 140145150CGGGACCTGCTGAAGAAATTCCGCATCCCTGTGGACACGATGGTGACA1252ArgAspLeuLeuLysLysPheArgIleProValAspThrMetValThr155 160165170TACATGCTGACGCTGGAGGATCACTACCACGCTGACGTGGCCTACCAT1300TyrMetLeuThrLeuGluAspHisTyrHisAlaAspValAlaTyrHis 175180185AACAGCCTGCACGCAGCTGACGTGCTGCAGTCCACCCACGTACTGCTG1348AsnSerLeuHisAlaAlaAspValLeuGlnSerThrHisValLeuLe u190195200GCCACGCCTGCACTAGATGCAGTGTTCACGGACCTGGAGATTCTCGCC1396AlaThrProAlaLeuAspAlaValPheThrAspLeuGluIleLeuAl a205210215GCCCTCTTCGCGGCTGCCATCCACGATGTGGATCACCCTGGGGTCTCC1444AlaLeuPheAlaAlaAlaIleHisAspValAspHisProGlyValSer 220225230AACCAGTTCCTCATCAACACCAATTCGGAGCTGGCGCTCATGTACAAC1492AsnGlnPheLeuIleAsnThrAsnSerGluLeuAlaLeuMetTyrAsn235 240245250GATGAGTCGGTGCTCGAGAATCACCACCTGGCCGTGGGCTTCAAGCTG1540AspGluSerValLeuGluAsnHisHisLeuAlaValGlyPheLysLeu 255260265CTGCAGGAGGACAACTGCGACATCTTCCAGAACCTCAGCAAGCGCCAG1588LeuGlnGluAspAsnCysAspIlePheGlnAsnLeuSerLysArgGl n270275280CGCAGAGCCTACGCAAGATGGTCATCGACATGGTGCTGGCCACGGACA1636ArgArgAlaTyrAlaArgTrpSerSerThrTrpCysTrpProArgTh r285290295TGTCCAAGCACATGACCCTCCTGGCTGACCTGAAGACCATGGTGGAG1683CysProSerThrThrLeuLeuAlaAspLeuLysThrMetValGlu 300305310ACCAAGAAAGTGACCAGCTCAGGGGTCCTCCTGCTAGATAACTACTCC1731ThrLysLysValThrSerSerGlyValLeuLeuLeuAspAsnTyrSer31 5320325GACCGCATCCAGGTCCTCCGGAACATGGTGCACTGTGCCGACCTCAGC1779AspArgIleGlnValLeuArgAsnMetValHisCysAlaAspLeuSer330 335340345AACCCCACCAAGCCGCTGGAGCTGTACCGCCAGTGGACAGACCGCATC1827AsnProThrLysProLeuGluLeuTyrArgGlnTrpThrAspArgIle 350355360ATGGCCGAGTTCTTCCAGCAGGGTGACCGAGAGCGCGAGCGTGGCATG1875MetAlaGluPhePheGlnGlnGlyAspArgGluArgGluArgGlyMet 365370375GAAATCAGCCCCATGTGTGACAAGCACACTGCCTCCGTGGAGAAGTCT1923GluIleSerProMetCysAspLysHisThrAlaSerValGluLysSer 380385390CAGGTGGGTTTTATTGACTACATTGTGCACCCATTGTGGGAGACCTGG1971GlnValGlyPheIleAspTyrIleValHisProLeuTrpGluThrTrp39 5400405GCGGACCTTGTCCACCCAGATGCCCAGGAGATCTTGGACACTTTGGAG2019AlaAspLeuValHisProAspAlaGlnGluIleLeuAspThrLeuGlu410 415420425GACAACCGGGACTGGTACTACAGCGCCATCCGGCAGAGCCCATCTCCG2067AspAsnArgAspTrpTyrTyrSerAlaIleArgGlnSerProSerPro 430435440CCACCCGAGGAGGAGTCAAGGGGGCCAGGCCACCCACCCCTGCCTGAC2115ProProGluGluGluSerArgGlyProGlyHisProProLeuProAsp 445450455AAGTTCCAGTTTGAGCTGACGCTGGAGGAGGAAGAGGAGGAAGAAATA2163LysPheGlnPheGluLeuThrLeuGluGluGluGluGluGluGluIle 460465470TCAATGGCCCAGATACCGTGCACAGCCCAAGAGGCATTGACTGAGCAG2211SerMetAlaGlnIleProCysThrAlaGlnGluAlaLeuThrGluGln47 5480485GGATTGTCAGGAGTCGAGGAAGCTCTGGATGCAACCATAGCCTGGGAG2259GlyLeuSerGlyValGluGluAlaLeuAspAlaThrIleAlaTrpGlu490 495500505GCATCCCCGGCCCAGGAGTCGTTGGAAGTTATGGCACAGGAAGCATCC2307AlaSerProAlaGlnGluSerLeuGluValMetAlaGlnGluAlaSer 510515520CTGGAGGCCGAGCTGGAGGCAGTGTATTTGACACAGCAGGCACAGTCC2355LeuGluAlaGluLeuGluAlaValTyrLeuThrGlnGlnAlaGlnSer 525530535ACAGGCAGTGCACCTGTGGCTCCGGATGAGTTCTCGTCCCGGGAGGAA2403ThrGlySerAlaProValAlaProAspGluPheSerSerArgGluGlu 540545550TTCGTGGTTGCTGTAAGCCACAGCAGCCCCTCTGCCCTGGCTCTTCAA2451PheValValAlaValSerHisSerSerProSerAlaLeuAlaLeuGln55 5560565AGCCCCCTTCTCCCTGCTTGGAGGACCCTGTCTGTTTCAGAGCATGCC2499SerProLeuLeuProAlaTrpArgThrLeuSerValSerGluHisAla570 575580585CCGGGCCTCCCGGGCCTCCCCTCCACGGCGGCCGAGGTGGAGGCCCAA2547ProGlyLeuProGlyLeuProSerThrAlaAlaGluValGluAlaGln 590595600CGAGAGCACCAGGCTGCCAAGAGGGCTTGCAGTGCCTGCGCAGGGACA2595ArgGluHisGlnAlaAlaLysArgAlaCysSerAlaCysAlaGlyThr 605610615TTTGGGGAGGACACATCCGCACTCCCAGCTCCTGGTGGCGGGGGGTCA2643PheGlyGluAspThrSerAlaLeuProAlaProGlyGlyGlyGlySer 620625630GGTGGAGACCCTACCTGATCCCCAGACCTCTGTCCCTGTTCCCCTCCACTCCTCC2698GlyGlyAspProThr635CCTCACTCCCCTGCTCCCCCGACCACCTCCTCCTCT GCCTCAAAGACTCTTGTCCTCTTG2758TCCCTCCTGAGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACAACACAAATGAATG2818GGCCATTTTATTGATTTTTACCTCCTAATAGTGGATACAGGTTGCTGTGGTTTCCAGCAG2878GATCTCAGAT GCAAAGGGAAGTGAAGAAAACAGATGAATCCCTAGGGTACCCCGCCATGG2938AACCAAACACCACGTCAACTGGAACTCTTCTTGCAAACGAAGGCTGAAGATCAAGAATGA2998CATTCTCACACCACAGCACAGCTTAAATACTTCTTTGACAAAAATAATAATAAA TTATAT3058TTGACTCAGAAAATAAATTCTGTTCAGCAGAGTGACAGGAGGTAAAAATCAAATGAATGG3118GCAATGCGGCCGC3131(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 638 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:MetGlnThrTyrArgSerValSerGluMetAlaSerHisLysPheLys1510 15ArgMetLeuAsnArgGluLeuThrHisLeuSerGluMetSerArgSer202530GlyAsnGlnValSerGluTyrIleSerThrThrPheLeuAspLysGl n354045AsnGluValGluIleProSerProThrMetLysGluArgGluLysGln505560GlnAlaProArgProArg ProSerGlnProProProProProValPro65707580HisLeuGlnProMetSerGlnIleThrGlyLeuLysLysLeuMetHis85 9095SerAsnSerLeuAsnAsnSerAsnIleProArgPheGlyValLysThr100105110AspGlnGluGluLeuLeuAlaGlnGluL euGluAsnLeuAsnLysTrp115120125GlyLeuAsnIlePheCysValSerAspTyrAlaGlyGlyArgSerLeu130135140 ThrCysIleMetTyrMetIlePheGlnGluArgAspLeuLeuLysLys145150155160PheArgIleProValAspThrMetValThrTyrMetLeuThrLeuGlu 165170175AspHisTyrHisAlaAspValAlaTyrHisAsnSerLeuHisAlaAla180185190AspValLeu GlnSerThrHisValLeuLeuAlaThrProAlaLeuAsp195200205AlaValPheThrAspLeuGluIleLeuAlaAlaLeuPheAlaAlaAla2102 15220IleHisAspValAspHisProGlyValSerAsnGlnPheLeuIleAsn225230235240ThrAsnSerGluLeuAlaLeuMetTyrAsnA spGluSerValLeuGlu245250255AsnHisHisLeuAlaValGlyPheLysLeuLeuGlnGluAspAsnCys260265 270AspIlePheGlnAsnLeuSerLysArgGlnArgArgAlaTyrAlaArg275280285TrpSerSerThrTrpCysTrpProArgThrCysProSerThrThrLeu 290295300LeuAlaAspLeuLysThrMetValGluThrLysLysValThrSerSer305310315320GlyValLeuLeu LeuAspAsnTyrSerAspArgIleGlnValLeuArg325330335AsnMetValHisCysAlaAspLeuSerAsnProThrLysProLeuGlu340 345350LeuTyrArgGlnTrpThrAspArgIleMetAlaGluPhePheGlnGln355360365GlyAspArgGluArgGluArgGlyMetGluI leSerProMetCysAsp370375380LysHisThrAlaSerValGluLysSerGlnValGlyPheIleAspTyr385390395 400IleValHisProLeuTrpGluThrTrpAlaAspLeuValHisProAsp405410415AlaGlnGluIleLeuAspThrLeuGluAspAsnArgAspTrpTyrTy r420425430SerAlaIleArgGlnSerProSerProProProGluGluGluSerArg435440445GlyProGlyHis ProProLeuProAspLysPheGlnPheGluLeuThr450455460LeuGluGluGluGluGluGluGluIleSerMetAlaGlnIleProCys465470 475480ThrAlaGlnGluAlaLeuThrGluGlnGlyLeuSerGlyValGluGlu485490495AlaLeuAspAlaThrIleAlaTrpGluA laSerProAlaGlnGluSer500505510LeuGluValMetAlaGlnGluAlaSerLeuGluAlaGluLeuGluAla515520 525ValTyrLeuThrGlnGlnAlaGlnSerThrGlySerAlaProValAla530535540ProAspGluPheSerSerArgGluGluPheValValAlaValSerHis545 550555560SerSerProSerAlaLeuAlaLeuGlnSerProLeuLeuProAlaTrp565570575ArgThrLeu SerValSerGluHisAlaProGlyLeuProGlyLeuPro580585590SerThrAlaAlaGluValGluAlaGlnArgGluHisGlnAlaAlaLys595 600605ArgAlaCysSerAlaCysAlaGlyThrPheGlyGluAspThrSerAla610615620LeuProAlaProGlyGlyGlyGlySerGlyGlyAspP roThr625630635(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3186 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 139..2348(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:GCGGCCGCGGCGGTGCAGCAGAGGCGCCTCGGGCAGGAGGAGGGCGGCTTCTGCGAGGGC60AGCCTGAGGTATTAAAAAGTGTCAGCAAACTGCATTGAATAACAGACATCCTAAGAGGGG120ATATTTTCC ACCTCTATAATGAAGAAAAGCAGGAGTGTGATGACGGTGATG171MetLysLysSerArgSerValMetThrValMet1510GCTG ATGATAATGTTAAAGATTATTTTGAATGTAGCTTGAGTAAATCC219AlaAspAspAsnValLysAspTyrPheGluCysSerLeuSerLysSer152025TACA GTTCTTCCAGTAACACACTTGGGATCGACCTCTGGAGAGGGAGA267TyrSerSerSerSerAsnThrLeuGlyIleAspLeuTrpArgGlyArg303540AGGTGTT GCTCAGGAAACTTACAGTTACCACCACTGTCTCAAAGACAG315ArgCysCysSerGlyAsnLeuGlnLeuProProLeuSerGlnArgGln455055AGTGAAAGGGCAA GGACTCCTGAGGGAGATGGTATTTCCAGGCCGACC363SerGluArgAlaArgThrProGluGlyAspGlyIleSerArgProThr60657075ACACTGC CTTTGACAACGCTTCCAAGCATTGCTATTACAACTGTAAGC411ThrLeuProLeuThrThrLeuProSerIleAlaIleThrThrValSer808590CAGG AGTGCTTTGATGTGGAAAATGGCCCTTCCCCAGGTCGGAGTCCA459GlnGluCysPheAspValGluAsnGlyProSerProGlyArgSerPro95100105CTGG ATCCCCAGGCCAGCTCTTCCGCTGGGCTGGTACTTCACGCCACC507LeuAspProGlnAlaSerSerSerAlaGlyLeuValLeuHisAlaThr110115120TTTCCTG GGCACAGCCAGCGCAGAGAGTCATTTCTCTACAGATCAGAC555PheProGlyHisSerGlnArgArgGluSerPheLeuTyrArgSerAsp125130135AGCGACTATGACT TGTCACCAAAGGCGATGTCGAGAAACTCTTCTCTT603SerAspTyrAspLeuSerProLysAlaMetSerArgAsnSerSerLeu140145150155CCAAGCG AGCAACACGGCGATGACTTGATTGTAACTCCTTTTGCCCAG651ProSerGluGlnHisGlyAspAspLeuIleValThrProPheAlaGln160165170GTCC TTGCCAGCTTGCGAAGTGTGAGAAACAACTTCACTATACTGACA699ValLeuAlaSerLeuArgSerValArgAsnAsnPheThrIleLeuThr175180185AACC TTCATGGTACATCTAACAAGAGGTCCCCAGCTGCTAGTCAGCCT747AsnLeuHisGlyThrSerAsnLysArgSerProAlaAlaSerGlnPro190195200CCTGTCT CCAGAGTCAACCCACAAGAAGAATCTTATCAAAAATTAGCA795ProValSerArgValAsnProGlnGluGluSerTyrGlnLysLeuAla205210215ATGGAAACGCTGG AGGAATTAGACTGGTGTTTAGACCAGCTAGAGACC843MetGluThrLeuGluGluLeuAspTrpCysLeuAspGlnLeuGluThr220225230235ATACAGA CCTACCGGTCTGTCAGTGAGATGGCTTCTAACAAGTTCAAA891IleGlnThrTyrArgSerValSerGluMetAlaSerAsnLysPheLys240245250AGAA TGCTGAACCGGGAGCTGACACACCTCTCAGAGATGAGCCGATCA939ArgMetLeuAsnArgGluLeuThrHisLeuSerGluMetSerArgSer255260265GGGA ACCAGGTGTCTGAATACATTTCAAATACTTTCTTAGACAAGCAG987GlyAsnGlnValSerGluTyrIleSerAsnThrPheLeuAspLysGln270275280AATGATG TGGAGATCCCATCTCCTACCCAGAAAGACAGGGAGAAAAAG1035AsnAspValGluIleProSerProThrGlnLysAspArgGluLysLys285290295AAAAAGCAGCAGC TCATGACCCAGATAAGTGGAGTGAAGAAATTAATG1083LysLysGlnGlnLeuMetThrGlnIleSerGlyValLysLysLeuMet300305310315CATAGTT CAAGCCTAAACAATACAAGCATCTCACGCTTTGGAGTCAAC1131HisSerSerSerLeuAsnAsnThrSerIleSerArgPheGlyValAsn320325330ACTG AAAATGAAGATCACCTGGCCAAGGAGCTGGAAGACCTGAACAAA1179ThrGluAsnGluAspHisLeuAlaLysGluLeuGluAspLeuAsnLys335340345TGGG GTCTTAACATCTTTAATGTGGCTGGATATTCTCACAATAGACCC1227TrpGlyLeuAsnIlePheAsnValAlaGlyTyrSerHisAsnArgPro350355360CTAACAT GCATCATGTATGCTATATTCCAGGAAAGAGACCTCCTAAAG1275LeuThrCysIleMetTyrAlaIlePheGlnGluArgAspLeuLeuLys365370375ACATTCAGAATCT CATCTGACACATTTATAACCTACATGATGACTTTA1323ThrPheArgIleSerSerAspThrPheIleThrTyrMetMetThrLeu380385390395GAAGACC ATTACCATTCTGACGTGGCATATCACAACAGCCTGCACGCT1371GluAspHisTyrHisSerAspValAlaTyrHisAsnSerLeuHisAla400405410GCTG ATGTAGCCCAGTCGACCCATGTTCTCCTTTCTACACCAGCATTA1419AlaAspValAlaGlnSerThrHisValLeuLeuSerThrProAlaLeu415420425GACG CTGTCTTCACAGATTTGGAGATCCTGGCTGCCATTTTTGCAGCT1467AspAlaValPheThrAspLeuGluIleLeuAlaAlaIlePheAlaAla430435440GCCATCC ATGACGTTGATCATCCTGGAGTCTCCAATCAGTTTCTCATC1515AlaIleHisAspValAspHisProGlyValSerAsnGlnPheLeuIle445450455AACACAAATTCAG AACTTGCTTTGATGTATAATGATGAATCTGTGTTG1563AsnThrAsnSerGluLeuAlaLeuMetTyrAsnAspGluSerValLeu460465470475GAAAATC ATCACCTTGCTGTGGGTTTCAAACTGCTGCAAGAAGAACAC1611GluAsnHisHisLeuAlaValGlyPheLysLeuLeuGlnGluGluHis480485490TGTG ACATCTTCATGAATCTCACCAAGAAGCAGCGTCAGACACTCAGG1659CysAspIlePheMetAsnLeuThrLysLysGlnArgGlnThrLeuArg495500505AAGA TGGTTATTGACATGGTGTTAGCAACTGATATGTCTAAACATATG1707LysMetValIleAspMetValLeuAlaThrAspMetSerLysHisMet510515520AGCCTGC TGGCAGACCTGAAGACAATGGTAGAAACGAAGAAAGTTACA1755SerLeuLeuAlaAspLeuLysThrMetValGluThrLysLysValThr525530535AGTTCAGGCGTTC TTCTCCTAGACAACTATACCGATCGCATTCAGGTC1803SerSerGlyValLeuLeuLeuAspAsnTyrThrAspArgIleGlnVal540545550555CTTCGCA ACATGGTACACTGTGCAGACCTGAGCAACCCCACCAAGTCC1851LeuArgAsnMetValHisCysAlaAspLeuSerAsnProThrLysSer560565570TTGG AATTGTATCGGCAATGGACAGACCGCATCATGGAGGAATTTTTC1899LeuGluLeuTyrArgGlnTrpThrAspArgIleMetGluGluPhePhe575580585CAGC AGGGAGACAAAGAGCGGGAGAGGGGAATGGAAATTAGCCCAATG1947GlnGlnGlyAspLysGluArgGluArgGlyMetGluIleSerProMet590595600TGTGATA AACACACAGCTTCTGTGGAAAAATCCCAGGTTGGTTTCATC1995CysAspLysHisThrAlaSerValGluLysSerGlnValGlyPheIle605610615GACTACATTGTCC ATCCATTGTGGGAGACATGGGCAGATTTGGTACAG2043AspTyrIleValHisProLeuTrpGluThrTrpAlaAspLeuValGln620625630635CCTGATG CTCAGGACATTCTCGATACCTTAGAAGATAACAGGAACTGG2091ProAspAlaGlnAspIleLeuAspThrLeuGluAspAsnArgAsnTrp640645650TATC AGAGCATGATACCTCAAAGTCCCTCACCACCACTGGACGAGCAG2139TyrGlnSerMetIleProGlnSerProSerProProLeuAspGluGln655660665AACA GGGACTGCCAGGGTCTGATGGAGAAGTTTCAGTTTGAACTGACT2187AsnArgAspCysGlnGlyLeuMetGluLysPheGlnPheGluLeuThr670675680CTCGATG AGGAAGATTCTGAAGGACCTGAGAAGGAGGGAGAGGGACAC2235LeuAspGluGluAspSerGluGlyProGluLysGluGlyGluGlyHis685690695AGCTATTTCAGCA GCACAAAGACGCTTTGTGTGATTGATCCAGAAAAC2283erTyrPheSerSerThrLysThrLeuCysValIleAspProGluAsn700705710715AGAGATT CCCTGGGAGAGACTGACATAGACATTGCAACAGAAGACAAG2331ArgAspSerLeuGlyGluThrAspIleAspIleAlaThrGluAspLys720725730TCCC CCGTGGATACATAATCCCCCTCTCCCTGTGGAGATGAACATTC2378SerProValAspThr735TATCCTTGATGAGCATGCCAGCTATGTGGTAGGGCCAGCCCACCATGGGGGCCAAGACCT2438GCACAGGACAAG GGCCACCTGGCCTTTCAGTTACTTGAGTTTGGAGTCAGAAAGCAAGAC2498CAGGAAGCAAATAGCAGCTCAGGAAATCCCACGGTTGACTTGCCTTGATGGCAAGCTTGG2558TGGAGAGGGCTGAAGCTGTTGCTGGGGGCCGATTCTGATCAAGACACATGGCTTGA AAAT2618GGAAGACACAAAACTGAGAGATCATTCTGCACTAAGTTTCGGGAACTTATCCCCGACAGT2678GACTGAACTCACTGACTAATAACTTCATTTATGAATCTTCTCACTTGTCCCTTTGTCTGC2738CAACCTGTGTGCCTTTTTTGTAAAACATTT TCATGTCTTTAAAATGCCTGTTGAATACCT2798GGAGTTTAGTATCAACTTCTACACAGATAAGCTTTCAAAGTTGACAAACTTTTTTGACTC2858TTTCTGGAAAAGGGAAAGAAAATAGTCTTCCTTCTTTCTTGGGCAATATCCTTCACTTTA2918CTACA GTTACTTTTGCAAACAGACAGAAAGGATACACTTCTAACCACATTTTACTTCCTT2978CCCCTGTTGTCCAGTCCAACTCCACAGTCACTCTTAAAACTTCTCTCTGTTTGCCTGCCT3038CCAACAGTACTTTTAACTTTTTGCTGTAAACAGAATAAAATTGAACAAA TTAGGGGGTAG3098AAAGGAGCAGTGGTGTCGTTCACCGTGAGAGTCTGCATAGAACTCAGCAGTGTGCCCTGC3158TGTGTCTTGGACCCTGCAATGCGGCCGC3186(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 736 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:MetLysLysSerArgSerValMetThrValMetAlaAspAspAsnVal1510 15LysAspTyrPheGluCysSerLeuSerLysSerTyrSerSerSerSer202530AsnThrLeuGlyIleAspLeuTrpArgGlyArgArgCysCys SerGly354045AsnLeuGlnLeuProProLeuSerGlnArgGlnSerGluArgAlaArg505560ThrProGluGlyA spGlyIleSerArgProThrThrLeuProLeuThr65707580ThrLeuProSerIleAlaIleThrThrValSerGlnGluCysPheAsp8 59095ValGluAsnGlyProSerProGlyArgSerProLeuAspProGlnAla100105110SerSerSerAlaGlyLeuValLe uHisAlaThrPheProGlyHisSer115120125GlnArgArgGluSerPheLeuTyrArgSerAspSerAspTyrAspLeu130135 140SerProLysAlaMetSerArgAsnSerSerLeuProSerGluGlnHis145150155160GlyAspAspLeuIleValThrProPheAlaGlnValLeuAlaSer Leu165170175ArgSerValArgAsnAsnPheThrIleLeuThrAsnLeuHisGlyThr180185190SerA snLysArgSerProAlaAlaSerGlnProProValSerArgVal195200205AsnProGlnGluGluSerTyrGlnLysLeuAlaMetGluThrLeuGlu210 215220GluLeuAspTrpCysLeuAspGlnLeuGluThrIleGlnThrTyrArg225230235240SerValSerGluMetAlaSerAsnLy sPheLysArgMetLeuAsnArg245250255GluLeuThrHisLeuSerGluMetSerArgSerGlyAsnGlnValSer260265 270GluTyrIleSerAsnThrPheLeuAspLysGlnAsnAspValGluIle275280285ProSerProThrGlnLysAspArgGluLysLysLysLysGlnGln Leu290295300MetThrGlnIleSerGlyValLysLysLeuMetHisSerSerSerLeu305310315320AsnAsnT hrSerIleSerArgPheGlyValAsnThrGluAsnGluAsp325330335HisLeuAlaLysGluLeuGluAspLeuAsnLysTrpGlyLeuAsnIle34 0345350PheAsnValAlaGlyTyrSerHisAsnArgProLeuThrCysIleMet355360365TyrAlaIlePheGlnGluArgAspLe uLeuLysThrPheArgIleSer370375380SerAspThrPheIleThrTyrMetMetThrLeuGluAspHisTyrHis385390395 400SerAspValAlaTyrHisAsnSerLeuHisAlaAlaAspValAlaGln405410415SerThrHisValLeuLeuSerThrProAlaLeuAspAlaVal PheThr420425430AspLeuGluIleLeuAlaAlaIlePheAlaAlaAlaIleHisAspVal435440445AspHisP roGlyValSerAsnGlnPheLeuIleAsnThrAsnSerGlu450455460LeuAlaLeuMetTyrAsnAspGluSerValLeuGluAsnHisHisLeu465470 475480AlaValGlyPheLysLeuLeuGlnGluGluHisCysAspIlePheMet485490495AsnLeuThrLysLysGlnArgGl nThrLeuArgLysMetValIleAsp500505510MetValLeuAlaThrAspMetSerLysHisMetSerLeuLeuAlaAsp515520 525LeuLysThrMetValGluThrLysLysValThrSerSerGlyValLeu530535540LeuLeuAspAsnTyrThrAspArgIleGlnValLeuArgAsnMetVal5 45550555560HisCysAlaAspLeuSerAsnProThrLysSerLeuGluLeuTyrArg565570575GlnT rpThrAspArgIleMetGluGluPhePheGlnGlnGlyAspLys580585590GluArgGluArgGlyMetGluIleSerProMetCysAspLysHisThr595 600605AlaSerValGluLysSerGlnValGlyPheIleAspTyrIleValHis610615620ProLeuTrpGluThrTrpAlaAspLeuValGl nProAspAlaGlnAsp625630635640IleLeuAspThrLeuGluAspAsnArgAsnTrpTyrGlnSerMetIle645650 655ProGlnSerProSerProProLeuAspGluGlnAsnArgAspCysGln660665670GlyLeuMetGluLysPheGlnPheGluLeuThrLeuAspGlu GluAsp675680685SerGluGlyProGluLysGluGlyGluGlyHisSerTyrPheSerSer690695700ThrLysThrLeuC ysValIleAspProGluAsnArgAspSerLeuGly705710715720GluThrAspIleAspIleAlaThrGluAspLysSerProValAspThr72 5730735(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1276 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS (B) LOCATION: 2..504(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:GCGGCCGCATTGCGTGGTGGCGGCGGCCGAGCCTCGCTTTGAGAGACAGAATGGACAGCA60AATTATGGATGAACCTATGGGAGAGGAGGAGATTAACCCACAAACTGAAGAAGTCAGTAT120CAAAGAAATTGCAATCACA CATCATGTAAAGGAAGGACATGAAAAGGCAGATCCTTCCCA180GTTTGAACTTTTAAAAGTATTAGGGCAGGGATCATTTGGAAAGGTTTTCTTAGTTAAAAA240AATCTCAGGCTCTGATGCTAGGCAGCTTTATGCCATGAAGGTATTGAAGAAGGCCACACT 300GAAAGTTCGAGACCGAGTTCGGACAAAAATGGAACGTGATATCTTGGTAGAGGTTAATCA360TCCTTTTATTGTCAAGTTGCATTATCTTTTCAAACTGAAGGGAAGTTGTATCTTATTTGG420ATTTTCTCAGGGGAGGAGATTTGTTTACACGCTTATC CAAAGAGGTGATGTTCACAGAAG480AAGATGTCAAATTCTACCTGGCTGAACTTGCACTTGCTTTAGACCATCTACNTAGCCTGG540GAATAATTTATAGAGACTTAAAACCAGAAAATATCTTCTTGATGAAGAAGGTCACATCAA600GTTAACAGATT TCGGCCTAAGTAAAGAGTCTATTGACCATGAAAAGAAGGCATATCTTTT660TGTGGAACTGTGGAGTATATGGCTCCAGAAGTAGTTAATCGTCGAGGTCATACTCAGAGT720GCTGACTGGTGGTCTTTTGGTGTGTTAATGTTTGAAATGCTTACTGGTACCACTC CCTTT780CCAAGGAAAAGATCGAAAAGAAACAATGACTATGATTCTTAAAGCCAAAACTTGGAATGC840CACAGTTTTTGAGTCCTGAAGCGCAGAGTCTTTTACGAATGCTTTTMAAGCGAAATCCTG900CAAACAGATTAGGTGCAGGACCAGATGGAG TTGAAGAAATTAAAAGACATTCATTTTTCT960CAACGATAGACTGGAATAAACTGTATAGAGAGAAATTCATCCGCCATTTAAACCTGCAAC1020GGGCAGGCCTGAAGATACATTCTATTTTGATCCTGAGTTTACTGCAAAAACTCCCAAAGA1080TTCA CCTGGCATTCCACCTAGTGCTAATGCACATCAGCTTTTTCGGGGGTTTAGTTTTGT1140TGCTATTACCTCAGATGATGAAAGCCAAGCTATGCAGACAGTTGGTGTACATTCAATTGT1200TCAGCAGTTACACAGGAACAGTATNCAGTTTACTGATGGATATGAAGT AAAAGAAGATAT1260TGGAGTTGGCTCCTAC1276(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 167 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:ArgProHisCysValValAlaAlaAlaGluProArgPheGluArgGln151015AsnGlyGlnGlnIleMetAspGluProMetGlyGluGluG luIleAsn202530ProGlnThrGluGluValSerIleLysGluIleAlaIleThrHisHis354045ValLysG luGlyHisGluLysAlaAspProSerGlnPheGluLeuLeu505560LysValLeuGlyGlnGlySerPheGlyLysValPheLeuValLysLys6570 7580IleSerGlySerAspAlaArgGlnLeuTyrAlaMetLysValLeuLys859095LysAlaThrLeuLysValArgAspA rgValArgThrLysMetGluArg100105110AspIleLeuValGluValAsnHisProPheIleValLysLeuHisTyr115120 125LeuPheLysLeuLysGlySerCysIleLeuPheGlyPheSerGlnGly130135140ArgArgPheValTyrThrLeuIleGlnArgGlyAspValHisArgArg145 150155160ArgCysGlnIleLeuProGly165(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2384 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: join(1..1539, 1859..2383)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:GCGGCCGCATTCGGGGACAGCGGCGGGCGGCTGGGACGGCGGGTGCGG48AlaAlaAlaPheGlyAspS erGlyGlyArgLeuGlyArgArgValArg151015CGGGGCCGAGCCCGCACGATGCCTCACTTCACCGTGGTGCCAGTGGAC96ArgGlyArgAlaArgT hrMetProHisPheThrValValProValAsp202530GGGCCGAGGCGCGGCGACTATGACAACCTCGAGGGGCTCAGTTGGGTG144GlyProArgArgGlyA spTyrAspAsnLeuGluGlyLeuSerTrpVal354045GACTACGGGGAGCGCGCCGAGCTGGATGACTCGGACGGACATGGCAAC192AspTyrGlyGluArgAlaG luLeuAspAspSerAspGlyHisGlyAsn505560CACAGAGAGAGCAGCCCTTTTCTTTCCCCCTTGGAGGCTTCCAGAGGA240HisArgGluSerSerProPheLeuS erProLeuGluAlaSerArgGly65707580ATTGACTACTATGACAGGAACCTGGCACTGTTTGAGGAAGAGCTGGAC288IleAspTyrTyrAspArgA snLeuAlaLeuPheGluGluGluLeuAsp859095ATCCGCCCAAAGGTATCGTCTCTTCTGGGAAAGCTCGTCAGCTACACC336IleArgProLysValS erSerLeuLeuGlyLysLeuValSerTyrThr100105110AACCTCACCCAGGGCGCCAAAGAGCATGAGGAGGCCGAGAGTGGGGAG384AsnLeuThrGlnGlyA laLysGluHisGluGluAlaGluSerGlyGlu115120125GGCACCCGCCGGAGGGCAGCCGAGGCACCCAGCATGGGCACCCTCATG432GlyThrArgArgArgAlaA laGluAlaProSerMetGlyThrLeuMet130135140GGGGTGTACCTGCCCTGCCTGCAGAATATCTTTGGGGTTATCCTCTTC480GlyValTyrLeuProCysLeuGlnA snIlePheGlyValIleLeuPhe145150155160CTGCGGCTGACCTGGATGGTGGGCACAGCAGGTGTGCTACAGGCCCTC528LeuArgLeuThrTrpMetV alGlyThrAlaGlyValLeuGlnAlaLeu165170175CTCATCGTGCTTATCTGCTGCTGTTGTACCCTGCTGACGGCCATCTCC576LeuIleValLeuIleC ysCysCysCysThrLeuLeuThrAlaIleSer180185190ATGAGTGCCATCGCCACCAACGGTGTGGTTCCAGCTGGGGGCTCCTAT624MetSerAlaIleAlaT hrAsnGlyValValProAlaGlyGlySerTyr195200205TTCATGATCTCTCGTTCACTGGGGCCAGAATTTGGAGGTGCTGTGGGC672PheMetIleSerArgSerL euGlyProGluPheGlyGlyAlaValGly210215220CTGTGCTTCTACCTGGGAACAACATTCGCAGCAGCCATGTACATCCTG720LeuCysPheTyrLeuGlyThrThrP heAlaAlaAlaMetTyrIleLeu225230235240GGGGCCATCGAGATCTTGCTGACCTACATTGCCCCACCAGCTGCCATT768GlyAlaIleGluIleLeuL euThrTyrIleAlaProProAlaAlaIle245250255TTTTACCCATCGGGTGCTCATGACACGTCGAATGCCACTTTGAACAAT816PheTyrProSerGlyA laHisAspThrSerAsnAlaThrLeuAsnAsn260265270ATGCGTGTGTATGGGACCATTTTCCTGGCCTTCATGACCCTGGTGGTG864MetArgValTyrGlyT hrIlePheLeuAlaPheMetThrLeuValVal275280285TTTGTGGGGGTCAAGTATGTGAACAAATTTGCCTCGCTCTTCCTGGCC912PheValGlyValLysTyrV alAsnLysPheAlaSerLeuPheLeuAla290295300TGTGTGATCATCTCCATCCTCTCCATCTATGCTGGGGGCATAAAGTCT960CysValIleIleSerIleLeuSerI leTyrAlaGlyGlyIleLysSer305310315320ATATTTGACCCTCCCGTGTTTCCGGTATGCATGCTGGGCAACAGGACC1008IlePheAspProProValP heProValCysMetLeuGlyAsnArgThr325330335CTGTCCCGGGACCAGTTTGACATCTGTGCCAAGACAGCTGTAGTGGAC1056LeuSerArgAspGlnP heAspIleCysAlaLysThrAlaValValAsp340345350AATGAGACAGTGGCCACCCAGCTATGGAGTTTCTTCTGCCACAGCCCC1104AsnGluThrValAlaT hrGlnLeuTrpSerPhePheCysHisSerPro355360365AACCTTACGACCGACTCCTGTGACCCCTACTTCATGCTCAACAATGTG1152AsnLeuThrThrAspSerC ysAspProTyrPheMetLeuAsnAsnVal370375380ACCGAGATCCCTGGCATCCCCGGGGCAGCTGCTGGTGTGCTCCAGGAA1200ThrGluIleProGlyIleProGlyA laAlaAlaGlyValLeuGlnGlu385390395400AACCTGTGGAGCGCCTACCTGGAGAAGGGTGACATCGTGGAGAAGCAT1248AsnLeuTrpSerAlaTyrL euGluLysGlyAspIleValGluLysHis405410415GGGCTGCCCTCCGCAGATGCCCCGAGCCTGAAGGAGAGCCTGCCTCTG1296GlyLeuProSerAlaA spAlaProSerLeuLysGluSerLeuProLeu420425430TACGTGGTCGCTGACATCGCCACATCCTTCACCGTGCTGGTCGGCATC1344TyrValValAlaAspI leAlaThrSerPheThrValLeuValGlyIle435440445TTCTTCCCTTCTGTAACAGGTATGGCGATGGTGTCAGCAGGAACTTGG1392PhePheProSerValThrG lyMetAlaMetValSerAlaGlyThrTrp450455460TGGTGGGCACACTGGCCTGGCCTTCACCCTGGGTCATCGTCATCGGCT1440TrpTrpAlaHisTrpProGlyLeuH isProGlySerSerSerSerAla465470475480CCTTCTTTTCAACGTGTGGCGCTGGCCTCCAGAGCCTCACAGGGGCAC1488ProSerPheGlnArgValA laLeuAlaSerArgAlaSerGlnGlyHis485490495CACGCCTATTGCAGGCCATTGCCAAGGACAACATCATCCCCTTCCTCC1536HisAlaTyrCysArgP roLeuProArgThrThrSerSerProSerSer500505510GGGTGAGCCCCTCTGCACTCCCCCATGGCCTGGCTGCTCCCAGGCCCTCGCCC1589GlyGGCTGGGGAGAGA GATAGGGAACACAGATGCAGCACGTCCTGCCCTTATTGCCCCCGGGC1649CAGGCGGCCATCCATGAGGAGCTACTGAGAAGTGCCCTGGGCCTGGCACTCACCTGGGCC1709TGGAGCTGCCTGGACCCAGAATCTTCATGGCCTGTTTAGGGCTCATCCAAAGGAGAG AGG1769CCTGGTGAGGTGGAATCAGGGAGACTGGTGACACCCATAGGGATAGACACAGGGGCGGCC1829TGAGCCCCCAAGGCGGGCCCTGGGGGTGAGGGAGGCCAGGCTGGGGTCTGGGG1882Gly ArgProGlyTrpGlyLeuGly515520CCCAAGGTGTGGAATGGGGGTGACAGGACCCAGCTTCCTTCCTGGTGC1930ProLysValTrpAsnGlyGlyAsp ArgThrGlnLeuProSerTrpCys525530535ACACAGGTGTTTGGCCACGGGAAGGTGAATGGTGAACCCACATGGGCA1978ThrGlnValPheGlyHisGlyLys ValAsnGlyGluProThrTrpAla540545550CTCCTCCTGACGGCACTCATCGCCGAGCTGGGCATCCTCATCGCCTCC2026LeuLeuLeuThrAlaLeuIleAlaGlu LeuGlyIleLeuIleAlaSer555560565CTCGACATGGTGGCCCCCATCTTATCCATGTTCTTTCTGATGTGCTAC2074LeuAspMetValAlaProIleLeuSerMetPhe PheLeuMetCysTyr570575580585CTGTTCGTGAACCTCGCCTGTGCGGTGCAGACACTCCTGAGGACCCCC2122LeuPheValAsnLeuAlaCysAlaVal GlnThrLeuLeuArgThrPro590595600AACTGGCGGCCCCGGTTCAAGTACTATCACTGGGCGCTGTCCTTCCTG2170AsnTrpArgProArgPheLysTyr TyrHisTrpAlaLeuSerPheLeu605610615GGCATGAGTCTCTGCCTGGCCCTTATGTTTGTCTCCTCCTGGTACTAT2218GlyMetSerLeuCysLeuAlaLeu MetPheValSerSerTrpTyrTyr620625630GCCCTGGTGGCCATGCTCATCGCCGGCATGATCTACAAATACATCGAG2266AlaLeuValAlaMetLeuIleAlaGly MetIleTyrLysTyrIleGlu635640645TACCAAGGGGCTGAGAAGGAGTGGGGTGACGGGATCCGAGGCCTGTCC2314TyrGlnGlyAlaGluLysGluTrpGlyAspGly IleArgGlyLeuSer650655660665CTGAGCGCTGCCCGCTACGCGCTGTTGCGGCTGGAGGAGGGGCCTCCT2362LeuSerAlaAlaArgTyrAlaLeuLeu ArgLeuGluGluGlyProPro670675680CACACCAAGAACTGGCGGCCGC2384HisThrLysAsnTrpArgPro 685(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 688 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:AlaAlaAlaPheGlyAspSerGlyGlyArgLeuGlyArgArgValArg 151015ArgGlyArgAlaArgThrMetProHisPheThrValValProValAsp202530GlyProArg ArgGlyAspTyrAspAsnLeuGluGlyLeuSerTrpVal354045AspTyrGlyGluArgAlaGluLeuAspAspSerAspGlyHisGlyAsn50 -560HisArgGluSerSerProPheLeuSerProLeuGluAlaSerArgGly65707580IleAspTyrTyrAspArgAsnLeuAlaLeuP heGluGluGluLeuAsp859095IleArgProLysValSerSerLeuLeuGlyLysLeuValSerTyrThr100105 110AsnLeuThrGlnGlyAlaLysGluHisGluGluAlaGluSerGlyGlu115120125GlyThrArgArgArgAlaAlaGluAlaProSerMetGlyThrLeuMet 130135140GlyValTyrLeuProCysLeuGlnAsnIlePheGlyValIleLeuPhe145150155160LeuArgLeuThr TrpMetValGlyThrAlaGlyValLeuGlnAlaLeu165170175LeuIleValLeuIleCysCysCysCysThrLeuLeuThrAlaIleSer180 185190MetSerAlaIleAlaThrAsnGlyValValProAlaGlyGlySerTyr195200205PheMetIleSerArgSerLeuGlyProGluP heGlyGlyAlaValGly210215220LeuCysPheTyrLeuGlyThrThrPheAlaAlaAlaMetTyrIleLeu225230235 240GlyAlaIleGluIleLeuLeuThrTyrIleAlaProProAlaAlaIle245250255PheTyrProSerGlyAlaHisAspThrSerAsnAlaThrLeuAsnAs n260265270MetArgValTyrGlyThrIlePheLeuAlaPheMetThrLeuValVal275280285PheValGlyVal LysTyrValAsnLysPheAlaSerLeuPheLeuAla290295300CysValIleIleSerIleLeuSerIleTyrAlaGlyGlyIleLysSer305310 315320IlePheAspProProValPheProValCysMetLeuGlyAsnArgThr325330335LeuSerArgAspGlnPheAspIleCysA laLysThrAlaValValAsp340345350AsnGluThrValAlaThrGlnLeuTrpSerPhePheCysHisSerPro355360 365AsnLeuThrThrAspSerCysAspProTyrPheMetLeuAsnAsnVal370375380ThrGluIleProGlyIleProGlyAlaAlaAlaGlyValLeuGlnGlu385 390395400AsnLeuTrpSerAlaTyrLeuGluLysGlyAspIleValGluLysHis405410415GlyLeuPro SerAlaAspAlaProSerLeuLysGluSerLeuProLeu420425430TyrValValAlaAspIleAlaThrSerPheThrValLeuValGlyIle435 440445PhePheProSerValThrGlyMetAlaMetValSerAlaGlyThrTrp450455460TrpTrpAlaHisTrpProGlyLeuHisProGlySerS erSerSerAla465470475480ProSerPheGlnArgValAlaLeuAlaSerArgAlaSerGlnGlyHis485490 495HisAlaTyrCysArgProLeuProArgThrThrSerSerProSerSer500505510GlyGlyArgProGlyTrpGlyLeuGlyProLysValTrpAsnGlyGl y515520525AspArgThrGlnLeuProSerTrpCysThrGlnValPheGlyHisGly530535540LysValAsnGlyGluPro ThrTrpAlaLeuLeuLeuThrAlaLeuIle545550555560AlaGluLeuGlyIleLeuIleAlaSerLeuAspMetValAlaProIle565 570575LeuSerMetPhePheLeuMetCysTyrLeuPheValAsnLeuAlaCys580585590AlaValGlnThrLeuLeuArgThrProA snTrpArgProArgPheLys595600605TyrTyrHisTrpAlaLeuSerPheLeuGlyMetSerLeuCysLeuAla610615620 LeuMetPheValSerSerTrpTyrTyrAlaLeuValAlaMetLeuIle625630635640AlaGlyMetIleTyrLysTyrIleGluTyrGlnGlyAlaGluLysGlu 645650655TrpGlyAspGlyIleArgGlyLeuSerLeuSerAlaAlaArgTyrAla660665670LeuLeuArg LeuGluGluGlyProProHisThrLysAsnTrpArgPro675680685(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1675 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 492..1330(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:AAGCTTGCGGCCGCATTGCGAGAACGAGAACGGGAGCGAGAGAGAGAGCGAGAGAGGGAA60CGGGAGCGAGAAAGAGAAAAAGACAAAAAA CGGGACCGAGAAGAAGATGAAGAAGATGCA120TACGAACGAAGAAAACTTGAAAGAAAACTCCGAGAGAAAGAAGCTGCTTATCAAGAGCGC180CTTAAGAATTGGGAAATCAGAGAACGAAAGAAAACCCGGGAATATGAGAAAGAAGCTGAA240AGAG AAGAAGAAAGAAGAAGAGAAATGGCCAAAGAAGCTAAACGACTAAAAGAATTCTTA300GAAGACTATGATGATGATAGAGATGACCCCAAATATTACAGAGGAAGTGCTCTTCAGAAA360AGGTTGCGTGATAGAGAAAAGGAAATGGAAGCAGATGAACGAGATAGG AAGAGAGAGAAG420GAGGAGCTTGAGGAAATCAGGCAGCGCTTCTGGCAGAAGGGCATCCAGATCCAGATGCAG480AGCTCCAGAGGATGGAACAAGAGGCTGAGAGGCGCAGGCAGCCACAAATA530MetGluGln GluAlaGluArgArgArgGlnProGlnIle1510AAGCAAGAGCCAGAATCAGAAGAGGAGGAAGAAGAAAAGCAAGAAAAA578LysGlnGluProGluSerGlu GluGluGluGluGluLysGlnGluLys152025GAAGAAAAACGAGAAGAACCCATGGAAGAGGAAGAGGAGCCAGAGCAA626GluGluLysArgGluGluProMetGlu GluGluGluGluProGluGln30354045AAGCCTTGTCTGAAACCTACTCTGAGGCCCATCAGCTCTGCTCCATCT674LysProCysLeuLysProThr LeuArgProIleSerSerAlaProSer505560GTTTCCTCTGCCAGTGGCAATGCAACACCTAACACTCCTGGGGATGAG722ValSerSerAlaSerGly AsnAlaThrProAsnThrProGlyAspGlu657075TCTCCCTGTGGTATTATTATTCCTCATGAAAACTCACCAGATCAACAG770SerProCysGlyIleIle IleProHisGluAsnSerProAspGlnGln808590CAACCTGAGGAGCATAGGCCAAAAATAGGACTAAGTCTTAAACTGGGT818GlnProGluGluHisArgPro LysIleGlyLeuSerLeuLysLeuGly95100105GCTTCCAATAGTCCTGGTCAGCCTAATTCTGTGAAGAGAAAGAAACTA866AlaSerAsnSerProGlyGlnProAsn SerValLysArgLysLysLeu110115120125CCTGTAGATAGTGTCTTTAACAAATTTGAGGATGAAGACAGTGATGAC914ProValAspSerValPheAsn LysPheGluAspGluAspSerAspAsp130135140GTACCCCGAAAAAGGAAACTGGTTCCCTTGGATTATGGTGAAGATGAT962ValProArgLysArgLys LeuValProLeuAspTyrGlyGluAspAsp145150155AAAAATGCAACCAAAGGCACTGTAAACACTGAAGAAAAGCGTAAACAC1010LysAsnAlaThrLysGly ThrValAsnThrGluGluLysArgLysHis160165170ATTAAGAGTCTCATTGAGAAAATCCCTACAGCCAAACCTGAGCTCTTC1058IleLysSerLeuIleGluLys IleProThrAlaLysProGluLeuPhe175180185GCTTATCCCCTGGATTGGTCTATTGTGGATTCTATACTGATGGAACGT1106AlaTyrProLeuAspTrpSerIleVal AspSerIleLeuMetGluArg190195200205CGAATTAGACCATGGATTAATAAGAAAATCATAGAATATATAGGTGAA1154ArgIleArgProTrpIleAsn LysLysIleIleGluTyrIleGlyGlu210215220GAAGAAGCTACATTAGTTGATTTTGTTTGTTCTAAGGTTATGGCTCAT1202GluGluAlaThrLeuVal AspPheValCysSerLysValMetAlaHis225230235AGTTCACCCCAGAGCATTTTAGATGATGTTGCCATGGTACTTGATGAA1250SerSerProGlnSerIle LeuAspAspValAlaMetValLeuAspGlu240245250GAAGCAGAAGTTTTTATAGTCAAAATGTGGAGATTATTGATATATGAA1298GluAlaGluValPheIleVal LysMetTrpArgLeuLeuIleTyrGlu255260265ACAGAAGCCAAGAAAATTGGTCTTGTGAAGTAAAACTTTTTATATTTAGAGT1350ThrGluAlaLysLysIleGlyLeuVal Lys270275TCCATTTCAGATTTCTTCTTTGCCACCCTTTTAAGGACTTTGAATTTTTCTTTGTCTTTG1410AAGACATTGTGAGATCTGTAATTTTTTTTTTTTGTAGAAAATGTGAATTTTTTGGTCCTC1470TAATTTGTTGTTG CCCTGTGTACTCCCTTGGTTGTAAAGTCATCTGAATCCTTGGTTCTC1530TTTATACTCACCAGGTACAAATTACTGGTATGTTTTATAAGCCGCAGCTACTGTACACAG1590CCTATCTGATATAATCTTGTTCTGCTGATTTGTTTCTTGTAAATATTAAAACGACTC CCC1650AATTAAAAAAAAAAAATGCGGCCGC1675(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 279 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:MetGluGlnGluAlaGluArgArgArgGlnProGlnIleLysGlnGlu151015ProGluSerGluGluGluGluGluGluLysGlnGluLysGluGluLys 202530ArgGluGluProMetGluGluGluGluGluProGluGlnLysProCys354045LeuLysProThrLeu ArgProIleSerSerAlaProSerValSerSer505560AlaSerGlyAsnAlaThrProAsnThrProGlyAspGluSerProCys6570 7580GlyIleIleIleProHisGluAsnSerProAspGlnGlnGlnProGlu859095GluHisArgProLysIleGlyLeuSerLeu LysLeuGlyAlaSerAsn100105110SerProGlyGlnProAsnSerValLysArgLysLysLeuProValAsp1151201 25SerValPheAsnLysPheGluAspGluAspSerAspAspValProArg130135140LysArgLysLeuValProLeuAspTyrGlyGluAspAspLysAsnAla145 150155160ThrLysGlyThrValAsnThrGluGluLysArgLysHisIleLysSer165170175LeuIleGluLys IleProThrAlaLysProGluLeuPheAlaTyrPro180185190LeuAspTrpSerIleValAspSerIleLeuMetGluArgArgIleArg195 200205ProTrpIleAsnLysLysIleIleGluTyrIleGlyGluGluGluAla210215220ThrLeuValAspPheValCysSerLysValMetAlaHisS erSerPro225230235240GlnSerIleLeuAspAspValAlaMetValLeuAspGluGluAlaGlu245250 255ValPheIleValLysMetTrpArgLeuLeuIleTyrGluThrGluAla260265270LysLysIleGlyLeuValLys275(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3073 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 3..1111(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:GCGGCCGCGCGCCGCATTCGGAGAGCGGA CCCCAGAGAGCCCTGAGC47GlyArgAlaProHisSerGluSerGlyProGlnArgAlaLeuSer151015AGCCCCACCGCCGCCGCCGGCCTA GTTACCATCACACCCCGGGAGGAG95SerProThrAlaAlaAlaGlyLeuValThrIleThrProArgGluGlu202530CCGCAGCTGCCGCAGCCGGCC CCAGTCACCATCACCGCAACCATGAGC143ProGlnLeuProGlnProAlaProValThrIleThrAlaThrMetSer354045AGCGAGGCCGAGACCCAGCAG CCGCCCGCCGCCCCCCCCGCCGCCCCC191SerGluAlaGluThrGlnGlnProProAlaAlaProProAlaAlaPro505560GCCCTCAGCGCCGCCGACACCAAG CCCGGCACTACGGGCAGCGGCGCA239AlaLeuSerAlaAlaAspThrLysProGlyThrThrGlySerGlyAla657075GGGAGCGGTGGCCCGGGCGGCCTCACATCG GCGGCGCCTGCCGGCGGG287GlySerGlyGlyProGlyGlyLeuThrSerAlaAlaProAlaGlyGly80859095GACAAGAAGGTCATCGCAACGAAG GTTTTGGGAACAGTAAAATGGTTC335AspLysLysValIleAlaThrLysValLeuGlyThrValLysTrpPhe100105110AATGTAAGGAACGGATATGGT TTCATCAACAGGAATGACACCAAGGAA383AsnValArgAsnGlyTyrGlyPheIleAsnArgAsnAspThrLysGlu115120125GATGTATTTGTACACCAGACT GCCATAAAGAAGAATAACCCCAGGAAG431AspValPheValHisGlnThrAlaIleLysLysAsnAsnProArgLys130135140TACCTTCGCAGTGTAGGAGATGGA GAGACTGTGGAGTTTGATGTTGTT479TyrLeuArgSerValGlyAspGlyGluThrValGluPheAspValVal145150155GAAGGAGAAAAGGGTGCGGAGGCAGCAAAT GTTACAGGTCCTGGTGGT527GluGlyGluLysGlyAlaGluAlaAlaAsnValThrGlyProGlyGly160165170175GTTCCAGTTCAAGGCAGTAAATAT GCAGCAGACCGTAACCATTATAGA575ValProValGlnGlySerLysTyrAlaAlaAspArgAsnHisTyrArg180185190CGCTATCCACGTCGTAGGGGT CCTCCACGCAATTACCAGCAAAATTAC623ArgTyrProArgArgArgGlyProProArgAsnTyrGlnGlnAsnTyr195200205CAGAATAGTGAGAGTGGGGAA AAGAACGAGGGATCGGAGAGTGCTCCC671GlnAsnSerGluSerGlyGluLysAsnGluGlySerGluSerAlaPro210215220GAAGGCCAGGCCCAACAACGCCGG CCCTACCGCAGGCGAAGGTTCCCA719GluGlyGlnAlaGlnGlnArgArgProTyrArgArgArgArgPhePro225230235CCTTACTACATGCGGAGACCCTATGGGCGT CGACCACAGTATTCCAAC767ProTyrTyrMetArgArgProTyrGlyArgArgProGlnTyrSerAsn240245250255CCTCCTGTGCAGGGAGAAGTGATG GAGGGTGCTGACAACCAGGGTGCA815ProProValGlnGlyGluValMetGluGlyAlaAspAsnGlnGlyAla260265270GGAGAACAAGGTAGACCAGTG AGGCAGAATATGTATCGGGGATATAGA863GlyGluGlnGlyArgProValArgGlnAsnMetTyrArgGlyTyrArg275280285CCACGATTCCGCAGGGGCCCT CCTCGCCAAAGACAGCCTAGAGAGGAC911ProArgPheArgArgGlyProProArgGlnArgGlnProArgGluAsp290295300GGCAATGAAGAAGATAAAGAAAAT CAAGGAGATGAGACCCAAGGTCAG959GlyAsnGluGluAspLysGluAsnGlnGlyAspGluThrGlnGlyGln305310315CAGCCACCTCAACGTCGGTACCGCCGCAAC TTCAATTACCGACGCAGA1007GlnProProGlnArgArgTyrArgArgAsnPheAsnTyrArgArgArg320325330335CGCCCAGAAAACCCTAAACCACAA GATGGCAAAGAGACAAAAGCAGCC1055ArgProGluAsnProLysProGlnAspGlyLysGluThrLysAlaAla340345350GATCCACCAGCTGAGAATTCG TCCGCTCCCGAGGCTGAGCAGGGCGGG1103AspProProAlaGluAsnSerSerAlaProGluAlaGluGlnGlyGly355360365GCTGAGTAAATGCCGGCTTACCAT CTCTACCATCATCCGGTTTAGTCATCCAACAAGA1161AlaGluAGAAATATGAAATTCCAGCAATAAGAAATGAACAAAAGATTGGAGCTGAAGACCTAAAGT1221GCTTGCTTTTTGCCCGTTGACCAGATAAATAGAACTATCTGCATTATCTATGCAGCATGG 1281GGTTTTTATTATTTTTACCTAAAGACGTCTCTTTTTGGTAATAACAAACGTGTTTTTTAA1341AAAAGCCTGGTTTTTCTCAATACGCCTTTAAAGGTTTTTAAATTGTTTCATATCTGGTCA1401AGTTGAGATTTTTAAGAACTTCATTTTTAATTTGT AATAAAAGTTTACAACTTGATTTTT1461TCAAAAAAGTCAACAAACTGCAAGCACCTGTTAATAAAGGTCTTAAATAATTGTCTTTGT1521GTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCTTGGTATTCATTACTTCAT1581GTATATCAAG CACAGCAGTAAAACAAAAACCCATGTATTTAACTTTTTTTTAGGATTTTT1641GCTTTTGTGATTTTTTTTTTTTTTTTTTTGATACTTGCCTAACATGCATGTGCTGTAAAA1701ATAGTTAACAGGGAAATAACTTGAGATGATGGCTAGCTTTGTTTAATGTCTTA TGAAATT1761TTCATGAACAATCCAAGCATAATTGTTAAGAACACGTGTATTAAATTCATGTAAGTGGAA1821TAAAAGTTTTATGAATGGACTTTTCAACTACTTTCTCTACAGCTTTTCATGTAAATTAGT1881CTTGGTTCTGAAACTTCTCTAAAGGAAA TTGTACATTTTTTGAAATTTATTCCTTATTCC1941CTCTTGGCAGCTAATGGGCTCTTACCAAGTTTAAACACAAAATTTATCATAACAAAAATA2001CTACTAATATAACTACTGTTTCCATGTCCCATGATCCCCTCTCTTCCTCCCCACCCTGAA2061AA AAATGAGTTCCTATTTTTTCTGGGAGAGGGGGGGATTGATTAGAAAAAAATGTAGTGT2121GTTCCATTTAAAATTTTGGCATATGGCATTTTCTAACTTAGGAAGCCACAATGTTCTTGG2181CCCATCATGACATTGGGTAGCATTAACTGTAAGTTTTGTGCTTCCA AATCACTTTTTGGT2241TTTTAAGAATTTCTTGATACTCTTATAGCCTGCCTTCAATTTTGATCCTTTATTCTTTCT2301ATTTGTCAGGTGCACAAGATTACCTTCCTGTTTTAGCCTTCTGTCTTGTCACCAACCATT2361CTTACTTGGTGGCCATGTAC TTGGAAAAAGGCCGCATGATCTTTCTGGCTCCACTCAGTG2421TCTAAGGCACCCTGCTTCCTTTGCTTGCATCCCACAGACTATTTCCCTCATCCTATTTAC2481TGCAGCAAATCTCTCCTTAGTTGATGAGACTGTGTTTATCTCCCTTTAAAACCCTACCTA25 41TCCTGAATGGTCTGTCATTGTCTGCCTTTAAAATCCTTCCTCTTTCTTCCTCCTCTATTC2601TCTAAATAATGATGGGGCTAAGTTATACCCAAAGCTCACTTTACAAAATATTTCCTCAGT2661ACTTTGCAGAAAACACCAAACAAAAATGCCATTTTAAAA AAGGTGTATTTTTTCTTTTAG2721AATGTAAGCTCCTCAAGAGCAGGGACAATGTTTTCTGTATGTTCTATTGTGCCTAGTACA2781CTGTAAATGCTCAATGAATATTATCCCTAATACCTGCCACCCCACTCTTAATCAGTGGTG2841GAAGAACGGTCTC AGAACTGTTTGTTTCAATTGGCCATTTAAGTTTAGTAGTAAAAGACT2901GGTTAATGATAACAATGCATCGTAAAACCTTCAGAAGGAAAGGAGAATGTTTTGTGGACC2961ACTTTGGTTTTCTTTTTTGCGTGTGGCAGTTTTAAGTTATTAGTTTTTAAAATCAGT ACT3021TTTTAATGGAAACAACTTGACCAAAAATTTGTCACAGAATTTTGGCGGCCGC3073(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 369 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:GlyArgAlaProHisSerGluSerGlyProGlnArgAlaLeuSerSer151015ProThrAlaAlaAlaGlyLeuValThrIleThrProArgGluGluPro 202530GlnLeuProGlnProAlaProValThrIleThrAlaThrMetSerSer354045GluAlaGluThrGln GlnProProAlaAlaProProAlaAlaProAla505560LeuSerAlaAlaAspThrLysProGlyThrThrGlySerGlyAlaGly6570 7580SerGlyGlyProGlyGlyLeuThrSerAlaAlaProAlaGlyGlyAsp859095LysLysValIleAlaThrLysValLeuGly ThrValLysTrpPheAsn100105110ValArgAsnGlyTyrGlyPheIleAsnArgAsnAspThrLysGluAsp1151201 25ValPheValHisGlnThrAlaIleLysLysAsnAsnProArgLysTyr130135140LeuArgSerValGlyAspGlyGluThrValGluPheAspValValGlu145 150155160GlyGluLysGlyAlaGluAlaAlaAsnValThrGlyProGlyGlyVal165170175ProValGlnGly SerLysTyrAlaAlaAspArgAsnHisTyrArgArg180185190TyrProArgArgArgGlyProProArgAsnTyrGlnGlnAsnTyrGln195 200205AsnSerGluSerGlyGluLysAsnGluGlySerGluSerAlaProGlu210215220GlyGlnAlaGlnGlnArgArgProTyrArgArgArgArg PheProPro225230235240TyrTyrMetArgArgProTyrGlyArgArgProGlnTyrSerAsnPro245250 255ProValGlnGlyGluValMetGluGlyAlaAspAsnGlnGlyAlaGly260265270GluGlnGlyArgProValArgGlnAsnMetTyrArgGlyTyrArgPro 275280285ArgPheArgArgGlyProProArgGlnArgGlnProArgGluAspGly290295300AsnGluGluAspLysGluAsn GlnGlyAspGluThrGlnGlyGlnGln305310315320ProProGlnArgArgTyrArgArgAsnPheAsnTyrArgArgArgArg325 330335ProGluAsnProLysProGlnAspGlyLysGluThrLysAlaAlaAsp340345350ProProAlaGluAsnSerSerAlaProGlu AlaGluGlnGlyGlyAla355360365Glu(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1811 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:GAATTCCTGGTAGGGCCAGCCCACCATGGGGGCCAAGACCTGCACAGGACAAGGGCCACC60TGGCCTTTCAGTTACTTGAGTTTGGAGTCAGAAAGCAAGACCAGGAAGCAAATAGCAGCT120CAGGAAATCCCACGGTTGACTTGC CTTGATGGCAAGCTTGGTGGAGAGGGCTGAAGCTGT180TGCTGGGGGCCGATTCTGATCAAGACACATGGCTTGAAAATGGAAGACACAAAACTGAGA240GATCATTCTGCACTAAGTTTCGGGAACTTATCCCCGACAGTGACTGAACTCACTGACTAA300TAACTTCATTTATGAATCTTCTCCCTTGTCCCTTTGTCTGCCAACCTGTGTGCCTTTTTT360GTAAAACATTTTCATGTCTTTAAAATGCCTGTTGAATACCTGGAGTTTAGTATCAACTTC420TACACAGATAAGCTTTCAAAGTTGACAAACTTTTTTGACTCT TTCTGGAAAAGGGAAAGA480AAATAGTCTTCCTTCTTTCTTGGGCAATATCCTTCACTTTACTACAGTTACTTTTGCAAA540CAGACAGAAAGGATACACTTCTAACCACATTTTACTTCCTTCCCCTGTTGTCCAGTCCAA600CTCCACAGTCACTCTTA AAACTTCTCTCTGTTTGCCTGCCTCCAACAGTACTTTTAACTT660TTTGCTGTAAACAGAATAAAATTGAACAAATTAGGGGGTAGAAAGGAGCAGTGGTGTCGT720TCACCGTGAGAGTCTGCATAGAACTCAGCAGTGTGCCCTGCTGTGTCTTGGACCCTGCCC 780CCCACAGGAGTTGTACAGTCCCTGGCCCTGTTCCCTACCTCCTCTCTTCACCCCGTTAGG840CTGTTTTCAATGTAATGCTGCCGTCCTTCTCTTGCACTGCCTTCTGCGCTAACACCTCCA900TTCCTGTTTATAACCGTGTATTTATTACTTAATGT ATATAATGTAATGTTTTGTAAGTTA960TTAATTTATATATCTAACATTGCCTGCCAATGGTGGTGTTAAATTTGTGTAGAAAACTCT1020GCCTAAGAGTTACGACTTTTTCTTGTAATGTTTTGTATTGTGTATTATATAACCCAAACG1080TCACTTAGTA GAGACATATGGCCCCCTTGGCAGAGAGGACAGGGGTGGGCTTTTGTTCAA1140AGGGTCTGCCCTTTCCCTGCCTGAGTTGCTACTTCTGCACAACCCCTTTATGAACCAGTT1200TTGGAAACAATATTCTCACATTAGATACTAAATGGTTTATACTGAGCTTTTAC TTTTGTA1260TAGCTTGATAGGGGCAGGGGGCAATGGGATGTAGTTTTTACCCAGGTTCTATCCAAATCT1320ATGTGGGCATGAGTTGGGTTATAACTGGATCCTACTATCATTGTGGCTTTGGTTCAAAAG1380GAAACACTACATTTGCTCACAGATGATT CTTCTGAATGCTCCCGAACTACTGACTTTGAA1440GAGGTAGCCTCCTGCCTGCCATTAAGCAGGAATGTCATGTTCCAGTTCATTACAAAAGAA1500AACAATAAAACAATGTGAATTTTTATAATAAAATGTGAACTGATGTAGCAAATTACGCAA1560AT GTGAAGCCTCTTCTGATAACACTTGTTAGGCCTCTTACTGATGTCAGTTTCAGTTTGT1620AAAATATGTTTCATGCTTTCAGTTCAGCATTGTGACTCAGTAATTACAGAAAATGGCACA1680AATGTGCATGACCAATGGGTTTGTATGTCTATGAACACTGCATTGT TTCAGGTGGACATT1740TTATCATTTTCAAATGTTTCTCACAATGTATGTTATAGTATTATTATTATATATTGTGTT1800CAAATGCATTC1811(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1672 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:GAATTCCCCACCATGGGGGCCAAGACCTGCACAGGACAAGGCCACCTGGCCTTTCAGTTA60CTTGAGTTTGG AGTCAGAAAGCAAGACCAGGAAGCAAATAGCAGCTCAGGAAATCCCACG120GTTGACTTGCCTTGATGGCAAGCTTGGTGGAGAGGGCTGAAGCTGTTGCTGGGGGCCGTT180CTGATCAAGACACATGGCTTGAAAATGGAAGACACAAAACTGAGAGATCATTCTG CACTA240AGTTTCGGGAACTTATCCCCGACAGTGACTGAACTCACTGACTAATAACTTCATTTATGA300ATCTTCTCCCTTGTCCCTTTGTCTGCCAACCTGTGTGCCTTTTTTGTAAAACATTTCAGT360CTTTAAAATGCCTGTTGAATACCTGGAGTT AGATCAACTTCTACACAGATAAGCTTTCAA420AGTTGACAAACTTTTTTGACTCTTCTGGAAAAGGGAAAGAAAATAGTCTTCCTTCTTTCT480TGGGCAATATCCTTCACTTTACTACAGTTACTTTTGCAAACAGACAGAAAGGATACACTT540CTAA CCACATTTTACTTCCTTCCCCTGTTGTCCAGTCCAACTCCACAGTCACTCTTAAAA600CTTCTCTCTGTTTGCCTGCCTCCAACAGTACTTTTAACTTTTAACTTTTTGCTGTAAACA660GAATAAAATTGAACAAATTAGGGGGTAGAAAGGAGCAGTGGTGTCGTT CACCGTGAGAGT720CTGCATAGAACTCAGCAGTGTGCCCTGCTGTGTCTTGGACCCTGCCCCCCACAGGAGTTG780TACAGTCCCTGGCCCTGTTCCCTACCTCCTCTCTTCACCCCGTTAGGCTGTTTTCAATGT840AATGCTGCCGTCCTTCTCTTGC ACTGCCTTCTGCGCTAACACCTCCATTCCTGTTTATAA900CCGTGTATTTATTACTTAATGTATATAATGTAATGTTTTGTAAGTTATTAATTTATATAT960CTAACATTGCCTGCCAATGGTGGTGTTAAATTTGTGTAGAAAACTCTGCCTAAGAGTTAC1020GACTTTTTCTTGTAATGTTTTGTATTGTGTATTATATAACCCAAACGTCACTTAGTAGAG1080ACATATGGCCCCCTTGGCAGAGAGGACAGGGGTGGGCTTTTGTTCAAAGGGTCTGCCCTT1140TCCCTGCCTGAGTTGCTACTTCTGCACAACCCCTTTATGA ACCAGTTTTGGAAACAATAT1200TCTCACATTAGATACTAAATGGTTTATACTGAGCTTTTACTTTTGTATAGCTTGATAGGG1260GCAGGGGGCAATGGGATGTAGTTTTTACCCAGGTTCTATCCAAATCTATGTGGGCATGAG1320TTGGGTTATAACTGG ATCCTACTATCATTGTGGCTTTGGTTCAAAAGGAAACACTACATT1380TGCTCACAGATGATTCTTCTGAATGCTCCCGAACTACTGACTTTGAAGAGGTAGCCTCCT1440GCCTGCCATTAAGCAGGAATGTCATGTTCCAGTTCATTACAAAAGAAAACAATAAAACA A1500TGTGAATTTTTATAATAAAATGTGAACTGATGTAGCAAATTACGCAAATGTGAAGCCTCT1560TCTGATAACACTTGTTAGGCCTCTTACTGATGTCAGTTTCAGTTTGTAAAATATGTTTCA1620TGCTTTCAGTTCAGCATTGTGACTCAGTAATTA CAGAAAAAAAAAAGAATTC1672(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1649 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 210..1018(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:GAATTCCTTCTGACGTGGCATATCACAACAGCCTGCACGCTGCTGATGTAGCCCAGTCGA60CCCATGTTCTCCTTTCTACACCAGCATTAGACGCTGTCTTCACAGATTTGGAAATCCTGG120CTGCCATTTTTGCAGCTGCCATC CATGACGTTGATCATCCTGGAGTCTCCAATCAGTTTC180TCATCAACACAAATTCAGAACTTGCTTTGATGTATAATGATGAATCTGTGTTG233MetTyrAsnAspGluSerValLeu 15GAAAATCATCACCTTGCTGTGGGTTTCAAACTGCTGCAAGAAGAACAC281GluAsnHisHisLeuAlaValGlyPheLysLeuLeuGlnGluGluHis10 1520TGTGACATCTTCATGAATCTCACCAAGAAGCAGCGTCAGACACTCAGG329CysAspIlePheMetAsnLeuThrLysLysGlnArgGlnThrLeuArg25 303540AAGATGGTTATTGACATGGTGTTAGCAACTGATATGTCTAAACATATG377LysMetValIleAspMetValLeuAlaThrAspMetSerLysHisMet 455055AGCCTGCTGGCAGACCTGAAGACAATGGTAGAAACGAAGAAAGTTACA425SerLeuLeuAlaAspLeuLysThrMetValGluThrLysLysValThr 606570AGTTCAGGCGTTCTTCTCCTAGACAACTATACCGATCGCATTCAGGTC473SerSerGlyValLeuLeuLeuAspAsnTyrThrAspArgIleGlnVal 758085CTTCGCAACATGGTACACTGTGCAGACCTGAGCAACCCCACCAAGTCC521LeuArgAsnMetValHisCysAlaAspLeuSerAsnProThrLysSer90 95100TTGGAATTGTATCGGCAATGGACAGACCGCATCATGGAGGAATTTTTC569LeuGluLeuTyrArgGlnTrpThrAspArgIleMetGluGluPhePhe105 110115120CAGCAGGGAGACAAAGAGCGGGAGAGGGGAATGGAAATTAGCCCAATG617GlnGlnGlyAspLysGluArgGluArgGlyMetGluIleSerProMet 125130135TGTGATAAACACACAGCTTCTGTGGAAAAATCCCAGGTTGGTTTCATC665CysAspLysHisThrAlaSerValGluLysSerGlnValGlyPheIle 140145150GACTACATTGTCCATCCATTGTGGGAGACATGGGCAGATTTGGTACAG713AspTyrIleValHisProLeuTrpGluThrTrpAlaAspLeuValGln 155160165CCTGATGCTCAGGACATTCTCGATACCTTAGAAGATAACAGGAACTGG761ProAspAlaGlnAspIleLeuAspThrLeuGluAspAsnArgAsnTrp170 175180TATCAGAGCATGATACCTCAAAGTCCCTCACCACCACTGGACGAGCAG809TyrGlnSerMetIleProGlnSerProSerProProLeuAspGluGln185 190195200AACAGGGACTGCCAGGGTCTGATGGAGAAGTTTCAGTTTGAACTGACT857AsnArgAspCysGlnGlyLeuMetGluLysPheGlnPheGluLeuThr 205210215CTCGATGAGGAAGATTCTGAAGGACCTGAGAAGGAGGGAGAGGGACAC905LeuAspGluGluAspSerGluGlyProGluLysGluGlyGluGlyHis 220225230AGCTATTTCAGCAGCACAAAGACGCTTTGTGTGATTGATCCAGAAAAC953SerTyrPheSerSerThrLysThrLeuCysValIleAspProGluAsn 235240245AGAGATTCCCTGGGAGAGACTGACATAGACATTGCAACAGAAGACAAG1001ArgAspSerLeuGlyGluThrAspIleAspIleAlaThrGluAspLys250 255260TCCCCCGTGGATACATAATCCCCCTCTCCCTGTGGAGATGAACATTC1048SerProValAspThr265TATCCTTGATGAGCATGCCAGCTATGTGGTAGGGCCAGCCCACCATGGGGGC CAAGACCT1108GCACAGGACAAGGGCCACCTGGCCTTTCAGTTACTTGAGTTTGGAGTCAGAAAGCAAGAC1168CAGGAAGCAAATAGCAGCTCAGGAAATCCCACGGTTGACTTGCCTTGATGGCAAGCTTGG1228TGGAGAGGGCTGAAGCTGTTGCTGGGG GCCGATTCTGATCAAGACACATGGCTTGAAAAT1288GGAAGACACAAAACCGAGAGATCATTCTGCACTAAGTTTCGGGAACTTATCCCCGACAGT1348GACTGAACTCACTGACTAATAACTTCATTTATGAATCTTCTCCCTTGTCCCTTTGTCTGC1408C AACCTGTGTGCCTTTTTTGTAAAACATTTTCATGTCTTTAAAATGCCTGTTGAATACCT1468GGAGTTTAGTATCAACTTCTACACAGATAAGCTTTCAAAGTTGACAAACTTTTTTGACTC1528TTTCTGGAAAAGGGAAAGAAAATAGTCTTCCTTCTTTCTTGGGCA ATATCCTTCACTTTA1588CTACAGTTACTTTTGCAAACAGACAGAAAGGATACACTTCTAACCACATTTTACGGAATT1648C1649(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 269 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:MetTyrAsnAspGluSerValLeuGluAsnHisHisLeuAlaValGly1510 15PheLysLeuLeuGlnGluGluHisCysAspIlePheMetAsnLeuThr202530LysLysGlnArgGlnThrLeuArgLysMetValIleAsp MetValLeu354045AlaThrAspMetSerLysHisMetSerLeuLeuAlaAspLeuLysThr505560MetValGluT hrLysLysValThrSerSerGlyValLeuLeuLeuAsp65707580AsnTyrThrAspArgIleGlnValLeuArgAsnMetValHisCysAla 859095AspLeuSerAsnProThrLysSerLeuGluLeuTyrArgGlnTrpThr100105110AspArgIleMetGluGluPh ePheGlnGlnGlyAspLysGluArgGlu115120125ArgGlyMetGluIleSerProMetCysAspLysHisThrAlaSerVal130135 140GluLysSerGlnValGlyPheIleAspTyrIleValHisProLeuTrp145150155160GluThrTrpAlaAspLeuValGlnProAspAlaGlnAspIle LeuAsp165170175ThrLeuGluAspAsnArgAsnTrpTyrGlnSerMetIleProGlnSer180185190P roSerProProLeuAspGluGlnAsnArgAspCysGlnGlyLeuMet195200205GluLysPheGlnPheGluLeuThrLeuAspGluGluAspSerGluGly210 215220ProGluLysGluGlyGluGlyHisSerTyrPheSerSerThrLysThr225230235240LeuCysValIleAspProGluAs nArgAspSerLeuGlyGluThrAsp245250255IleAspIleAlaThrGluAspLysSerProValAspThr260265(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 609 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 2..606(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:GAATTCCAACATTCCCCGA TTTGGGGTGAAGACCGATCAAGAAGAG46AsnSerAsnIleProArgPheGlyValLysThrAspGlnGluGlu151015CTCCTGGCCCAAGAAC TGGAGAACCTGAACAAGTGGGGCCTGAACATC94LeuLeuAlaGlnGluLeuGluAsnLeuAsnLysTrpGlyLeuAsnIle202530TTTTGCGTGTCGG ATTACGCTGGAGGCCGCTCACTCACCTGCATCATG142PheCysValSerAspTyrAlaGlyGlyArgSerLeuThrCysIleMet354045TACATGATATTCC AGGAGCGGGACCTGCTGAAGAAATTCCGCATCCCT190TyrMetIlePheGlnGluArgAspLeuLeuLysLysPheArgIlePro505560GTGGACACGATGGTGAC ATACATGCTGACGCTGGAGGATCACTACCAC238ValAspThrMetValThrTyrMetLeuThrLeuGluAspHisTyrHis657075GCTGACGTGGCCTACCATAACAG CCTGCACGCAGCTGACGTGCTGCAG286AlaAspValAlaTyrHisAsnSerLeuHisAlaAlaAspValLeuGln80859095TCCACCCACGTACTGCT GGCCACGCCTGCACTAGATGCAGTGTTCACG334SerThrHisValLeuLeuAlaThrProAlaLeuAspAlaValPheThr100105110GACCTGGAGATTCT CGCCGCCCTCTTCGCGGCTGCCATCCACGATGTG382AspLeuGluIleLeuAlaAlaLeuPheAlaAlaAlaIleHisAspVal115120125GATCACCCTGGGGT CTCCAACCAGTTCCTCATCAACACCAATTCGGAG430AspHisProGlyValSerAsnGlnPheLeuIleAsnThrAsnSerGlu130135140CTGGCGCTCATGTACAA CGATGAGTCGGTGCTCGAGAATCACCACCTG478LeuAlaLeuMetTyrAsnAspGluSerValLeuGluAsnHisHisLeu145150155GCCGTGGGCTTCAAGCTGCTGCA GGAGGACAACTGCGACATCTTCCAG526AlaValGlyPheLysLeuLeuGlnGluAspAsnCysAspIlePheGln160165170175AACCTCAGCAAGCGCCA GCGGCAGAGCTACGCAAGATGGTCATCGACA574AsnLeuSerLysArgGlnArgGlnSerTyrAlaArgTrpSerSerThr180185190TGGTGCTGGCCACG GACATGTCCAAGCACATGACC609TrpCysTrpProArgThrCysProSerThr195200(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 201 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:AsnSerAsnIleProArgPheGlyValLysThrAspGlnGluGluLeu151015LeuAlaGlnGluL euGluAsnLeuAsnLysTrpGlyLeuAsnIlePhe202530CysValSerAspTyrAlaGlyGlyArgSerLeuThrCysIleMetTyr35 4045MetIlePheGlnGluArgAspLeuLeuLysLysPheArgIleProVal505560AspThrMetValThrTyrMetLeuThrLeuGluAspHisTyr HisAla65707580AspValAlaTyrHisAsnSerLeuHisAlaAlaAspValLeuGlnSer8590 95ThrHisValLeuLeuAlaThrProAlaLeuAspAlaValPheThrAsp100105110LeuGluIleLeuAlaAlaLeuPheAlaAlaAlaIleHisAspValAsp 115120125HisProGlyValSerAsnGlnPheLeuIleAsnThrAsnSerGluLeu130135140AlaLeuMetTyrAsnAspGluSe rValLeuGluAsnHisHisLeuAla145150155160ValGlyPheLysLeuLeuGlnGluAspAsnCysAspIlePheGlnAsn165 170175LeuSerLysArgGlnArgGlnSerTyrAlaArgTrpSerSerThrTrp180185190CysTrpProArgThrCysProSerThr 195200(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1230 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 3..1156 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:ACATGGTGCACTGTGCCGACCTCAGCAACCCCACCAAGCCGCTGGAG47MetValHisCysAlaAspLeuSerAsnProThrLysProLeuGlu1510 15CTGTACCGCCAGTGGACAGACCGCATCATGGCCGAGTTCTTCCAGCAG95LeuTyrArgGlnTrpThrAspArgIleMetAlaGluPhePheGlnGln2025 30GGTGACCGAGAGCGCGAGCGTGGCATGGAAATCAGCCCCATGTGTGAC143GlyAspArgGluArgGluArgGlyMetGluIleSerProMetCysAsp3540 45AAGCACACTGCCTCCGTGGAGAAGTCTCAGGTGGGTTTTATTGACTAC191LysHisThrAlaSerValGluLysSerGlnValGlyPheIleAspTyr5055 60ATTGTGCACCCATTGTGGGAGACCTGGGCGGACCTTGTCCACCCAGAT239IleValHisProLeuTrpGluThrTrpAlaAspLeuValHisProAsp6570 75GCCCAGGAGATCTTGGACACTTTGGAGGACAACCGGGACTGGTACTAC287AlaGlnGluIleLeuAspThrLeuGluAspAsnArgAspTrpTyrTyr808590 95AGCGCCATCCGGCAGAGCCCATCTCCGCCACCCGAGGAGGAGTCAAGG335SerAlaIleArgGlnSerProSerProProProGluGluGluSerArg100105 110GGGCCAGGCCACCCACCCCTGCCTGACAAGTTCCAGTTTGACGTGACG383GlyProGlyHisProProLeuProAspLysPheGlnPheAspValThr115120 125CTGGAGGAGGAAGAGGAGGAAGAAATATCAATGGCCCAGATACCGTGC431LeuGluGluGluGluGluGluGluIleSerMetAlaGlnIleProCys130135 140ACAGCCCAAGAGGCATTGACTGCGCAGGGATTGTCAGGAGTCGAGGAA479ThrAlaGlnGluAlaLeuThrAlaGlnGlyLeuSerGlyValGluGlu145150 155GCTCTGGATGCAACCATAGCCTGGGAGGCATCCCCGGCCCAGGAGTCG527AlaLeuAspAlaThrIleAlaTrpGluAlaSerProAlaGlnGluSer160165170 175TTGGAAGTTATGGCACAGGAAGCATCCCTGGAGGCCGAGCTGGAGGCA575LeuGluValMetAlaGlnGluAlaSerLeuGluAlaGluLeuGluAla180185 190GNGTATTTGACACAGCAGGCACAGTCCACAGGCAGTGCACCTGTGGCT623ValTyrLeuThrGlnGlnAlaGlnSerThrGlySerAlaProValAla195200 205CCGGATGAGTTCTCGTCCCGGGAGGAATTCGTGGTTGCTGTAAGCCAC671ProAspGluPheSerSerArgGluGluPheValValAlaValSerHis210215 220AGCAGCCCCTCTGCCCTGGCTCTTCAAAGCCCCCTTCTCCCTGCTTGG719SerSerProSerAlaLeuAlaLeuGlnSerProLeuLeuProAlaTrp225230 235AGGACCCTGTCTGTTTCAGAGCATGCCCCGGGCCTCCCGGCCTCCCCT767ArgThrLeuSerValSerGluHisAlaProGlyLeuProAlaSerPro240245250 255CCACGGCGGCCTAGGTGGAACGAGAGCACCAGGCTGCCAAGAGGGCTT815ProArgArgProArgTrpAsnGluSerThrArgLeuProArgGlyLeu260265 270GCAGTGCCTGCGCAGGGACATTTGGGGAGGACACATCCGCACTCCCAG863AlaValProAlaGlnGlyHisLeuGlyArgThrHisProHisSerGln275280 285CTCCTGGTGGCGGGGGGTCAGGTGGAGACCCTACCTGATCCCCAGACC911LeuLeuValAlaGlyGlyGlnValGluThrLeuProAspProGlnThr290295 300TCTGTCCCTGTTCCCCTCCACTCCTCCCCTCACTCCCCTGCTCCCCCG959SerValProValProLeuHisSerSerProHisSerProAlaProPro305310 315ACCACCTCCTCCTCTGCCTCAAAGACTCTTGTCCTCTTGTCCCTCCTG1007ThrThrSerSerSerAlaSerLysThrLeuValLeuLeuSerLeuLeu320325330 335AGAAAAAAGAAAACGAAAAGTGGGGTTTTTTTCTGTTTTCTTTTTTTC1055ArgLysLysLysThrLysSerGlyValPhePheCysPheLeuPhePhe340345 350CCCTTTCCCCCTGCCCCCACCCACGGGGCCTTTTTTTGGAGGTGGGGG1103ProPheProProAlaProThrHisGlyAlaPhePheTrpArgTrpGly355360 365CTGGGGAATGAGGGGCTGAGGTCCCGGAAGGGATTTTATTTTTTTGAA1151LeuGlyAsnGluGlyLeuArgSerArgLysGlyPheTyrPhePheGlu370375 380TTTTAATTGTAACATTTTTAGAAAAAGAACAAAAAAAGAAAAAAAAAAGAAAGAA1206PheACACAAAAAAAAAAAAAGGAATTC1230(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 384 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:MetValHisCysAlaAspLeuSerAsnProThrLysProLeuGluLeu1510 15TyrArgGlnTrpThrAspArgIleMetAlaGluPhePheGlnGlnGly202530AspArgGluArgGluArgGlyMetGluIleSerProMetC ysAspLys354045HisThrAlaSerValGluLysSerGlnValGlyPheIleAspTyrIle505560ValHisProLeu TrpGluThrTrpAlaAspLeuValHisProAspAla65707580GlnGluIleLeuAspThrLeuGluAspAsnArgAspTrpTyrTyrSer 859095AlaIleArgGlnSerProSerProProProGluGluGluSerArgGly100105110ProGlyHisProProLeuPro AspLysPheGlnPheAspValThrLeu115120125GluGluGluGluGluGluGluIleSerMetAlaGlnIleProCysThr130135 140AlaGlnGluAlaLeuThrAlaGlnGlyLeuSerGlyValGluGluAla145150155160LeuAspAlaThrIleAlaTrpGluAlaSerProAlaGlnGluS erLeu165170175GluValMetAlaGlnGluAlaSerLeuGluAlaGluLeuGluAlaVal180185190Tyr LeuThrGlnGlnAlaGlnSerThrGlySerAlaProValAlaPro195200205AspGluPheSerSerArgGluGluPheValValAlaValSerHisSer210 215220SerProSerAlaLeuAlaLeuGlnSerProLeuLeuProAlaTrpArg225230235240ThrLeuSerValSerGluHisAla ProGlyLeuProAlaSerProPro245250255ArgArgProArgTrpAsnGluSerThrArgLeuProArgGlyLeuAla260265 270ValProAlaGlnGlyHisLeuGlyArgThrHisProHisSerGlnLeu275280285LeuValAlaGlyGlyGlnValGluThrLeuProAspProGlnT hrSer290295300ValProValProLeuHisSerSerProHisSerProAlaProProThr305310315320ThrSer SerSerAlaSerLysThrLeuValLeuLeuSerLeuLeuArg325330335LysLysLysThrLysSerGlyValPhePheCysPheLeuPhePhePro3 40345350PheProProAlaProThrHisGlyAlaPhePheTrpArgTrpGlyLeu355360365GlyAsnGluGlyLeuArgSerArgL ysGlyPheTyrPhePheGluPhe370375380(2) INFORMATION FOR SEQ ID NO:41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 798 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 3..798(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:GAATTCCTCTGACTAATTCAAGTATCCCAAGGTTTGGAGTTAAAACT47IleProLeuThrAsnSerSerIleProArgPheGlyValLys Thr151015GAACAAGAAGATGTCCTTGCCAAGGAACTAGAAGATGTGAACAAATGG95GluGlnGluAspValLeuAlaLysGluLeuGluAspVal AsnLysTrp202530GGTCTTCATGTTTTCAGAATAGCAGAGTTGTCTGGTAACCGGCCCTTG143GlyLeuHisValPheArgIleAlaGluLeuSerGly AsnArgProLeu354045ACTGTTATCATGCACACCATTTTTCAGGAACGGGATTTATTAAAAACA191ThrValIleMetHisThrIlePheGlnGluArgAsp LeuLeuLysThr505560TTTAAAATTCCAGTAGATACTTTAATTACATATCTTATGACTCTCGAA239PheLysIleProValAspThrLeuIleThrTyrLeuMet ThrLeuGlu657075GACCATTACCATGCTGATGTGGCCTATCACAACAATATCCATGCTGCA287AspHisTyrHisAlaAspValAlaTyrHisAsnAsnIleHisAla Ala80859095GATGTTGTCCAGTCTACTCATGTGCTATTATCTACACCTGCTTTGGAG335AspValValGlnSerThrHisValLeuLeuSerThrPro AlaLeuGlu100105110GCTGTGTTTACAGATTTGGAGATTCTTGCAGCAATTTTTGCCAGTGCA383AlaValPheThrAspLeuGluIleLeuAlaAlaIle PheAlaSerAla115120125ATACATGATGTAGATCATCCTGGTGTGTCCAATCAATTTCTGATCAAT431IleHisAspValAspHisProGlyValSerAsnGln PheLeuIleAsn130135140ACAAACTCTGAACTTGCCTTGATGTACAATGATTCCTCAGTCTTAGAG479ThrAsnSerGluLeuAlaLeuMetTyrAsnAspSerSer ValLeuGlu145150155AACCATCATTTGGCTGTGGGCTTTAAATTGCTTCAGGAAGAAAACTGT527AsnHisHisLeuAlaValGlyPheLysLeuLeuGlnGluGluAsn Cys160165170175GACATTTTCCAGAATTTGACCAAAAAACAAAGACAATCTTTAAGGAAA575AspIlePheGlnAsnLeuThrLysLysGlnArgGlnSer LeuArgLys180185190ATGGTCATTGACATCGTACTTGCAACAGATATGTCAAAACACATGAAT623MetValIleAspIleValLeuAlaThrAspMetSer LysHisMetAsn195200205CTACTGGCTGATTTGAAGACTATGGTTGAAACTAAGAAAGTGACAAGC671LeuLeuAlaAspLeuLysThrMetValGluThrLys LysValThrSer210215220TCTGGAGTTCTTCTTCTTGATAATTATTCCGATAGGATTCAGGTTCTT719SerGlyValLeuLeuLeuAspAsnTyrSerAspArgIle GlnValLeu225230235CAGAATATGGTGCACTGTGCAGATCTGAGCAACCCAACAAAGCCTCTC767GlnAsnMetValHisCysAlaAspLeuSerAsnProThrLysPro Leu240245250255CAGCTGTACCGCCAGTGGACGGACGGAATTC798GlnLeuTyrArgGlnTrpThrAspGlyIle 260265(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 265 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:IleProLeuThrAsnSerSerIleProArgPheGlyVal LysThrGlu151015GlnGluAspValLeuAlaLysGluLeuGluAspValAsnLysTrpGly202530LeuHisValPheArgIleAlaGluLeuSerGlyAsnArgProLeuThr354045ValIleMetHisThrIlePheGlnGluArgAspLeuLeuLysThrPhe50 5560LysIleProValAspThrLeuIleThrTyrLeuMetThrLeuGluAsp65707580HisTyrHisAlaAspValAl aTyrHisAsnAsnIleHisAlaAlaAsp859095ValValGlnSerThrHisValLeuLeuSerThrProAlaLeuGluAla100 105110ValPheThrAspLeuGluIleLeuAlaAlaIlePheAlaSerAlaIle115120125HisAspValAspHisProGlyValSerAsnGlnPheLeu IleAsnThr130135140AsnSerGluLeuAlaLeuMetTyrAsnAspSerSerValLeuGluAsn145150155160H isHisLeuAlaValGlyPheLysLeuLeuGlnGluGluAsnCysAsp165170175IlePheGlnAsnLeuThrLysLysGlnArgGlnSerLeuArgLysMet 180185190ValIleAspIleValLeuAlaThrAspMetSerLysHisMetAsnLeu195200205LeuAlaAspLeuLysThrMe tValGluThrLysLysValThrSerSer210215220GlyValLeuLeuLeuAspAsnTyrSerAspArgIleGlnValLeuGln225230235 240AsnMetValHisCysAlaAspLeuSerAsnProThrLysProLeuGln245250255LeuTyrArgGlnTrpThrAspGlyIle 260265(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1902 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 97..1256 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:GAATTCCTTTGTTCACATCTTCTAGTTCCTTGGCAAGGACATCTTCATGTTTTCAGAATA60GCAGAGTTGTCTGGTAACCGGCCCTTGACTGTTATCATGCACACCATTTTTCAG114 MetHisThrIlePheGln15GAACGGGATTTATTAAAAACATTTAAAATTCCAGTAGATACTTTAATT162GluArgAspLeuLeuLysThrPhe LysIleProValAspThrLeuIle101520ACATATCTTATGACTCTCGAAGACCATTACCATGCTGATGTGGCCTAT210ThrTyrLeuMetThrLeuGluAsp HisTyrHisAlaAspValAlaTyr253035CACAACAATATCCATGCTGCAGATGTTGTCCAGTCTACTCATGTGCTA258HisAsnAsnIleHisAlaAlaAspVal ValGlnSerThrHisValLeu404550TTATCTACACCTGCTTTGGAGGCTGTGTTTACAGATTTGGAGATTCTT306LeuSerThrProAlaLeuGluAlaValPheThr AspLeuGluIleLeu55606570GCAGCAATTTTTGCCAGTGCAATACATGATGTAGATCATCCTGGTGTG354AlaAlaIlePheAlaSerAlaIleHis AspValAspHisProGlyVal758085TCCAATCAATTTCTGATCAATACAAACTCTGAACTTGCCTTGATGTAC402SerAsnGlnPheLeuIleAsnThr AsnSerGluLeuAlaLeuMetTyr9095100AATGATTCCTCAGTCTTAGAGAACCATCATTTGGCTGTGGGCTTTAAA450AsnAspSerSerValLeuGluAsn HisHisLeuAlaValGlyPheLys105110115TTGCTTCAGGAAGAAAACTGTGACATTTTCCAGAATTTGACCAAAAAA498LeuLeuGlnGluGluAsnCysAspIle PheGlnAsnLeuThrLysLys120125130CAAAGACAATCTTTAAGGAAAATGGTCATTGACATCGTACTTGCAACA546GlnArgGlnSerLeuArgLysMetValIleAsp IleValLeuAlaThr135140145150GATATGTCAAAACACATGAATCTACTGGCTGATTTGAAGACTATGGTT594AspMetSerLysHisMetAsnLeuLeu AlaAspLeuLysThrMetVal155160165GAAACTAAGAAAGTGACAAGCTCTGGAGTTCTTCTTCTTGATAATTAT642GluThrLysLysValThrSerSer GlyValLeuLeuLeuAspAsnTyr170175180TCCGATAGGATTCAGGTTCTTCAGAATATGGTGCACTGTGCAGATCTG690SerAspArgIleGlnValLeuGln AsnMetValHisCysAlaAspLeu185190195AGCAACCCAACAAAGCCTCTCCAGCTGTACCGCCAGTGGACGGACCGG738SerAsnProThrLysProLeuGlnLeu TyrArgGlnTrpThrAspArg200205210ATAATGGAGGAGTTCTTCCGCCAAGGAGACCGAGAGAGGGAACGTGGC786IleMetGluGluPhePheArgGlnGlyAspArg GluArgGluArgGly215220225230ATGGAGATAAGCCCCATGTGTGACAAGCACAATGCTTCCGTGGAAAAA834MetGluIleSerProMetCysAspLys HisAsnAlaSerValGluLys235240245TCACAGGTGGGCTTCATAGACTATATTGTTCATCCCCTCTGGGAGACA882SerGlnValGlyPheIleAspTyr IleValHisProLeuTrpGluThr250255260TGGGCAGACCTCGTCCACCCTGACGCCCAGGATATTTTGGACACTTTG930TrpAlaAspLeuValHisProAsp AlaGlnAspIleLeuAspThrLeu265270275GAGGACAATCGTGAATGGTACCAGAGCACAATCCCTCAGAGCCCCTCT978GluAspAsnArgGluTrpTyrGlnSerT hrIleProGlnSerProSer280285290CCTGCACCTGATGACCCAGAGGAGGGCCGGCAGGGTCAAACTGAGAAA1026ProAlaProAspAspProGluGluGlyArgGlnG lyGlnThrGluLys295300305310TTCCAGTTTGAACTAACTTTAGAGGAAGATGGTGAGTCAGACACGGAA1074PheGlnPheGluLeuThrLeuGluGluA spGlyGluSerAspThrGlu315320325AAGGACAGTGGCAGTCAAGTGGAAGAAGACACTAGCTGCAGTGACTCC1122LysAspSerGlySerGlnValGluG luAspThrSerCysSerAspSer330335340AAGACTCTTTGTACTCAAGACTCAGAGTCTACTGAAATTCCCCTTGAT1170LysThrLeuCysThrGlnAspSerG luSerThrGluIleProLeuAsp345350355GAACAGGTTGAAGAGGAGGCAGTAGGGGAAGAAGAGGAAAGCCAGCCT1218GluGlnValGluGluGluAlaValGlyG luGluGluGluSerGlnPro360365370GAAGCCTGTGTCATAGATGATCGTTCTCCTGACACGTAACAGTGCAAA1266GluAlaCysValIleAspAspArgSerProAspT hr375380385AACTTTCATGCCTTTTTTTTTTTTAAGTAGAAAAATTGTTTCCAAAGTGCATGTCACATG1326CCACAACCACGGTCACACCTCACTGTCATCTGCCAGGACGTTTGTTGAACAAAACTGACC1386TTGACTACTCAGTCCAGCGCTCAGGAATATCGTAACCAGTTTTTTCACCTCCATGTCATC1446CGAGCAAGGTGGACATCTTCACGAACAGCGTTTTTAACAAGATTTCAGCTTGGTAGAGCT1506GACAAAGCAGATAAAATCTACTCCAAATTATTTTCAAGAG AGTGTGACTCATCAGGCAGC1566CCAAAAGTTTATTGGACTTGGGGTTTCTATTCCTTTTTATTTGTTTGCAATATTTTCAGA1626AGAAAGGCATTGCACAGAGTGAACTTAATGGACGAAGCAACAAATATGTCAAGAACAGGA1686CATAGCACGAATCTG TTACCAGTAGGAGGAGGATGAGCCACAGAAATTGCATAATTTTCT1746AATTTCAAGTCTTCCTGATACATGACTGAATAGTGTGGTTCAGTGAGCTGCACTGACCTC1806TACATTTTGTATGATATGTAAAACAGATTTTTTGTAGAGCTTACTTTTATTATTAAATG T1866ATTGAGGTATTATATTTAAAAAAAAAAAAGGAATTC1902(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 386 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:MetHisThrIlePheGlnGluArgAspLeuLeuLysThrPheLysIle151015ProValAspThrLeuIleThrTyrLeuMetThrLeuGluAspHisTyr 202530HisAlaAspValAlaTyrHisAsnAsnIleHisAlaAlaAspValVal354045GlnSerThrHisValL euLeuSerThrProAlaLeuGluAlaValPhe505560ThrAspLeuGluIleLeuAlaAlaIlePheAlaSerAlaIleHisAsp6570 7580ValAspHisProGlyValSerAsnGlnPheLeuIleAsnThrAsnSer859095GluLeuAlaLeuMetTyrAsnAspSerSerVa lLeuGluAsnHisHis100105110LeuAlaValGlyPheLysLeuLeuGlnGluGluAsnCysAspIlePhe115120125GlnAsnLeuThrLysLysGlnArgGlnSerLeuArgLysMetValIle130135140AspIleValLeuAlaThrAspMetSerLysHisMetAsnLeuLeuAla145 150155160AspLeuLysThrMetValGluThrLysLysValThrSerSerGlyVal165170175LeuLeuLeuAspA snTyrSerAspArgIleGlnValLeuGlnAsnMet180185190ValHisCysAlaAspLeuSerAsnProThrLysProLeuGlnLeuTyr195 200205ArgGlnTrpThrAspArgIleMetGluGluPhePheArgGlnGlyAsp210215220ArgGluArgGluArgGlyMetGluIleSerProMetCysAs pLysHis225230235240AsnAlaSerValGluLysSerGlnValGlyPheIleAspTyrIleVal245250 255HisProLeuTrpGluThrTrpAlaAspLeuValHisProAspAlaGln260265270AspIleLeuAspThrLeuGluAspAsnArgGluTrpTyrGlnSerThr 275280285IleProGlnSerProSerProAlaProAspAspProGluGluGlyArg290295300GlnGlyGlnThrGluLysPheG lnPheGluLeuThrLeuGluGluAsp305310315320GlyGluSerAspThrGluLysAspSerGlySerGlnValGluGluAsp325 330335ThrSerCysSerAspSerLysThrLeuCysThrGlnAspSerGluSer340345350ThrGluIleProLeuAspGluGlnValGluGl uGluAlaValGlyGlu355360365GluGluGluSerGlnProGluAlaCysValIleAspAspArgSerPro370375380Asp Thr385(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1155 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 95..762(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:GAAT TCCCTGGCTGTGGGCTTCAAGCTGCTGCAGGCAGAGAACTGCGATATCTTCCAGAA60CCTCAGCGCCAAGCAGCGACTGAGTCTGCGCAGGATGGTCATTGACATGGTG112MetValIleAsp MetVal15CTGGCCACAGACATGTCCAAACACATGAACCTCCTGGCCGACCTCAAG160LeuAlaThrAspMetSerLysHisMetAsnLeuLeuAlaA spLeuLys101520ACCATGGTGGAGACCAAGAAGGTGACAAGCCTCGGTGTCCTCCTCCTG208ThrMetValGluThrLysLysValThrSerLeuGlyValL euLeuLeu253035GACAACTATTCCGACCGAATCCAGGTCTTGCAGAACCTGGTGCACTGT256AspAsnTyrSerAspArgIleGlnValLeuGlnAsnLeuValH isCys404550GCTGATCTGAGCAACCCCACCAAGCCGCTGCCCCTGTACCGCCAGTGG304AlaAspLeuSerAsnProThrLysProLeuProLeuTyrArgGlnTrp 55606570ACGGACCGCATCATGGCCGAGTTCTTCCAGCAGGGAGACCGCGAGCGT352ThrAspArgIleMetAlaGluPhePheGlnGlnGlyAspArgG luArg758085GAGTCGGGCCTGGACATCAGTCCCATGTGTGACAAGCATACGGCCTCA400GluSerGlyLeuAspIleSerProMetCysAspLysHisT hrAlaSer9095100GTGGAGAAGTCCCAGGTGGGTTTCATTGACTACATTGCTCACCCACTG448ValGluLysSerGlnValGlyPheIleAspTyrIleAlaH isProLeu105110115TGGGAGACTTGGGCTGACCTGGTCCACCCAGATGCACAGGACCTGCTG496TrpGluThrTrpAlaAspLeuValHisProAspAlaGlnAspL euLeu120125130GACACGCTGGAGGACAATCGAGAGTGGTACCAGAGCAAGATCCCCCGA544AspThrLeuGluAspAsnArgGluTrpTyrGlnSerLysIleProArg 135140145150AGTCCCTCAGACCTCACCAACCCCGAGCGGGACGGGCCTGACAGATTC592SerProSerAspLeuThrAsnProGluArgAspGlyProAspA rgPhe155160165CAGTTTGAACTGACTCTGGAGGAGGCAGAGGAAGAGGATGAGGAGGAA640GlnPheGluLeuThrLeuGluGluAlaGluGluGluAspG luGluGlu170175180GAAGAGGAGGGGGAAGAGACAGCTTTAGCCAAAGAGGCCTTGGAGTTG688GluGluGluGlyGluGluThrAlaLeuAlaLysGluAlaL euGluLeu185190195CCTGACACTGAACTCCTGTCCCCTGAAGCCGGCCCAGACCCTGGGGAC736ProAspThrGluLeuLeuSerProGluAlaGlyProAspProG lyAsp200205210TTACCCCTCGACAACCAGAGGACTTAGGGCCAGCCCTGCGTGAACT782LeuProLeuAspAsnGlnArgThr215220GCAGG GGCAATGGATGGTAAAGCCCTTTGGCTCTTGGCAGGCAGACTTTCCAGGAAGAGG842CTCCATGTGGCTCCTGCTTCACTTTCCCACCCATTTAGGGAGACAATCAAGCTCTTAGTT902ATAGGTGGCTCCCAGGGTCTAATTGGAGGCACCTGGCTGGGGTCCACTC TGACCCTAGAC962TTGCCTAAAAGAGCTCTCTAAGGGGCAGCCTCTTACGATGCCCTGGTGTCTTTCTCCTGG1022GCTTCTATCCCTGTGAGGAGAGGTGCTGTCTGCTGGAGCCTCTAGTCCACCCTCTCCAGT1082GGTCACTCTTGAGTCACATCTGT CACTTAATTATTTCCTTCTTTATCAAATATTTATTGC1142TCATCTGGAATTC1155(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 222 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:MetValIleAspMetValLeuAlaThrAspMetSerLysHisMetAsn151015LeuLeuAlaAspLeuLysThr MetValGluThrLysLysValThrSer202530LeuGlyValLeuLeuLeuAspAsnTyrSerAspArgIleGlnValLeu3540 45GlnAsnLeuValHisCysAlaAspLeuSerAsnProThrLysProLeu505560ProLeuTyrArgGlnTrpThrAspArgIleMetAlaGluPhePheGln 65707580GlnGlyAspArgGluArgGluSerGlyLeuAspIleSerProMetCys859095Asp LysHisThrAlaSerValGluLysSerGlnValGlyPheIleAsp100105110TyrIleAlaHisProLeuTrpGluThrTrpAlaAspLeuValHisPro115 120125AspAlaGlnAspLeuLeuAspThrLeuGluAspAsnArgGluTrpTyr130135140GlnSerLysIleProArgSerProSerAsp LeuThrAsnProGluArg145150155160AspGlyProAspArgPheGlnPheGluLeuThrLeuGluGluAlaGlu165170 175GluGluAspGluGluGluGluGluGluGlyGluGluThrAlaLeuAla180185190LysGluAlaLeuGluLeuProAspThrGluLeuLeuSerP roGluAla195200205GlyProAspProGlyAspLeuProLeuAspAsnGlnArgThr210215220(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:TACGAAGCTTTGATGGGGTCTACTGCTAC29(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:TACGAAGCTTTGATGGTTGGCTTGGCATATC31(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(iv) ANTI-SENSE: YES(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:ATTAACCCTCATAAAG16(2) INFORMATION FOR SEQ ID NO:50:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:TACGAAGCTTTGATGCGCCGACAGCCTGC29(2) INFORMATION FOR SEQ ID NO:51:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:GGTCTCCTGTTGCAGATATTG21(2) INFORMATION FOR SEQ ID NO:52:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:TTYAARTCTNYTNCARGRNGA21(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:ACNATRTCTRATNACCATYTT21(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:PheLysLeuLeuGlnGluGluAsn15(2) INFORMATION FOR SEQ ID NO:55:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:PheLysLeuLeuGlnGlyGluAsn15(2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:AspMetValIleAspMetVal15(2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(x i) SEQUENCE DESCRIPTION: SEQ ID NO:57:AspMetValIleAspIleVal15__________________________________________________________________________

Cloning by complementation and related processes

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Colicelli, J., et al., Proc. Natl. Acad. Sci. (USA), 86:3599-3603 (1989).
DeFeo-Jones, D., et al., Science, 228:179-184 (1985).
Field, J., et al., Molecular and Cellular Biology, 8:2159-2165 (1988).
Henikoff, S., et al., Nature 289:33-37 (1981).
Kataoka, T., et al., Cell, 43:493-505 (1985).
Kataoka, T., et al., Cell, 40:19-26 (1985).
Nikawa, J-I., et al., Molecular and Cellular Biology, 7:3629-3636 (1987).
Powers, S., et al., Molecular and Cellular Biology, 9:390-395 (1989).
Sass, P., et al., Proc. Natl. Acad. Sci. (USA), 83:9303-9307 (1986).
Swinnen, J. V., et al., Proc. Natl. Acad. Sci. (USA) 86:5325-5329 (1989).
Wigler, M., et al., Cold Spring Harbor Symposia, LIII:649-655 (1988).
Kataoka et al, Cell, V. 40, Jan. 1985, 19-26.
Colicelli, et al, PNAS, V. 86, May 1989, 3599-3603.