WO1994017189A2 - Protein kinases - Google Patents

Abstract

Protein kinase mutant and wild-type genes encoding polypeptides of the class heretofore designated 'casein kinase I' and useful in screening compositions which may effect DNA double-strand break repair activity are disclosed. Also disclosed are methods using the polynucleotides in cell-proliferative disorders.

Description

PROTEIN KINASES

This application is a Continuation-in-Partof U.S. Application Serial No. 08/008,001, filed January 21, 1993, which is a Continuation-in-Part of U.S. Application Serial No. 728,783, filed July 3, 1991.

FIELD OF THE INVENTION

The present invention relates to novel polynucleotides encoding polypeptides which correspond to the class of protein kinase isolates heretofore referred to as casein kinase I and which possess protein kinase and/or DNA recombination/repair promoting functional capabilities.

BACKGROUND OF THE INVENTION

Protein Kinases

The protein kinases comprise an exceptionally large family of eukaryotic proteins which mediate the responses of cells to external stimuli and are related by amino acid sequence homology within the so-called "catalytic domain" of the enzymes. To date, in excess of 100 unique members of the protein kinase family from a wide variety of eukaryotic organisms have been described and characterized at the amino acid sequence level. See, e.g., Hanks, et al. (Science, 241-42-52, 1988) which presents a sequence alignment of 65 protein kinase catalytic domains which range in size from about 250 to 300 amino acids and Hanks, et al. (Methods in Enzymol, 200:38-62, 1991) presenting a catalytic domain sequence alignment for 117 distinct protein kinase family members including a variety of vertebrate, invertebrate, higher plant and yeast species enzymes. The location of the catalytic domain within a protein kinase is not fixed. In most single subunit enzymes, the domain is near the carboxy terminus of the polypeptide while in multimeric protein kinases the catalytic domain takes up almost the entirety of the subunit polypeptide.

Protein kinases are generally classified into a protein- serine/threonine subfamily or a protein-tyrosine subfamily on the basis of phosphorylation substrate specificity. Among the many classes of enzymes within the protein-serine/threonine kinase subfamily are two distinct classes which have been designated casein kinase I and casein kinase II based on the order of their elution from DEAE-cellulose. The casein kinases are distinguished from other protein kinases by their ability to phosphorylate serine or threonine residues within acidic recognition sequences such as found in casein. Tuazon, et al. , (Adv. in Second Messenger and Phosphoprotein Res. , 23:123-164, 1991) presents a review of over 200 publications related to casein kinase I and II, addressing the physicochemical characterization, recognition sequences, substrate specificity and effects on metabolic regulation for these two classes of enzymes. Casein kinase II is active as a heterotetramer and the complete amino acid sequences of human, rat, Drosophila and yeast species catalytic regions have been determined. Despite the fact that partially purified casein kinase I preparations have been obtained from cell nuclei, cytoplasm, and cell membranes of various plant and animal species, prior to the present invention, nothing was known concerning the primary structure of its enzymatically active monomeric subunit.

As of the time of the present invention, therefore, there existed a significant need in the art for information concerning the primary structure (amino acid sequence) of protein-serine/threonine kinase enzymes of the casein kinase I class. Such information, provided in the form of DNA sequences encoding one or more of these kinases (from which primary structures could be deduced), would allow for the large scale production of kinases by recombinant techniques as well as for determination of the distribution and function of these enzymes, the structural distinctions between membrane-bound and non-membranous forms, the potential ligand-receptor interactions in which these kinases interact, and the identification of agents capable of modulating ligand-receptor binding, kinase, and other activities.

DNA Recombination And Repair

Chromosomes experience single-stranded or double-stranded breaks as a result of energy-rich radiation, chemical agents, as well as spontaneous breaks occurring during replication among others. Although genes present in the chromosomes undergo continuous damage, repair, exchange, transposition, and splicing, certain enzymes protect or restore the specific base sequences of the chromosome.

The repair of DNA damage is a complex process that involves the coordination of a large number of gene products. This complexity is in part dependent upon both the form of DNA damage and cell cycle progression. For example, in response to ultraviolet (UV) irradiation, cells can employ photoreactivation or excision repair functions to correct genetic lesions. The repair of strand breaks, such as those created by X-rays, can proceed through recombinational mechanisms. For many forms of DNA damage, the cell is induced to arrest in the G2 phase of the cell cycle. During this G2 arrest, lesions are repaired to ensure chromosomal integrity prior to mitotic segregation.

Since the transfer of genetic information from generation to generation is dependent on the integrity of DNA, it is important to identify those gene products which affect or regulate genetic recombination and repair. Through the use of organisms with specific genetic mutations, the normal functional gene can be obtained, molecularly cloned, and the gene products studied.

In eukaryotes such as Saccharomyces cerevisiae, genetic studies have defined repair-deficient mutants which have allowed the identification of more than 30 radiation-sensitive (RAD) mutants (Haynes, et al., in Molecular Biology of the Yeast Saccharomyces, pp. 371, 1981; J. Game in Yeast Genetics: Fundamental and Applied Aspects, pp. 109, 1983). These mutants can be grouped into three classes depending upon their sensitivities. These classes broadly define excision-repair, error-prone repair, and recombinational-repair functions. The molecular characterization of yeast RAD genes has increased the understanding of the enzymatic machinery involved in excision repair, as well as the arrest of cell division by DNA damage.

The understanding of RAD genes and their expression products has become increasingly important as research continues to develop more effective therapeutic compositions. Often these new compositions appear quite effective against a particular disease condition, such as certain tumors, but prove to be too toxic for in vivo therapy in an animal having the disease. Indeed, these compositions can actually increase the likelihood of mutagenesis. Most agents that are mutagenic or carcinogenic are in themselves unreactive, but are broken down to reactive intermediates in vivo. It is these reactive intermediates which interact with DNA to produce a mutation. This event is thought to be the initial step in chemical carcinogenesis. Mutations in a large number of genes affect the cellular response to agents that damage DNA. In all likelihood, many of these mutated genes encode enzymes that participate in DNA repair systems. Consequently, when the repair system is compromised, the cells become extremely sensitive to toxic agents. Although the DNA may revert to normal when DNA repair mechanisms operate successfully, the failure of such mechanisms can result in a transformed tumor cell which continues to proliferate.

Although there are currently available tests to determine the toxicity or mutagenicity of chemical agents and compositions, there are limitations in both laboratory screening procedures and animal toxicity tests. These limitations include extrapolating laboratory data from animals to humans. There is often a large measure of uncertainty when attempting to correlate the results obtained in laboratory animals with effects in human subjects. In most cases, doses of the test drug have been used in the animal which are too high to be safely administered to humans. In addition, some types of toxicity can be detected if the drug is administered in a particular species, yet may be missed if the experiment is not done in the correct animal species. Moreover, many currently available laboratory tests are incapable of detecting certain types of toxic manifestations which occur in man.

Phenotypic complementation, as a way of identifying homologous normal functional genes, is widely used. For example, the human homologue of the yeast cell cycle control gene, cdc 2, was cloned by expressing a human cDNA library in Schizosaccharomyces pombe and selecting those clones which could complement a mutation in the yeast cdc 2 gene (Lee, et al, Nature, 327:31. 1987). A mammalian gene capable of reverting the heat shock sensitivity of the RAS2^va119 gene of yeast, has also been cloned by using complementation (Colicelli, et al, Proc.Nat'l.Acad.Sci. USA, 86:3599, 1989). A rat brain cDNA library was used to clone a mammalian cDNA that can complement the loss of growth control associated with the activated RAS2 gene in yeast. The gene, DPD (dunce-like phosphodiesterase), encodes a high-affinity CAMP phosphodiesterase. In summary, limitations and uncertainties of existing laboratory tests fail to provide an accurate method of examining the effects of a composition on DNA integrity. In view of this, a considerable need exists for screening methodologies which are inexpensive, rapid, and contain the relevant gene from the animal which is to be treated with the composition. Such methods provide a direct assay to determine if a composition interferes with the DNA repair system of a cell.

SUMMARY OF THE INVENTION

In one of its aspects, the present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts thereof) encoding eukaryotic protein kinases of the casein kinase I class herein designated as "HRR25-like" proteins and characterized by greater than 35% amino acid sequence homology with the prototypical yeast enzyme HRR25 through the protein kinase catalytic domain thereof. Polynucleotides provided by the invention include RNAs, mRNAs and DNAs, including antisense forms thereof. Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences and biological replicas thereof. Specifically illustrating the invention are Saccharomyces cerevisiae DNAs including those encoding HRR25 and NUFl, Schizosaccharomyces pombe DNAs including those encoding Hhpl + and Hhp2+, and human DNAs including those encoding CKIαlHu, CKIeώHu, CKIα3Hu, CKIγlHu, CKTy2Hu, and CKIδHu. Also provided are autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating such sequences and especially vectors wherein DNA encoding an HRR2J-like casein kinase I protein is linked to an endogenous or exogenous expression control DNA sequence.

According to another aspect of the invention, host cells, especially unicellular host cells such as procaryotic and eukaryotic cells, are stably transformed with DNA sequences of the invention in a manner allowing the desired polypeptides to be expressed therein. Host cells expressing such HRR25-like products can serve a variety of useful purposes. To the extent that the expressed products are "displayed" on host cell surfaces, the cells may constitute a valuable immunogen for the development of antibody substances specifically immunoreactive therewith.

Host cells of the invention are conspicuously useful in methods for the large scale production of HR#25-like proteins wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown. Also comprehended by the present invention are antibody substances (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) and other binding proteins which are specific for HRR25-like proteins (i.e., non-reactive with protein kinase molecules which are not related by at least 35% homology with HRR25 through the protein kinase catalytic domain). Antibody substances can be developed using isolated natural or recombinant HRR25-lϊke proteins or cells expressing such products on their surfaces. The antibody substances are useful, in turn, for purifying recombinant and naturally occurring HRR25-like polypeptides and identifying cells producing such polypeptides on their surfaces. The antibody substances and other binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting, or stimulating) ligand-receptor binding reactions involving HRR25-like proteins. Anti idiotypic antibodies specific for anti-HRR25-like antibody substances are also contemplated. Assays for the detection and quantification of HRR25-like proteins on cell surfaces and in fluids such as serum and cytoplasmic fractions may involve a single antibody substance or multiple antibody substances in a "sandwich" assay format.

Recombinant HR_?25-like protein products obtained according to the invention have been observed to display a number of properties which are unique among the eukaryotic protein kinases. As one example, the HRR25 protein possesses both protein-tyrosine kinase and protein-serine/threonine kinase activities. Moreover, HRR25 operates to promote repair of DNA strand breaks at a specific nucleotide sequence and is the only protein kinase known to have such recombination/repair promoting activity.

The DNA sequence information for yeast and mammalian

(including human) species HRR25-like proteins which is provided by the present invention makes possible the identification and isolation of DNAs encoding other

HRR25-)ik& proteins by such well-known techniques as DNA/DNA hybridization and polymerase chain reaction (PCR) cloning.

Recombinant HRR25-like proteins and host cells expressing the same are useful in screening methods designed to examine the effects of various compositions on DNA break repair and protein kinase activities of the proteins. Protein kinase inhibitory effects may be assessed by well-known screening procedures such as described in Ηidaka, et al. (Methods in Enzymology, 201:328- 339, 1991).

BRIEF DESCRIPTION OF THE DRAWING Further aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of presently preferred embodiments thereof, reference being made to the drawing wherein:

Figure 1 (A) presents an alignment of the predicted amino acid sequence of HRR25 with the catalytic domains of the yeast CDC28, yeast KSS1 and human RAFl protein kinases. Figure 1(B) shows a schematic representation of the structure of HRR25, and

Figure 2 presents an alignment of the predicted amino acid sequences of HRR25 with the sequences of three other Saccharomyces cerevisiae HRR25-lik& proteins (YCK1/CKI2, YCK2/CKI1, and NUF1), two HRR25-like proteins (Hhpl + and Hhp2+) from Schizosaccharomyces pombe and three putative isoforms (CKIαlHu, CKIo^*2Hu, and CKIα3Hu) of a human HRR2J-like protein. DETAELED DESCRIPTION OF THE INVENTION

In one of its aspects, the present invention relates to a DNA encoding a recombination/repair promoting polypeptide which can be used in an assay system to examine the effects of various compositions on DNA integrity. These functional sequences, which can be characterized by their ability to promote restoration of DNA strand breaks, permit the screening of compositions to determine whether a particular composition has an effect on the restoration of such repair activity. The invention also provides a DNA sequence encoding a polypeptide which promotes normal mitotic recombination, but is defective in protein kinase activity and essentially unable to repair DNA strand breaks. This defective DNA sequence is highly useful for identifying other DNA sequences which encode proteins with functional protein kinase activity. In addition, the present invention relates to the polypeptide encoded by the defective DNA sequence, as well as the polypeptide encoded by the functional wild-type DNA. In order to identify a DNA sequence encoding a polypeptide with protein kinase activity, a method is provided whereby a DNA library is screened for nucleotide sequences capable of restoring DNA strand break repair in a mutant lacking such activity. A method is further provided for identifying a composition which affects the activity of a mammalian polypeptide having protein kinase activity, wherein the polypeptide is capable of restoring DNA double-strand break repair activity in a mutant lacking such activity.

In general, the defective protein kinase can be characterized by its ability to promote normal mitotic recombination, while being essentially unable to repair DNA double-strand break including that which occurs at the cleavage site:

I CAACAG

GTTGTC t

The DNA double-strand breaks which the defective protein kinase is essentially unable to repair can be induced by various means, including endonucleases, x- rays, or radiomimetic agents including alkylating agents. Preferred endonucleases are those which recognize the same nucleotide cleavage site as endonuclease HO. Radiomimetic alkylating agents having methylmethane sulfonate activity are preferred. Those of skill in the art will be able to identify other agents which induce the appropriate DNA strand breaks without undue experimentation. The present invention specifically discloses mutants sensitive to continuous expression of the DNA double-strand endonuclease HO, which codes for a 65 kDa site-specific endonuclease that initiates mating type interconversion (Kostriken, et al , Cold Spring Harbor Symp. Quant. BioL , 49:89, 1984). These mutants are important to understanding the functions involved in recognizing and repairing damaged chromosomes. This invention also discloses a yeast wild-type DNA recombination and repair gene called HRR25 (HO and/or radiation repair). Ηomozygous mutant strains, hrr25-l, are sensitive to methylmethane sulfonate and X-rays, but not UV irradiation. The wild-type gene encodes a novel protein kinase, homologous to other serine/threonine kinases, which appears critical in activation of DNA repair functions by phosphorylation.

The HRR25 kinase is important for normal cell growth, nuclear segregation, DNA repair and meiosis, and deletion of HRR25 results in cell cycle defects. These phenotypes, coupled with the sequence similarities between the HRR25 kinase and the Raf/c-mos protein kinase subgroup suggest that HRR25 might play a similar role in S. cerevisiae growth and development. The defects in DNA strand break repair and the aberrant growth properties revealed by mutations in HRR25 kinase, expands the role that protein kinases may play and places HRR25 in a functional category of proteins associated with DNA metabolism. The development of specific DNA sequences encoding protein kinase polypeptides of the invention can be accomplished using a variety of techniques. For example, methods which can be employed include (1) isolation of a double-stranded DNA sequence from the genomic DNA of the eukaryote; (2) chemical synthesis of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double stranded DNA complement of MRNA is eventually formed which is generally referred to as CDNA.

The novel DNA sequences of the invention include all sequences useful in providing for expression in prokaryotic or eukaryotic host cells of polypeptides which exhibit the functional characteristics of the novel protein kinase of the invention. These DNA sequences comprise: (a) the DNA sequences as set forth in SEQ. I.D. No. 1 or their complementary strands; (b) DNA sequences which encode an amino acid sequence with at least about 35% homology in the protein kinase domain with the amino acid sequences encoded by the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences defined in (a) and (b) above. Specifically embraced in (b) are genomic DNA sequences which encode allelic variant forms. Part (c) specifically embraces the manufacture of DNA sequences which encode fragments of the protein kinase and analogs of the protein kinase wherein the DNA sequences thereof may incorporate codons which facilitate translation of mRNA. Also included in part (c) are DNA sequences which are degenerate as a result of the genetic code.

The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue.

Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.

With the DNA sequences of the invention in hand, it is a routine matter to prepare, subclone, and express smaller DNA fragments from this or a corresponding DNA sequences. The term "polypeptide" denotes any sequence of amino acids having the characteristic activity of the mutant or wild-type protein kinase of the invention, wherein the sequence of amino acids is encoded by all or part of the DNA sequences of the invention.

The polypeptide resulting from expression of the DNA sequence of the invention can be further characterized as being free from association with other eukaryotic polypeptides or other contaminants which might otherwise be associated with the protein kinase in its natural cellular environment. Isolation and purification of microbially expressed polypeptides provided by the invention may be by conventional means including, preparative chromatographic separations and immunological separations involving monoclonal and/or polyclonal antibody preparation. In general, recombinant expression vectors useful in the present invention contain a promotor sequence which facilitates the efficient transcription of the inserted eukaryotic genetic sequence. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The polypeptides of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.

The DNA sequences of the present invention can be expressed in vt^'vo in either prokaryotes or eukaryotes. Methods of expressing DNA sequences containing eukaryotic coding sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors used to incorporate DNA sequences of the invention, for expression and replication in the host cell are well known in the art. For example, DNA can be inserted in yeast using appropriate vectors and introducing the product into the host cells. Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, et al , Nature, 340:205, 1989; Rose, et al , Gene, 60:237, 1987). Those of skill in the art will know of appropriate techniques for obtaining gene expression in both prokaryotes and eukaryotes, or can readily ascertain such techniques, without undue experimentation.

Hosts include microbial, yeast, insect and mammalian host organisms. Thus, the term "host" is meant to include not only prokaryotes, but also such eukaryotes such as yeast, filamentous fungi, as well as plant and animal cells which can replicate and express an intron-free DNA sequence of the invention. The term also includes any progeny of the subject cell. It is understood that not all progeny are identical to the parental cell since there may be mutations that occur at replication. However, such progeny are included when the terms above are used.

Transformation with recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl could be used in the reaction. Transformation can also be performed after forming a protoplast of the host cell. Where the host is a eukaryote, various methods of DNA transfer can be used. These include transfection of DNA by calcium phosphate- precipitates, conventional mechanical procedures such as microinjection, insertion of a plasmid encased in liposomes, spheroplast electroporation, salt mediated transformation of unicellular organisms or the use of virus vectors. Analysis of eukaryotic DNA has been greatly simplified since eukaryotic DNA can be cloned in prokaryotes using vectors well known in the art. Such cloned sequences can be obtained easily in large amounts and can be altered in vivo by bacterial genetic techniques and in vitro by specific enzyme modifications. To determine the effects of these experimentally induced changes on the function and expression of eukaryotic genes, the rearranged sequences must be taken out of the bacteria in which they were cloned and reintroduced into a eukaryotic organism. Since there are still many functions in eukaryotic cells which are absent in prokaryotes, (e.g., localization of ATP-generating systems to mitochondria, association of DNA with histones, mitosis and meiosis, and differentiation of cells), the genetic control of such functions must be assessed in a eukaryotic environment. Cloning genes from other eukaryotes in yeast has been useful for analyzing the cloned eukaryotic genes as well as other yeast genes. A number of different yeast vectors have been constructed for this purpose. All vectors replicate in E. coli, which is important for amplification of the vector DNA. All vectors contain markers, e.g., LEU 2, HIS 3, URA 3, that can be selected easily in yeast. In addition, these vectors also carry antibiotic resistance markers for use in E. coli. Many strategies for cloning human homologues of known yeast genes are known in the art. These include, but are not limited to: 1) low stringency hybridization to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared structural features; and 3) complementation of mutants to detect genes with similar functions.

For purposes of the present invention, protein kinases which are homologous can be identified by structural as well as functional similarity. Structural similarity can be determined, for example, by assessing amino acid homology or by screening with antibody, especially a monoclonal antibody, which recognizes a unique epitope present on the protein kinases of the invention. When amino acid homology is used as criteria to establish structural similarity, those amino acid sequences which have homology of at least about 35 % in the protein kinase domain with the prototypical HRR25 protein are considered to uniquely characterize polypeptides. Conserved regions of amino acid residues in HRR25 can be used to identify HRR25-like genes from other species. Conserved regions which can be used as probes for identification and isolation of HRR25-like genes (homologues) include the nucleotides encoding amino acid sequences GPSLED (amino acids 86 to 91 in SEQ ID NO: 2), RDIKPDNFL (amino acids 127 to 135 in SEQ ID NO: 2), ΗIPYRE (amino acids 164 to 169 in SEQ ID NO: 2), and SVN (amino acids 181 to 183 in SEQ ID NO: 2), for example. These conserved motifs can be used, for example, to develop nucleotide primers to detect other HRR25-like genes by methods well known to those skilled in the art, such as polymerase chain reaction (PCR). When homologous amino acid sequences are evaluated based on functional characteristics, then a homologous amino acid sequence is considered equivalent to an amino acid sequence of the invention when the homologous sequence is essentially unable to repair (in the case of the repair defective mutant gene) or able to repair (in the case of the natural gene), DNA double-strand breaks, including that which occurs at a nucleotide cleavage site I CAACAG

GTTGTC t

and when the homologous amino acid sequence allows normal mitotic recombination.

This invention provides screening methods whereby genes are cloned from plasmid libraries by complementation of a recessive marker. A recipient strain such as Saccharomyces cerevisiae is constructed that carries a recessive mutation in the gene of interest. This strain is then transformed with a plasmid, for example, pYES2 (Invitrogen, San Diego, CA) containing the wild- type genomic DNA or cDNA. The clone carrying the gene of interest can then be selected by replica plating to a medium that distinguishes mutant from wild- type phenotypes for the gene of interest. The plasmid can then be extracted from the clone and the DNA studied. Several yeast vectors allow the application of complementation systems to go beyond isolation of yeast genes. Genes from a wide variety of species can be isolated using these vectors. In such systems, DNA sequences from any source are cloned into a vector and can be screened directly in yeast for activities that will complement specific yeast mutations. In a preferred embodiment, the present invention uses a mutation in yeast, the hrr25 mutation, which was identified by sensitivity to DNA double- strand breaks induced by the HO endonuclease. The genomic DNA which complements this mutation was isolated by transforming the hrr25 strain with a DNA library and subsequently screening for methylmethane sulfonate (MMS) resistance. Alternately, functional genes from a variety of mammalian species can now be cloned using the system described.

Yeast genes can be cloned by a variety of techniques, including use of purified RNA as hybridization probes, differential hybridization of regulated RNA transcripts, antibody screening, transposon mutagenesis, cross suppression of mutant phenotypes, cross hybridization with heterologous CDNA or oligonucleotide probes, as well as by complementation in E. coli. Minor modifications of the primary amino acid sequence may result in proteins which have substantially equivalent or enhanced activity as compared to the sequence set forth in SEQ. I.D. NO. 2. The modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous by HRR25 producing organisms. All of these modifications are included in the invention as long as HRR25 activity is retained. Substitution of an aspartic acid residue for a glycine acid residue at position 151 in the sequence shown in SEQ. I.D. NO. 2 identifies the mutant hrr25.

Antibodies provided by the present invention are immunoreactive with the mutant polypeptides and/or the naturally occurring protein kinase. Antibody which consist essentially of numerous monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibody is made from antigen containing fragments of the polypeptide by methods well known in the art (Kohler,G. et al. , Nature 256:495, 1975; Current Protocols in Molecular Biology , Ausubel, F. et al, ed.,1989).

The invention also discloses a method for identifying a composition which affects the activity of a polypeptide having tyrosine kinase activity. The polypeptide is capable of promoting restoration of DNA double-strand break repair activity in host cells containing the hrr25 gene. The composition and the polypeptide are incubated in combination with host cells for a period of time and under conditions sufficient to allow the components to interact, then subsequently monitoring the change in protein kinase activity, for example, by decreased repair of DNA double-strand breaks. The DNA strand breaks are induced, for example, by a radiomimetic agent, such as methylmethane sulfonate, x-rays, or by endonuclease like HO. Other means of inducing double-strand breaks that are well known in the art may be employed as well.

One embodiment of the invention provides a method of treating a cell proliferative disorder associated with or HRR25 or an HRR25-like protein comprising administering to a subject with the disorder, a therapeutically effective amount of reagent which modulates an HRR25 -like protein activity. The term "cell proliferative disorder" denotes malignant as well as non-malignant cell populations which differ from the surrounding tissue both morphologically and/or genotypically. Such disorders may be associated, for example, with abnormal expression of HRR25-like protein genes. "Abnormal expression" encompasses both increased or decreased levels of expression as well as expression of mutant forms such that the normal function of HRR2J-like genes is altered. Abnormal expression also includes inappropriate temporal expression during the cell cycle or expression in an incorrect cell type. Antisense polynucleotides of the invention are useful in treating malignancies of the various organ systems. Essentially, any disorder which is etiologically linked to altered expression of HRR25-like genes is a candidate for treatment with a reagent of the invention. "Treatment" of cell proliferative disorder refers to increasing or decreasing populations of malignant or non-malignant cells.

As used herein, the term "modulate" envisions the suppression of HRR25-like protein expression or the augmentation of expression. When a cell proliferative disorder is associated with HRR25-like gene overexpression, appropriate reagents such as antisense or binding antibody can be introduced to a cell. This approach utilizes, for example, antisense nucleic acid and ribozymes to block translation of a specific HRR25-like protein mRNA, either by masking that mRNA with an antisense nucleic acid or by cleaving it with a ribozyme. Alternatively, when a cell proliferative disorder is associated with insufficient HRR25-like protein, a sense polynucleotide sequence (the DNA coding strand) or HRR25-like polypeptide can be introduced into the cell by methods known in the art.

As used herein, the term "therapeutically effective" refers to that amount of polynucleotide, antibody or polypeptide that is sufficient to ameliorate the HRR25-associated disorder. "Ameliorate" denotes a lessening of the detrimental effect of the HRR25-associated disorder in the subject receiving therapy.

Antisense nucleic^* acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule. This interferes with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause non¬ specific interference with translation than larger molecules when introduced into the target HRR25 producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem. , 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn. , 260:3030, 1988). A major advantage of this approach is that, because ribosomes are sequence-specific, only mRNAS with particular sequences are inactivated. There are two basic types of ribozymes namely, tetrahymena-type and "hammerhead"-type. Tetrahymena-ty^~~e ribozymes recognize sequences which are four bases in length, while "hammerhead "-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-ty_pe ribozymes for inactivating a specific mRNA species and longer recognition sequences are preferable to shorter recognition sequences.

The present invention also provides gene therapy for the treatment of cell proliferative disorders which are mediated by HRR25-like polypeptides. Such therapy comprises introducing into cells of subjects having the proliferative disorder, the HRR25-like antisense polynucleotide. Delivery of antisense polynucleotide can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Disorders associated with under-expression of HRR25 can similarly be treated using gene therapy with nucleotide coding sequences.

Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting an HRR25-like sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the HRR25-like antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include but are not limited to 2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for HRR25-like antisense polynucleotides comprises a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al. Trends Biochem. Scl, 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al, Biotechniques, 6:682, 1988).

The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and "home in" on the desired cells. A ligand may be any compound of interest which will bind to another compound, such as a receptor.

The present invention will be better understood upon consideration of the following illustrative examples wherein: Example 1 addresses isolation of hrr25 mutant strains of Saccharomyces cerevisiae; Example 2 describes the isolation of HRR25 DNA by complementation screening; Example 3 is drawn to characterization of the DNA and putative amino acid sequence of HRR25; Example 4 addresses microscopic analysis of HRR25 wild type and hrr25 mutant yeast morphology; Example 5 addresses the relationship of the amino acid sequence of HRR25 and three exemplary protein kinases which are not HRR25- like; Example 6 describes the isolation of DNAs encoding two Schizosaccharomyces pombe HRR25-like protein kinases; Example 7 is directed to isolation of DNA encoding another Saccharomyces cerevisiae protein, NUF1; Example 8 is drawn to isolation of DNAs encoding various eukaryotic species HRR25-like proteins including three human isoforms, CKIcdHu, CKIα2Hu, and CKIαθHu; Examples 9 and 10 are respectively directed to determination of casein kinase and both serine-threonine kinase and tyrosine kinase activities for HRR25; Example 11 is drawn to the recombinant expression of HRR25 products and the generation of antibodies thereto; Example 12 relates to the isolation of human CKI isoforms, CKtylHu and CKI 2HU; Example 13 addresses isolation of another human isoform CKIδHu; Example 14 describes complementation of yeast CKI mutants with human CKI isoforms; and Example 15 is directed to generation of monoclonal antibodies against peptide fragments of human CKIαHu isoforms. The following examples are intended to illustrate but not limit the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.

Example 1 Isolation of hrr25

S. cerevisiae strain K264-5B (MAT ho ura3 canl^R tyrl his7 lys2 ade5 metlS trp5 leul ade5) was employed for the mutant isolation. The yeast were transformed according to standard procedures with a Z7R_43-based integrating plasmid that contained a GAL1, 10-regulated HO endonuclease and a transformant was mutagenized to approximately 50% survival with ethyl methanesulfonate (EMS), as described (Current Protocols in Molecular Biology, supra). The culture was spread onto glycerol-containing rich medium (YPG, to avoid petites), colonies were allowed to form at 30°C, and plates were replicated to glucose (HO repressing) and galactose (HO inducing) media. Mutants were identified by their inability to grow on galactose. Approximately 200 mutants were chosen for initial characterization and 62 maintained the gal- phenotype through repeated single colony purification. Among these, many were not complemented by various gal mutants. The remainder (25 mutants) were surveyed for overlapping DNA repair defects by determining sensitivity to ultraviolet (UV) irradiation and to methyl methane sulfonate (MMS) . This screening method identified five alleles of known rod mutations and one new mutation. This new mutation hrr25-l (HO and/or radiation repair), presented severe defects and was studied further.

A recessive DNA repair defect is conferred by hrr25-l that includes sensitivity to MMS. Hrr25-1 strains also show sensitivity at 5-20 Krad X- irradiation similar to that observed with mutations in the radiation repair genes RAD50 and RAD52 (Cole, et al , Mol. Cell.Biol. , 9:3101, 1989). The hrr25-l strains are no more sensitive to UV irradiation than wild type and are not temperature sensitive for growth at 37 °C. Unlike hypo- and hyper-rec rod mutants which have several of the hrr25-l phenotypes, hrr25-l strains undergo normal mitotic recombination (Cole, et al, Mol. Cell.Biol , 9:3101, 1989). Spontaneous gene conversion and crossing-over were the same for homozygous hrr25-l and wild type strains. However, HRR25 is required for the correct completion of meiosis. The hrr25-l homozygotes showed less than 1% spores (tetranucleate cells) under conditions that produced 75-80% spores in an isogenic wild type strain. The hrr25-l mutation could be complemented by a number of radiation sensitive mutations (radό, 50, 52, 54, and 57) that present some of the hrr25 phenotypes, suggesting that hrr25-l is a newly uncovered rad-lϊke mutation and not one of these previously described genes. These results also indicate that HRR25 plays a role in DNA repair and meiosis, but is not specifically required for the repair of spontaneous mitotic lesions by recombination.

Example 2

Isolation of HRR25 The HRR25 gene was obtained by complementing for MMS sensitivity using a yeast genomic library constructed in the plasmid YCp50 (Rose, et al. , Gene, 60:237, 1987). An hrr25-l strain, MHML 3-36d (ura3 hrr25), was transformed by standard methods (Nickoloff, et al, J.Mol.BioL, 207:527, 1989) to uracil prototrophy, transformants were amplified on media without uracil and replicated to media containing 0.01 % MMS. Among 1200 transformants, a single MMS resistant isolate was identified. Complementation for MMS sensitivity was found to segregate with the plasmid as determined by methods known in the art. A 12 kb genomic fragment was identified and complementing activity was localized to a 3.1 kb Bar Αl-Sa . fragment by transposon mutagenesis and subcloning. This region complemented DNA repair defects as well as meiotic deficiencies. Gene targeting experiments linked this cloned region to hrr25-l. Transposon insertion mutations within the BamHI-Sali fragment replaced into the cognate HRR25 genomic locus did not complement hrr25-l for MMS sensitivity, whereas adjacent chromosomal insertions outside the complementing region segregated in repulsion when crossed against hrr25-l .

Mini-Tni 0L K transposons (Huisman, et al , Genetics, 116: 191. 1987) were used to delineate the approximate location of HRR25 on the 12 kb BamΑl-Satl fragment. Insertions located to the left hand 9 kb (of the 12 kb genomic fragment) did not inactivate complementation of hrr25-l MMS resistance compared with the un-mutagenized plasmid. Two insertions, located near an EcoRV site in the right hand 2 kb inactivated complementation. HRR25 complementation activity was localized to a 3.4 kb Sail fragment. Approximately 300 bp of this fragment (right hand side of the 12 kb) were part of the pBR322 tetracycline resistance gene (between the Bam l site of PBR322-based YCp50). The HRR25 open reading frame spans an internal region across an EcoRV site and two Bglϊl sites within the right terminal 3 kb.

The DNA sequence of the 3.1 kb fragment revealed a centrally located open reading frame of 1482 nucleotide. A transposon insertion mutation in this open reading frame inactivated HRR25 complementation whereas insertions elsewhere in the 12 kb clone did not affect HRR25 complementation. Transposon- mediated disruption of HRR25 also revealed several phenotypes not seen with hrr25-l. As expected, a Tn70-based LUK transposon insertion (Huisman, et al. , Genetics, 116: 191, 1987) into the middle of plasmid-borne HRR25 coding region inactivated complementation for MMS sensitivity. Transplacement of this insertion into the genomic HRR25 gene revealed a severe growth defect in addition to MMS sensitivity and meiotic inviability. This severe growth defect was not observed with hrr25-l strains. Wild type HRR25 strains doubled in rich media at 30 °C every 80-90 minutes whereas isogenic hrr25::LUK strains and hrr25A doubled every 9-12 hours. hrr25-l had a doubling time of 2-4 hours.

To determine whether the mutant phenotypes revealed by the hrr::LUK disruption allele represent a null phenotype, the entire HRR25 coding sequence was deleted. Briefly, deletion of the HRR25 coding sequence employed a hisG::URA3::hisG cassette (Alani, et al, Genetics, 116:541, 1988). The 3.1 kb HRR25 Sail fragment was cloned into pBluescript (Stratagene, La Jolla, CA). This plasmid was digested with BgRl and the two BglR fragments that span the entire HRR25 gene and its flanking sequences were deleted. Into this deletion was introduced the 3.8 kb BamHI-figffl hisG::URA3::hisG fragment from pNKY51 to create the hrr25A allele. Sail digestion yielded a linearized fragment that deleted the entire HRR25 locus. Yeast carrying the deletion-disruption allele (hrr25A) showed phenotypes identical to those with the hrr25::LUK allele for all properties examined, including MMS sensitivity, slow growth, and the sporulation defect, indicating that wild-type HRR25 protein is associated with these processes and that the hrr25::LUK allele does not indirectly interfere with DNA repair, growth or sporulation. In direct parallel comparisons, the hrr25::LUK and hrr25A alleles behaved identically. Yeast strain MFH14 (MATa/MATot ura3/ura3) was transformed with JSgfll-linearized YCp50-HRR25: :LUK to uracil prototrophy, heterozygous disruption of HRR25 was verified by Southern blot analysis, the diploid was sporulated by starvation for nitrogen and fermentable carbon sources, tetrads dissected and cells allowed to germinate at 30 °C for 7 days. After a normal germination period of 2 days, the severe growth defect of hrr25: :LUK suggested that the deletion of HRR25 was lethal. However, microscopic examination of segregants revealed that hrr25: :LUK germinating cells grew slowly and in every case examined (20/20 tetrads), slow growth, MMS sensitivity, and uracil prototrophy co-segregated. A color variation was seen with diploid MFH14 segregants, due to mutations in adenine biosynthesis. MFH14 is ade5/ADE5 ade2/ade2. An ade5/ade2 strain was white, while an ADE5/ade2 strain was red.

Example 3

Sequence and Structure of the HRR25 GENE

DNA sequencing of both strands of the HRR25 gene was done by uni-directional deletions employing Sequenase (USB, Cleveland, OH) and Exo-

Meth (Stratagene, La Jolla, CA) procedures as described by the manufacturers.

DNA and deduced amino acid sequences are set out respectively in SEQ. I.D.

NOs. 1 and 2. Figure 1A, shows the alignment of the amino acid sequences for

HRR25, CDC28, KSS1, and RAFl. Figure IB shows a schematic representation of the structure of HRR25. The protein kinase homology is represented by a shaded region while the P/Q rich region is indicated by cross-hatchings. The mutant, hrr25, can be distinguished from HRR25 by one amino acid substitution.

At position 151, an aspartic acid is substituted for glycine.

The predicted translation product of HRR25 revealed an unexpected feature for a rad-lϊke, DNA repair function. HRR25 contains the hallmark signatures of sequence homology with the catalytic domain of serine/threonine protein kinase superfamily members (Hanks, et al , Science, 241:42. 1988). For comparison, the HRR25 translation product was aligned with the catalytic domains for two subgroups of yeast protein kinases, the CDC28/cdc2 group and the KSS1/FUS3 group. Located between amino acids 15 and 30 is a region that contains the conserved GXGXXG region. Just C-terminal to this region is a conserved lysine and glutamic acid present in most known kinases. These regions are thought to function in the nucleotide binding and phosphotransfer steps of the kinase reaction (Hanks, et al, Science, 24*1:42,1988). Between amino acid residues 120 to 150 are regions containing the HRD and DFG motifs, also found in most protein kinase family members. In addition, sequence examination of all known serine/threonine kinases indicates that HRR25 shares some additional similarities with the Raf/PKS/mos subgroup (Hanks, et al, Science, 241:42. 1988). The strongest homologies can be found in areas around the GXGXXG, DFG, and DXXSXG conserved regions in protein kinase catalytic domains. The functional relevance of the observed sequence similarity between HRR25 and protein kinases was studied by altering specific residues within the HRR25 kinase domain and examining the phenotypic consequences of these changes. A lysine at position 38 (Lys ³⁸) was mutated to an arginine residue by site directed mutagenesis, by methods known in the art. The mutagenic oligonucleotide SEQ. I.D. NO. 22 was:

5 '-CCTGATCGATTCCAGCCTGATCGCTACTTCTTCACCACT-3 ' .

Lys³⁸ in HRR25 corresponds to the lysine found in all known protein kinases, and this subdomain is involved in ATP binding. Mutations at the conserved lysine in protein kinases such as v-src, v-mos, and DBF2 inactivate these proteins. The mutant /zrr25-Lys³⁸ allele was incapable of complementing hrr25-l , hrr25::LUK, and hrr25A alleles for all properties examined, an indication that the HRR25 kinase domain is required for in vivo function of HRR25.

The predicted HRR25 translation product (SEQ. I.D. NO. 2) has a number of notable features outside the region of homology to protein kinase catalytic domains. For example, the last 100 amino acids is proline and glutamine rich, containing 50 of these residues. Other proteins with regions rich in these two amino acids include the transcription factors Spl, jun, and HAP2, steroid hormone receptors, the S. pombe rani kinase, and mak-m^~ & germ cell-associated kinase (Courey, et al , Cell, 55:887, 1988; Bohmann, et al , Science, 238: 1386, 1987; Roussou, et al, Mol. Cell.Biol , 8:2132, 1988; Arriza, et al, Science, 237:268, 1987; Matsushime, et al, Mol. Cell.Biol , 10:2261, 1990). In the case of Spl and jun, the proline-glutamine regions are involved in transactivation, whereas the P/Q region in the human mineralocorticoid receptor is thought to serve as an intramolecular bridge. This proline-glutamine region in HRR25 might function as a structural feature for substrate interaction, or for subcellular localization. Also, the glutamine richness of this region is similar to the opa or M-repeat seen in the Drosophila and Xenopus Notch/Xotch proteins (Wharton, et al , Cell, 40:55, 1985; Coffman, et al, Science, 249: 1438, 1990). The function of the opa repeat is not certain, but it is found in several Drosophila genes. Lastly, the sequence TKKQKY at the C-terminal end of the region homologous to protein kinases is similar to the nuclear localizing signal of SV40 large T antigen and yeast histone H2B (Silver, et al , J. Cell.Biol , 109:983, 1989; Moreland, et al , Mol. Cell.Biol , 7:4048, 1987).

Example 4 Microscopic Analysis of Germinating and

Proliferating hrr25 Cells Photomicrographs of HRR25 and hrr25::L UK colonies were taken after germination on rich medium. An MFH14 hrr25::LUK heterozygous transformant was dissected onto a thin film of YPD rich medium on a sterilized microscope slide and segregants were allowed to germinate under a coverslip by incubating the slide in a moist 30 °C chamber. Photographs of colonies were taken after 2 days of growth. Phase contrast and DAPI staining of proliferating HRR25A and hrr25::LUK cells were compared. Cells were inoculated into YPD rich medium and grown at 30°C to a mid-log density of 1-3 X 10⁷ cells/ml, briefly sonicated to disrupt clumps, fixed with formaldehyde, and stained with DAPI (Williamson, et al, Meth. Cell.Biol , 12:335, 1975). Many cells with hrr25::LUK lacked DAPI stainable nuclei.

Microscopic examination of germinating and actively growing mid- log phase hrr25::LUK cells revealed aberrant cellular morphologies. Transposon disruption of HRR25 resulted in large cells, and 25-40% of cells were filamentous or extended. DAPI nuclear staining (Williamson, et al. , Meth. Cell.Biol. , 12:335, 1975) of mid-log populations showed that orderly cell cycle progression in hrr25 mutants was lost. There were a large number of cells lacking DAPI-stainable nuclei which, by single cell manipulations proved to be inviable. Consistent with this nuclear segregation defect, the plating efficiency of hrr25::LUK haploids was also reduced to 75-80% of wild type. However, this reduction in plating efficiency is insufficient to account for the severe growth rate reduction. Plating efficiency was measured from mid-log phase cells by comparing the efficiency of colony formation on rich medium relative to the total number of cells determined by hemocytometer count. Cell populations were analyzed for DNA content distribution by flow cytometric analysis following staining with propidium iodide as described (Hutter, et al. J.Gen.Microbiol , 113:369, 1979). Cell sorting analysis showed that a large number of the cells in a haploid hrr25::LUK population were delayed in the cell cycle and exhibited G2 DNA content, but the population was not arrested uniformly in the cell cycle.

Example 5 Sequence Comparison of HRR25 with CDC28. KSS1. and RAFl The predicted translation product of HRR25 (SEQ. I.D. NO. 2) was compared with the catalytic domains of several members of the serine/threonine protein kinase superfamily. Initial sequence comparisons employed the UWGCG programs (Devereux, et al, Nuc. Acids. Res. , 12:387, 1984), whereas subgroup comparisons used the methods of Hanks, et al. , supra. HRR25 contains all eleven subdomains described by Hanks, et al , supra. Structurally similar groupings were compared in the sequence comparisons. These included nonpolar chain R groups, aromatic or ring-containing R groups, small R groups with near neutral polarity, acidic R groups, uncharged polar R groups, and basic polar R groups. CDC28 and KSSl represent members of two subgroups of serine/threonine protein kinases in yeast. CDC28 is involved in cell cycle regulation while KSSl acts in the regulation of the yeast mating pathway. HRR25 shows 21 % identity and 41 % similarity to CDC28 and 19% identity and 43% similarity to KSSl (Figure 1A). HRR25 shows highest similarity to members of the Raβ/PKS/Mos family of protein kinases. Through the catalytic domain, HRR25 shows 30% identity and 49% similarity to Rafl.

Example 6

Identification. Isolation, and Analysis of Sc. pombe Hhpl+ and Hhp2+ Genes

A. Isolation of the Hhpl+ and Hhp2+ Genes

The clones were isolated by a two-pronged approach: i) DNA- based screening methods; and ii) direct complementation in S. cerevisiae hrr25 mutant strains. Two genes were identified (Hhpl + and Hhp2+ - so named for HRR25 Homologue from Schizosaccharomyces pombe). Expression of Hhpl + in S. cerevisiae hrr25 mutants fully rescued all mutant defects. Expression of Hhp2+ in S. cerevisiae also rescued, to varying degrees, the defects associated with hrr25 mutations.

DNA-based amplification of HRR25-like DNAs from Sc. pombe genomic and CDNA sequences prepared according to Fikes, et al. (Nature, 346:291-293. 1990) was conducted using polymerase chain reaction with the following partially degenerate oligonucleotide primers:

(1) Primer No. 4583 (SEQ. ID. NO. 13) representing top strand DNA encoding residues 16 through 23 of HRR25; [1 nmol/5 μl], T_m = 52°C; (2) Primer No. 4582 (SEQ. ID. NO. 14) representing top strand DNA encoding residues 126 through 133 of HRR25; [1.5 nmol/5 μl], T_m = 54°C;

(3) Primer No. 4589 (SEQ. ID. NO. 15) representing bottom strand DNA encoding residues 126 through 133 of HRR25;

[0.5 nmol/5 μl], T_m = 54°C;

(4) Primer No. 4590 (SEQ. ID. NO. 16) representing bottom strand DNA encoding residues 194 through 199 of HRR25; [2 nmol/5 μl], T_m = 38°C.

Two series of amplifications were conducted using Perkin Elmer

Automated apparatus; a first series using HRR2J-based primer Nos. 4583 and 4589 and a second series employing all four of the primers. In the first series, 30 cycles of denaturation (94 °C, 1 min), annealing (48 °C, 1 min), and extension (66°C, 3 min) were performed and in a final cycle, the extension time was extended to 5 min. Reaction products were sized on an agarose gel revealing a prominent band of the expected size of about 306 bp. In the second series of amplifications, 30 cycles were carried out as above except that annealing and extension were carried out at 35 °C and 60°C, respectively. Three major products of the expected sizes (513 bp, 180 bp, and 306 bp) were developed in both genomic and CDNA libraries and were purified by preparative agarose gel electrophoresis.

Products were cloned into M13mpl9 and sequenced by the dideoxy method (Maniatis, et al , Molecular Cloning: A Laboratory Manual, 1982). Two classes of sequences were identified. A representative clone from each class was radiolabelled with ³²P by random primed cut labeling to a specific activity of 10⁶ cpm/μg (Maniatis, et al. , supra) and used as a hybridization probe to isolate full length CDNA clones and to prove yeast genomic DNA in Southern blots and total RNA on Northern blots. Hybridization was carried out for 16 hours in a buffer containing 6 x SSPE, 0.1 % SDS, 5% dextran sulfate. Two genes were identified and designated Hhpl + and Hhp2+ for HRR25 Homologues from Sc. pombe.

For Hhpl+, 1 clones were identified (6 partial and 1 full length clone). For Hhp2+, 2 full length clones were identified. Both Southern and Northern analysis confirmed that these clones were from separate genes. These genes were sequenced using standard dideoxy method (Maniatis, et al , supra). The nucleotide and deduced amino acid sequences for Hhpl + are set out in SEQ. ID. NOS. 3 and 4; the nucleotide and deduced amino acid sequences for Hhp2+ are set out in SEQ. ID. NOS. 5 and 6.

B. Functional analysis of Hhpl + and Hhp2+ in S. cerevisiae hrr25 mutants.

Sc. pombe Hhpl+ and Hhp2+ cDNAs were cloned in a location which placed them under the control of the S. cerevisiae alcohol dehydrogenase-1

(ADH1) promoter in a URA3-based vector pDB20 to allow for expression in S. cerevisiae (Fikes, et al , supra). These resulting clones were analyzed for their ability to alter/modify the suppress phenotypes associated with the hrr25-l mutation and the hrr25t. mutation following transformation into appropriate yeast strains by standard methods (Ito, et al , J. Bacteriol 153: 163, 1983). Transformants were analyzed for their ability to overcome defects associated with the hrr25 mutations (Hoekstra, et al , Science, 253: 1031, 1991). Hhpl -Y expression fully complemented /zrr25-associated defects and was indistinguishable from wild type HRR25 in all analyses. Complementation was analyzed for the effect on DNA repair, cell cycle progression, cellular morphology, and sporulation. Hhp2+ complemented to a lesser degree than Hhpl + (its complementation level was 50% -75% that of bonafide HRR25). The alteration of zrr25-associated phenotypes was dependent upon the transformed yeast strains containing both a complementing Sc. pombe Hhp plasmid and having hrr25 mutations.

The degree of amino acid homology between HRR25 protein and Hhpl + protein is 73% through the kinase domain. The degree of similarity, which considers the presence of similar as well as identical amino acids, is greater than 85 % . The amino acid identity of HRR25 protein and Hhp 2 + protein is 63 % with a percent similarity score of 80% . The intraspecies comparison of Hhpl + protein to Hhp2+ protein is 72% identity. This structural and complementation analysis clearly indicates that these Sc. pombe clones are functional homologues of the S. cerevisiae HRR25. Such a high degree of relatedness is not seen with any other group of protein kinases. As a measure of comparison here, bonafide functional homologues (i.e., cdc2 protein kinases from S. cerevisiae, Sc. pombe, and humans) show 40% -45% identity. Any two randomly compared protein kinases, regardless of whether the comparison is inter-or intra-species show a degree of identity of about 20% -25%.

C. Disruption and mutation of Hhpl + and Hhp2+ in Sc. pombe

Mutations that inactivate or reduce the protein kinase activity of HRR25 in S. cerevisiae result in a wide variety of phenotypes including: sensitivity to various forms of DNA damage, severe cell cycle delay, sensitivity to drugs that affect cell cycle progression (e.g., caffeine), sensitivity to agents that affect microtubule integrity (e.g., benomyl), and sensitivity to agents that affect the integrity of replicating DNA (e.g., hydroxyurea).

Similarity, in Sc. pombe, inactivation of the Hhpl + and the Hhp2+ genes to reduce or abolish the encoded protein kinase activity resulted in cellular phenotypes that mimicked hrr25 mutations. For example, deletion of the Hhpl + gene resulted in a cell cycle delay and aberrant cellular morphology, in sensitivity to DNA damaging agents like MMS, and in sensitivity to benomyl and hydroxyurea. Deletion of the Hhp2+ gene resulted in caffeine sensitivity, benomyl sensitivity, and hydroxyurea sensitivity, amongst other defects. The Hhpl + gene was disrupted as follows: CDNA was subcloned into the Sc. pombe vector pHSS19 (Hoekstra et al , Meth. Enzymol , 194:329. 1991), which was digested with Nhel-EcoRI. The Sc. pombe URA4 gene was inserted resulting in deletion of the Hhpl + kinase domain. Sc. pombe was transformed by standard methods (Moreno, et al , Meth. Enzymol , 194:795. 1991) with the linearized DNA from the resulting plasmid construction. Stable transformants were identified and haploid hhplh. strains were verified by standard methods (Moreno, et al , Maniatis, et al).

The Hhp2+ gene was disrupted as follows: the Hhp2+ CDNA was cloned into the Sc. pombe based vector, plasmid pHSS19, and was disrupted by transposon shuttle mutagenesis using the mini-Tn3 transposon mTn3Leu2 (Hoekstra, et al , Meth. Enzymol supra.). Sc. pombe was transformed by standard methods with the linearized DNA from the resulting plasmid construction. Stable transformants were identified and haploid hhp2t. strains were verified by standard methods (see above). Standard physiological methods as described for S. cerevisiae

HRR25 (Hoekstra, et al , Science 253:1031, 1991) were employed to characterize hhp mutant strains. Phenotypic analysis revealed that both hhpl and hhp2 mutants showed defects previously seen in hrr25 mutants, including sensitivity to various DNA damaging treatments that include MMS treatment and X-ray treatment. The foregoing substantiates that Hhpl + and Hhp2+ are isoforms of S. cerevisiae HRR25 protein kinase. These three protein kinases show high levels of sequence identity. In addition, mutations that inactivate these kinases result in very similar defects in widely divergent organisms.

D. Complementation of Sc. pombe mutant strains with the S. cerevisiae HRR25 gene.

To show that Sc. pombe hhp mutants prepared as described above, were identical to S. cerevisiae hrr25 mutants and to show that HRR25-like protein kinases with greater than 35 % amino acid identity are functional homologues, the S. cerevisiae HRR25 gene was introduced into a Sc. pombe expression vector and transformed into Sc. pombe hhp mutants. The DNA sequence at the HRR25 initiating methionine was changed into an Ndel site, (a silent coding alteration that maintains the reading frame but allows the HRR25 gene to be introduced into appropriate Sc. pombe plasmids). This was done by a site-directed DNA change was made in the S. cerevisiae HRR25 gene by standard methods using a commercially available system (Bio-Rad, Cambridge, MA). The altered HRR25 gene was ligated into the Sc. pombe expression plasmid, pREP 1 (Maundrell, K. J., Biol. Chem. 265: 10857, 1990), at an Ndel site and the resulting construction was transformed by standard methods into Sc. pombe hhp mutants. Expression of HRR25 in Sc. pombe mutant strains resulted in complementation of the mutant defects as evaluated by physiological methods described by Hoekstra, et al. (Science, supra).

Example 7

Isolation and Characterization of Yeast HRR25-like Genes Isolation of additional HRR25-like genes from S. cerevisiae was accomplished by performing DNA-based amplification of genomic DNA from an S. cerevisiae strain lacking HRR25 coding sequences [Strain 7D of DeMaggio, et al. (Proc. Natl. Acad. Sci. , USA, 89:7008-7012, 1992, incorporated herein by reference) thereby eliminating the chance of obtaining HRR25 sequences from the amplification. The primers and amplification conditions were as in Example 6. The resulting amplification products were cloned in M13mpl9 and sequenced by dideoxy chain termination methods. Three unique classes of amplified products were identified. Two of these products respectively corresponded to the YCK1/CKI2 and YCK2/CKI1 genes of Robinson, et al (Proc. Natl. Acad. Sci. USA, 89:28-32, 1992) and Wang, et al (Molecular Biology of the Cell, 3:275-286, 1992). The third gene product was designated "NUFl" (for Number Four). The amplified products corresponding to NUFl were radiolabelled as described in Example 6 and used to screen a yeast YCp50- based genomic library (ATCC, Rockville, MD). Eight clones were identified and one of these clones included approximately 4 Kb Hindlll fragment containing the NUFl hybridizing gene. Southern analysis revealed that NUFl is a separate gene from HRR2J, YCK1/CKI2, and YCK2/CKI1. The Hindlll fragment was sequenced and revealed a protein kinase with about 65% identity to HRR25 through its protein kinase domain. The DNA and deduced amino acid sequences for NUFl are set out in SEQ. ID. NOS. 23 and 24. To further characterize the NUFl gene, the Hindlll fragment was subcloned into the yeast plasmid YEplacll2 [Gietz and Sugino, Gene 74:521-541 (1988)]. The resulting construct was transformed into the hrr25Δ deletion strain 7d and NUFl was found to complement for 1__T25Δ mitotic defects (e.g., NUFl complemented for slow growth defect, aberrant morphology defect, DNA damaging agent sensitivities). Furthermore, a null mutant allele of NUFl was constructed by transposon shuttle mutagenesis and strains lacking the NUFl gene product were found to have hrr25Δ mutant-like defects. In particular, like hrr25Δ mutants, NUFl mutants showed slower mitotic growth rates and increased sensitivity to DNA damaging agents like MMS, UV, and X-irradiation.

Example 8 Identification and Isolation of

Human HRR25-like Genes Oligonucleotides derived from amino acid sequences described above in Example 6 A were used to amplify cDNAS from the following sources: Arabidopsis thaliana, Drosophila melanogaster, Xenopus, chicken, mouse, rat, and human ΗeLa cells. These cDNAS were obtained from reverse transcribed mRNA (Maniatis, et al , supra) or from commercially-available cDNA libraries (Stratagene, La Jolla, CA, and Clonetech, Palo Alto, CA) Amplification products of similar migration size to those obtained from S. cerevisiae HRR25 and Sc. pombe, Hhpl - and Hhp2+ genes were observed in 1.0% Agarose gels (Maniatis, et al. , supra). This result indicated that HRR25-like genes exist in all species examined.

Isolation of full length DNAs encoding human HRR25-like protein kinases was accomplished by PCR amplification of human genomic DNA using unique sequence oligonucleotide primers based on portions of a bovine brain casein kinase I cDNA which had been reported in Rowles, et al. (Proc. Natl. Acad. Sci. USA, 88:9548-9552, 1991) to encode a mammalian protein that was 60% homologous to HRR25 over its catalytic domain.

A variety of primers were prepared and used in pairwise fashion including: (1) Primer JH21 (SEQ. ID. NO. 17) representing bovine top strand DNA bases 47-67;

(2) Primer JH22 (SEQ. ID. NO. 18) representing bovine top strand DNA bases 223-240;

(3) Primer JH29 (SEQ. ID. NO. 19) representing bovine top strand DNA bases 604-623;

(4) Primer JH30 (SEQ. ID. NO. 20) representing bovine top strand DNA bases 623-604; and

(5) Primer JH31 (SEQ. ID. NO. 21) representing bovine top strand DNA bases 835-817.

DNA amplification with combination of oligonucleotides JH21/JH30, JH22/JH30, and JH29/JH31 were carried out for 30 cycles with denaturation performed at 94 °C for 4 min for the first cycle and for 1 min for the remaining cycle annealing at 50°C for 2 min and extension at 72°C for 4 min. Products of the expected size from the three amplifications were purified on preparative acrylamide gels and labeled with ³²P using random nick translation (to a specific activity between 7 x 10⁶ cpm/μg and 1.4 x 10⁷ cpm/μg. The labelled probes were employed as a group to screen a commercial human fetal brain cDNA library (Stratagene). Hybridization was carried out for 16 hours at 65°C in a hybridization buffer containing 3 x SSC, 0.1 % Sarkosyl, 10 x Denhart's solution and 20 mM sodium phosphate (Ph 6.8). Three washes at 65 °C in 2 x SSC, 0.1 % SDS were performed. Approximately 1.5 x 10⁶ plaques were screened on 30 plates using duplicate filters. Six strong positive clones were isolated, purified and converted to plasmid form according to procedures recommended by the supplier of the library. Restriction digestion revealed the following insert sizes for the six clones: clone 35A1, lkb; clone 35B1, 1.4kb; clone 41A1, 3.7kb; clone 42A1, >4kb; clone 47A1, 3.35kb; and clone 51A1, 2.75kb. All six inserts contained sequences which could be aligned with both the DNAs and deduced protein sequence of the bovine CKIα gene. The abbreviated, partial cDNA clones 35A1 and 35B1 were not further analyzed. Clones 41A1 and 42 A 1 were identical except for size. Clones 42A1, 51A1, and 47 A 1 were redesignated as CKIαlHu, CKIα2Hu, and CKIαθHu. The DNA and deduced amino acid sequences of the inserts are set out in SEQ. ID. NOS. 7 and 8; 9 and 10; and 11 and 12, respectively. The deduced amino acid sequence for CKIαlHu was identical to the reported bovine CKIα sequence. Table 1, below sets out differences in nucleotides between the bovine and human DNAs, numbered from the first base in the initiation codon, ATG.

TABLE 1

The CKIα3Hu DNA also includes an insertion of 84 bases at position +454 in the coding sequence providing an intermediate extension of the CKIα2Hu expression product by 28 amino acids. This DNA insert is not present in the bovine gene, but it encodes an amino acid sequence insert which Rowles, et al. designated as CKI-alpha-L. The CKIα^*2Hu and CKIα3Hu DNAs insertion at position +971 of the CKIαlHu DNA. This insertion is not found in any of the bovine sequences and encodes an extension of the 13 amino acids adjacent the carboxy terminal. The last two codons of the CKIαθHu sequences differ from any of the bovine sequences or the sequences of CKIαlHu and CKIα2Hu, causing the CKIα3Hu expression product to terminate with a lysine, rather than a phenylalanine as found in all the other bovine and human casein kinase I sequences. The 3' flanking sequence of CKIαθHu DNA differs significantly from that of CKIαlHu and CKIα2Hu.

FIGURE 2 provides an alignment of the catalytic domain amino acid sequences of HRR25-like proteins whose DNAs were isolated in the above illustrative examples, including HRR25, Hhpl + , Hhp2 + , CKIαlΗu, CKIα*2Ηu, and CKIα3Hu as well as YCK1/CKI2, and YCK2/CKI1. Note that with the exception of the CKIα3Hu intermediate insert and the carboxy terminal region inserts of CKIα2Hu and CKIα3Hu, the sequences of the three human products are identical. "Common" residues are indicated in the Figure where at least 3 of the seven residues are identical at the corresponding position (the human sequences being taken as a single sequence). Like Hhpl + and Hhp2+, the three human HRR25-like protein kinases showed very high degrees of amino acid identity to the HRR25 gene product (68%), establishing that these human clones were enzymatic isoforms of the yeast HRR25 gene. The alignment of HRR25, Hhpl+, Hhp2+, and the human complementing-like kinase isoforms show that these enzymes share a number of primary structural features that indicate that these enzymes provide comparable activities in different species. This conclusion is reached based on several lines of evidence. First, all enzymes share the common primary sequence identifiers characteristic of protein kinases. Second, the enzymes share high degrees of amino acid identity in regions of the protein kinase domain that are not conserved in unrelated protein kinases. Finally, these enzymes share regions of identity in the kinase domain which regions differ in primary sequence from other protein kinases, but are identical among the members of this isoform grouping. For example, greater than 95 % of all known protein kinases have a so-called A-P- E sequence (Alanine-Proline-Glutamate) approximately two-thirds of the way through the kinase domain. HRZ?25-like protein kinases lack the A-P-E sequence and have instead a S-I/V-N sequence (Serine-Isoleucine or Valine-Asparagine). Based on this primary sequence comparison, between known protein kinases and the protein kinases of the invention from evolutionarily divergent organisms, these enzymes of the invention are isoforms of HRR25 protein kinase.

Example 9

Comparison of HRR25 with a Casein Kinase In all eukaryotes examined, two of the major protein kinases are casein kinase I and II (CKI and CKII, respectively). These enzymes have been found in all cell types and species examined. Both enzymes recognize Ser/Thr residues in an acidic environment in the substrate. These two protein kinases are found throughout the cell and their activities have been purified from or found to be associated with cytoplasmic fractions, membranes, nuclei, mitochondria, and cytoskeleton. CKII is predominantly a nuclear enzyme, but similar studies have yet to be described for CKI.

To determine whether HRR25 gene product might function as a casein kinase, the ability of HRR25-containing immunoprecipitates to phosphorylate casein was studied. HR/?2J-containing immunoprecipitates from yeast were incubated with casein and phosphorylated proteins were examined.

Yeast extracts were prepared by physical disruption. Equal volumes of a cells were suspended in lysis buffer and acid-washed 0.5 mm beads were mixed, 30 second bursts were interspersed with 1 min on ice, and the extent of disruption was followed microscopically. Lysis buffer contained 10 Mm sodium phosphate (Ph 7.2), 150 Mm NaCl, 1% Nonidet P-40, 1 % Trasylol, 1 Mm DTT, 1 Mm benzamidine, 1 Mm phenylmethyl sulfonyl fluoride, 5 Mm EDTA, pepstatin (1 ug/ml), Pepstatin A (2 ug/ml), leupeptin (1 ug/ml), lOOmM sodium vanadate, and 50 Mm NaF. Extracts were clarified by a 100,000 x g centrifugation for 30 min. , made to 50% (vol/vol) with glycerol, frozen in liquid nitrogen, and stored at -70 degrees C. Little loss in protein kinase activity was seen in frozen extracts over several months.

Immune complex protein kinase assays were performed on the extracts according to the methods described in Lindberg, et al. (Mol. Cell. Biol 10:6316, 1991). Frozen extracts were diluted to 25% glycerol with lysis buffer or fresh extracts were used directly. Extracts were precleared with preimmune serum and protein A-Sepharose, and then treated with immune serum (obtained as described in Example 11, infra, from immunization of rabbits with E. coli- derived type-HRR2J fusion products). HRR25 kinase-containing immune complexes were precipitated with protein A-Sepharose. Immune complexes were washed four times with lysis buffer and twice with kinase buffer containing 15 Mm Ηepes (Ph 7.4), 100 Mm NaCl, and 10 Mm MgCl₂

Reaction mixtures of HRR25 immunoprecipitates and heat-treated casein (300 ng/20ul reaction volume) were incubated at 30 degrees C for 5-10 min and contained 10 uCi of gamma-³²P-ATP per 20 ul reaction volume. Reactions were stopped by the addition of SDS and EDTA, boiled in SDS/PAGE sample buffer and resolved in 10 % gels. Phosphoamino acid analysis was as described (Hunter et al , Proc. Natl.Acad. Sci. USA 77:1311, 1980).

Immunoprecipitates from HRR+ strains were able to phosphorylate casein. To verify that the appropriate amino acids were phosphorylated, the phosphoamino acid composition of the HRK25-phosphorylated casein was examined by phosphoamino acid analysis. Samples were resolved by two- dimensional electrophoresis at Ph 1.9 and Ph 3.5. Consistent with mammalian CKI specificity, serine and threonine residues were phosphorylated. HRR25 phosphorylated serine residues on casein 3-fold greater than threonine residues. Similarly, the autophosphorylation of HRR25 in immune complexes in vitro occurred on serine and threonine residues. Coupled with the high degree of sequence identity, these results suggest that HRR25 might be a CKI isoform.

To extend and confirm that HRR25 immunoprecipitates from yeast could phosphorylate casein, several experiments were performed. HRR25 immunoprecipitated from E. coli strains expressing HRR25 (See Example 11) also showed casein kinase activity, whereas E. coli extracts lacking HRR25 protein did not phosphorylate casein. HRR25-containing baculovirus constructs produced casein kinase activity in immunoprecipitates. Wild-type baculovirus-infected cells showed (0.5 % casein kinase activity under comparable conditions. The protein kinase activity from S19 cells expressing HRR25 protein was sensitive to the same conditions that reduced or inactivated the HRR25 protein activity from yeast extracts. The bservations that HR/?25-dependent casein kinase activity was present in immunoprecipitates from E. coli cells expressing wild-type HRR25, in insect cells infected with HRR25-containing baculovirus, and in wild-type but not hrr25A mutants indicated that the HRR25 gene product could function as a casein kinase and that the casein kinase activity in HRR25 protein-containing immunoprecipitates was due to HRR25 gene product.

Example 10

Analysis of Protein Kinase Activity of HRR25-\ike Proteins Because the predominant protein kinase activity in E. coli is histidine kinase, rather than serine/threonine or tyrosine kinase, those procaryotic cells provide a system for examination of HRR25-like protein kinase activities which is not compromised by presence of endogenous kinases. Both HRR25 and Ηhpl + DNAs were, therefore, expressed in the IPTG-inducible T7 gene 10-based commercial expression system (Invitrogen, San Diego, CA) using E. coli strain BL21 (DE3) which contains an IPTG-inducible T7 RNA polymerase and T7 lysozyme gene. See, DeMaggio, et al , Proc. Natl Acad. Sci. USA, 89:7008- 7012, (1991). In a first series of experiments, __.. coli ly sates were prepared by inducing mid-log phase cells with IPTG for 2 hours, pelleting the cells, and preparing extracts by a freeze-thaw method using buffers described in DeMaggio, et al, supra. Extracts were electrophoresed in polyacrylamide gels, transferred to nylon-based support membranes, and probed by Western analysis with antibodies directed against phosphotyrosine (UBI, Lake Placid, NY). These procedures revealed that HRR25 and Hhpl-Y expressing cells contained novel tyrosine phosphorylated proteins not observed in control cells (transformed with the vector alone or with kinase inactive mutants). In a second experiment, the HRR25 and Hhpl-Y -containing E. coli strains were examined for tyrosine- phosphorylated protein by a sensitive and accurate radiolabelling and phosphoamino acid procedure. To do this experiment, cells were induced with IPTG and grown in the presence of ³²P-orthophosphate. Radiolabelled extracts were prepared by the freeze-thaw method, electrophoresed in polyacrylamide gels, and the gels were examined by autoradiographic methods. Novel phosphoproteins were observed in the strains expressing HRR25 and Hhpl + , but not in the above controls. Phosphoproteins were examined by extracting and hydrolyzing the proteins from the gels using standard methods (Boyle, et al , Meth. Enzymol, 201: 110, 1991). These experiments verified that HRR25 and Hhpl+ could phosphorylate tyrosine, serine, and threonine residues on protein substrates.

Example 11

Recombinant Expression of HRR25 Products and Generation of Antibodies Thereto Two different plasmid constructions were developed for expression of HRR25 DNA in E. coli to generate immunogens useful in preparation of anti- HRR25 antibodies.

The first plasmid construction involved plasmid pATH according to Koerner et al , Meth. Enzymol. , 194:477-491 (1991). An approximately [606] base pair DNA fragment was isolated from the HRR25 open reading frame by Bgl II digestion and this fragment (which encodes amino acid residues 275-476) was ligated into pATH which had been digested with BamBI. The resulting plasmid encoded a fusion protein comprising the E. coli TrpE gene product at its amino terminus and a carboxy terminal fragment of HRR25 at its carboxyl terminus. Inclusion bodies were isolated from E. coli DH5α (Bethesda

Research Laboratories, Bethesda, MD) host cells transformed the plasmid using lysis buffers as described in Koerner et al , supra, and were purified by polyacrylamide gel electrophoresis. The gel purified materials were then employed in the immunization of rabbits by subcutaneous injection as recommended by Harlow, et al. , Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988), using gel purified products with complete Freund's adjuvant for primary injections and incomplete Freund's adjuvant for subsequent injections. Serum reactivity was followed by Western blotting against the gel purified antigen. Affinity purification of serum antibodies was effected using the E. cø/.-produced antigen immobilized on a nitrocellulose membrane support. Example 12

Isolation of CKLylHu and CKI_Υ2Hu

Additional human HRR25-like protein kinase encoding DNAs were isolated by combined DNA amplification and library screening methods. Oligonucleotides based on conserved regions in HRR25-like protein kinases were used to amplify DNA segments for use as probes in screening human a cDNA library. Redundant oligonucleotides of the sequence

5 '-GAR YTI MGI YTI GGI AAY YTI TA-3 ' (SEQ ID NO. 28) and 5 '-GTY TTR TTI CCI GGI CKI CCI AT-3 ' (SEQ ID NO. 29)

(where G, A, T, and C = standard nucleotides and R = A and G; Y = C and T; I = Inosine; M = A and C; and K = G and T) were used to amplify an approximately 540 nucleotide from a human fetal brain cDNA library (Clonetech). Amplification conditions used 200 Mm Tris.Hcl (Ph 8.2), 100 Mm KC1, 60 Mm (NH4)2SO4, 15 Mm MgC12, 1 % Triton X-100, 0.5 μM of each primer, 100 ng library DNA template, 200 μM dNTPs and 2.5 U polymerase. The reactions were performed for 30 cycles. Reactions were started with a 4 minute treatment at 94 °C and all cycles were 1 minute at 94 °C, 2 minutes at 5°C for annealing, and 4 minutes at 72 °C for extension. The amplification reaction was electrophoresed through a 1 % agarose gel and the region corresponding to approximately 540 base pairs was excised and DNA was eluted using a Nal extraction and glass powder binding (GeneClean, BiolOl, La Jolla, CA). The gel-purified fragment was ligated into Smal-digested Bluescript II SK(+) and the resulting plasmid contained a partial protein kinase domain that was used as a source of cDNA for library screening. Ten micrograms of this plasmid was digested with ZscøRI and BamHl to liberate the subcloned fragment and the reaction was electrophoresed through a 1 % agarose gel. The approximately 540 nucleotide fragment was eluted from the gel and was radiolabelled by random primed oligonucleotide directed labelling (Amersham, Arlington Heights, IL) using ³²P-dCTP as the radioactive nucleotide. The radioactive probe was used to screen a human Manca B cell lymphoma library [Wiman, et al. , Proc. Natl Acad. Sci. (USA) δi:6798-6802 (1984)] prepared in phage cloning vector λgtlO prepared as follows. Polyd(A)⁺RNA was prepared from 2.8 x 10⁸ cells of the B-cell lymphoma Manca using the "Fast Track" kit (Invitrogen). 5 μg of RNA was used for oligo d(T) primed cDNA synthesis with the cDNA Synthesis System (Gibco BRL, Burlington, Ontario, Canada); the resulting cDNA was size selected by agarose gel electrophoresis and ligated to EcoRI adapters with the Ribo Clone kit (Promega, Madison, WI). Varying amounts of the adapted cDNA were ligated to EcøRI-digested λgtlO with 1 unit of T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN) in a commercially prepared buffer supplied by the manufacturer with the enzyme. The ligations were packaged with Gigapack packaging extracts (Stratagene) and the resulting phage pool (1.5 x 10⁶ phage) was amplified in the C600 Hfi strain. A total of 1 x 10⁶ phage plaques were screened by standard hybridization methods (Maniatis, et al , supra). Hybridizations were at 65°C for 18 hours in 6X SSPE (20X SSPE is 175.3 g/1 NaCl, 27.6 g/1 NaH₂PO₄.H₂O), 7.4 g/1 EDTA, pH 7.4), 100 μg/ml salmon sperm carrier DNA, 5X Denhardt Reagent (50X Denhardts is 5% ficoll, 5 % polyvinyl pyrolidone, 5% bovine serum albumin), 0.1 % SDS and 5% sodium dextran sulfate. Filters were washed four times in 0.1X SSPE, 1 % SDS. Each wash was at 65 °C for 30 minutes. Five clones were chosen for further analysis.

DNA from these phage clones was prepared using a Qiagen lambda DNA preparation kit (Qiagen, Chatsworth, CA) and human cDNA inserts were excised by EcøRI digestion. These inserts were subcloned into EcoRI-digested plasmid Bluescript II SK(+) (Stratagene) and the inserts were sequenced using an ABI 373A automated DNA sequencer. Two of the five cDNA contained near full-length cDNAS with a polyA tail and a protein kinase open reading frame. These protein kinases were most closely related to isoforms of casein kinase I were designated CKI7IHU and CKI 2HU. The DNA sequences of CKI7IHU and CKI72HU are set out in SΕQ ID NOS: 30 and 32, respectively; the deduced amino acid sequences of CKLylHu and CKI72HU are set out in SΕQ ID NOS: 31 and 33, respectively. Example 13

Isolation of CKIδHu

Human CKIδ was subcloned by first isolating the human gene from a human fetal brain library constructed in λZAPII (Stratagene). A 2.2 kb EcøRI fragment containing rat CKIδ was gel purified through 1 % agarose, isolated from the gel by Nal extraction with glass powder (BiolOl, La Jolla, CA), and radiolabelled by random primer methods (Boehringer Mannheim) using ³²P-dCTP.

This probe was used to screen 1 X 10⁶ plaques containing human fetal brain cDNA library. Plaque hybridization conditions were 3X SSC, 0.1% Sarkosyl, 10X Denhardts reagent, 50 μg/ml salmon sperm DNA carrier. Hybridization was allowed to proceed for 18 hours at 65 °C after which time the filters were washed

4 times for 30 minutes each at 65°C in 2X SSC, 1.0% SDS. Positive clones were identified by autoradiography at -70 °C with an enhancing screen and sequenced using an automated ABI373A DNA sequencer (Applied Biosy stems, Foster City, California)..

One clone was determined to encode a full length CKIδ isoform and was designated CKIδHu. The nucleotide sequence for CKIδHu is set out in SEQ

ID NO: 34, and the deduced amino acid sequence is set out in SEQ ID NO: 35.

Expression of the CKIδHu isoform was then determined in eight different human tissues using an approximately 1.2 kb EcøRI fragment as a probe.

, CKIδHu mRNA levels were highest in kidney, liver and placenta cells, in contrast to the testes-specific expression of rat CKIδ demonstrated by Graves, et al, [supra].

Table 2 - Sequence Homology Between CKI Isoforms

HRR25

HRR25 100

Human CKIαl

Human CKI7I

Human CKI 2

Human CKIδ

Example 14

Complementation of Yeast CKI Mutants by Human CKI Genes In order to determine if CKI7IHU was an isoform of yeast HRR25- like protein the gene was expressed in yeast protein kinase mutants. The cDNA was expressed under control of the yeast GALl promoter. The expression plasmid was a derivative of plasmid pRS305 (Stratagene) that contains the yeast GALl promoter. The parental plasmid with the GALl promoter was previously described [Davis et al , Cell 57:965-978 (1990)] and contained a Bglll site adjacent to the GALl promoter as well as Bamϋl and Sαcl sites adjacent to the BgRl site. This plasmid was modified by site-directed mutagenesis to contain a unique Ncøl site between the GALl promoter and the BgRl site. The Ncøl site was adjacent to the GALl promoter such that the order of genetic elements was GALl promoter-NcøI-_3gπi-j3flmHI-SαcI. Site-directed mutagenesis (MutaGene kit, BioRad) employed the oligonucleotide

5'-CTA GAT CTA GCT AGA CCA TGGTAG TTT TTT CTC CTT GAC G-3' (SEQID NO.36) and generated a unique Ncol site (underlined in SEQ ID NO: 36). The resulting plasmid was called pRS305(N) 2μ GALl.

To clone CKPylHu into pRS305(N) 2μ GALl , the CKI7IHU cDNA was amplified from cDNA with oligonucleotides that would introduce an Ncol site at the initiating ATG and a BamHl site in the 3' untranslated region. The sequence of the mutagenic oligonucleotide (with the Ncol site underlined) for the amino terminus was

5'-CAT GCC ATG GCA CGA CCT AGT-3' (SEQID NO: 37).

The oligonucleotide M13rev, purchased from Stratagene (Stratagene, La Jolla, CA) was used to introduce the BamRl site in the 3' untranslated region. Amplification conditions used 200 Mm Tris-HCl (Ph 8.2), 100 Mm KC1, 60 mM (NH₄)₂SO₄, 15 mM MgCl₂, 1 % Triton X-100, 0.5 μM of each primer, 100 ng template, 200 μM of each dNTP and 2.5 units polymerase. The reactions were performed for 30 cycles. Reactions were started with a 4 minute treatment at 94°C and all cycles were 1 minute at 94°C for denaturing, 2 minutes at 50°C for annealing, and 4 minutes at 72 °C for extension. The amplified product was digested with Ncol and BamHl and was cloned into NcoI BαmHI-digested pRS305(Ν) 2μ GALl.

Complementation of yeast CKI mutants employed yeast strains 7D (hrr 25 A, ura3-l, trpl-1, leu2-3, 112, his3-ll,15, canl-100, ade2-l) [DeMaggio, et al , (1992) supra] and YI227 (ckilD, cki2D, FOA^R, ade2-l, canl- 100, his3-ll,15, leu2-3,12, trpl-1, ura3-l, pRS415::Ckilts) Strain 7D lacked the HRR25 isoform of yeast CKI and strain YI227 contained a temperature sensitive allele of yeast CKII . Yeast strains were transformed by lithium acetate- mediated transformation methods and transformants were selected on SD-leucine medium (BiolOl). Controls for transformation were plasmids pRS305(N) 2μg GALl alone, plasmid pRS315 (Stratagene), and plasmid pRS315::HRR25, which contains a Sall-EcoRI genomic fragment that spans the genomic HRR25 fragment [Hoekstra et al, Science, supra]. Plasmid pRS315::HRR25 was constructed by ligating a SaR/EcόKL genomic fragment of HRR25 into Sα/I/£coRI-digested pRS315. Both HRR25 and CKJ ylHu, when expressed in yeast mutants, are capable of fully complementing for the temperature-sensitive growth defect of CKI. In addition, CKI7IHU partially suppressed a severe growth rate defect associated with HRR25 mutants. The partial suppression of HRR25 growth defects by CKI7IHU was detected by a 10-20 fold greater plating efficiency relative to pRS305(N) 2μ GALl.

To extend the complementation analysis to additional CKI family members, the ability of other human CKIαHu and CKIδHu genes to complement for the HRR25 mutant defects was examined. Human CKIcdHu was subcloned into plasmid pRS305(N) 2μ GALl by first introducing an Ncol site at the initiating methionine by site-directed mutagenesis. The mutagenic oligonucleotide (with the Ncol site underlined) was

5'-CTA GAT CTA GCTAGA CCATGGTAGTTTTTT CTC CTT GAC G-3' (SEQIDNO.38)

and mutagenesis was performed using the Mutagene kit (BioRad). The mutagenized cDNA was digested with Ncol and BgRl and the CKIc Hu fragment was ligated into pRS305(n) 2μ GALl.

Two constructs containing the CKIδHu cDΝA were examined for complementation. Plasmid pEC7B (containing CKIδHu cDΝA) was used as a template for site-directed mutagenesis (MutaGene, BioRad). The mutagenic oligonucleotide

5'-GAATCG GGC CGC CGA GAT CTC ATA TGG AGC TGA GAGTC-3' (SEQID NO: 39)

was used to introduce BgRl (underlined in SEQ ID NO: 39) and Ndel (in italics in SEQ ID NO: 39) sites at the initiating ATG of CKIδHu. One plasmid construction employed _3gZII/StzcI-digested CKI DNA from the mutagenized cDNA that was ligated into 5g/II/S cI-digested pRS305(N) 2μ GALl to produce pRS305(CKIδ). The second plasmid construct employed NcoI/SαcI-digested CKIδHu cDΝA from unmutagenized pEC7B cDΝA that was ligated into NcoI/SαcI-digested pRS305(Ν) 2μ GALl to produce pRS305(N)(CKIδ). Plasmid pRS305(CKIδ) contained the nucleotides

5 '-CCC GGA TCT AGC AGA TCT CAT-3 ' (SEQ ID NO: 40)

between the GALl promoter and the initiating methionine of CKIδ. Plasmid pRS305(N)(CKIδ) had a near-perfect fusion between the initiating methionine of CKIδHu and the 3' end of GALl . Near perfect fusion indicates that the promoter and initiating methionine codon have few or no intervening nucleic acid sequences, and therefore are approximately abutting.

The CKIαlHu and CKIδHu-containing plasmids were transformed into yeast strains 7D and YI227 and were examined for their ability to complement for their mutant defects. Like CKJγHu, CKIαlHu partially complemented the growth defect associated with HRR25 mutations. CKIδHu was able to complement for the growth defect of temperature-conditional CKI strains, for the growth defect of HRR25 mutants, and for the DNA repair defect of HRR25. The ability of CKIδHu to complement for mutant defects in these yeast strains was indistinguishable from yeast HRR25 or CKI genes only when the appropriate plasmid construct was employed. Plasmid pRS305(CKIδ), which contained the additional 21 bases was unable to complement for any mutant phenotypes, while the near-perfect fusion in pRS305(N)(CKIδ) was fully functional. This difference was attributed to the inability of yeast to translate extended and/or CG rich leader sequences. Example 15

Generation of Monoclonal Antibodies A. CKIc.Hu Peptides

Monoclonal antibodies were raised against the following peptides. SEQ ID NO: 41 was derived from the common amino terminus of CKIαlHu, CKIα2Hu, and CKIα3Hu, and SEQ ID NO: 42 was derived from an internal alternative splice region in CKIα3Hu.

NH₂-ASSSGSKAEFIVGGY-COOH (SEQ ID NO: 41) NH₂-RSMTVSTSQDPSFSGY-COOH (SEQ ID NO: 42)

These peptides were initially each coupled to bovine gamma globulin (Sigma, St Louis, MO). Five mg of gamma globulin and 5 mg of peptide were resuspended in 0.4 ml 100 mM K₂HPO₄ (pH 7.2) and to this mixture, 35 mg l-ethyl-3(3- dimethylamino propyl)-carbodiimide-HCl (EDC, Pierce) previously dissolved in 50 μl K₂HPO₄ (pH 7.2) was added. The reaction was allowed to proceed for 16 hr at 4°C and was quenched by addition of 0.25 ml 2 M ethanolamine and 0.25 ml acetic acid. The reaction mixture was then diluted to a final volume of 2.5 ml with PBS and desalted using Sephadex G-25M (Pharmacia) chromatography. Protein containing fractions were concentrated by centrifugal microconcentration (Amicon). Mice were then injected with 50 μg of the coupled peptide nine times over a period of 8 months. Antibody production was measured against the respective peptides by ELISA.

Fusions were performed by standard methods. Briefly, a single-cell suspension was formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640 media, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco). The cell suspension was filtered through a sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, NJ), and washed twice by centrifuging at 200 g for 5 minutes and the pellet resuspended in 20 ml serum free RPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in a similar manner. NS-1 myeloma cells, kept in log phase in RPMI with 11 % fetal bovine serum (FBS) (Hyclone, Laboratories, Inc., Logan,Utah) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the pellet was washed twice as described in the foregoing paragraph. After washing, each cell suspension was brought to a final volume of 10 ml in serum free RPMI, and 10 μl was diluted 1:100. From each dilution, 20 μl was removed, mixed with 20 μl 0.4% trypan blue stain in 0.85% saline (Gibco), loaded onto a hemacytometer (Baxter Healthcare Corp., Deerfield, IL) and cells counted.

Approximately 2 x 10⁸ spleen cells were combined with 4 x 10⁷ NS-1 cells, centrifuged, and the supernatant was aspirated. The cell pellet was dislodged by tapping the tube and 2 ml of 37°C PEG 1500 (50% in 75 mM Hepes, Ph 8.0) (Boehringer Mannheim) was added with stirring over the course of 1 minute, followed by adding 14 ml of serum free RPMI over 7 minutes. An additional 16 ml RPMI was added and the cells were centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Mallinckrodt, St. Louis, MO) and 1.5 x 10⁶ thymocytes/ml. The suspension was dispensed into ten 96-well flat bottom tissue culture plates (Corning, Essex, United Kingdom) at 200 μl/well. Cells in the plates were fed 2-3 times between fusing and screening by aspirating approximately half of the medium from each well with an 18 gauge needle (Becton Dickinson), and replenishing plating medium described above except containing 10 units/ ml IL-6 and lacking thymocytes.

Fusions were screened when cell growth reached 60-80% confluency (usually 7-9 days). Fusion 75 was screened by ELISA on either the common amino terminal peptide (SEQ ID NO: 41) or the internal peptide (SEQ ID NO: 42), and fusion 80 was screened on the amino terminal peptide (SEQ ID NO: 41) only. Immulon 4 plates (Dynatech, Cambridge, MA) were coated at 4°C overnight with 100 ng/well peptide in 50 mM carbonate buffer, Ph 9.6. Plates were washed three times with PBS containing 0.05 % Tween 20 (PBST) and 50 μl culture supernatant was added. After incubation at 37 °C for 30 minutes, and washing as above, 50 μl horseradish peroxidase conjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove, PA) diluted 1:3500 in PBST was added. Plates were incubated as above, washed four times with PBST and 100 μl substrate, consisting of 1 mg/ml o-phenylene diamine (Sigma) and 0.1 μl/ml 30% H₂O₂ in 100 mM citrate, pH 4.5, was added. The color reaction was stopped in 5 minutes with the addition of 50 μl of 15 % H₂S0₄. Absorbance at 490 nm was read on a plate reader (Dynatech).

Three wells from each fusion (designated 75D3G, 75C10H, 75C2g, 80G10H, 80H4F, and 80J9E) were cloned two to three times, successively, by doubling dilution in RPMI, 15% FBS, lOOμM sodium hypoxanthine, 16 μM thymidine and 10 units/ml IL-6. Wells of clone plates were scored visually after 4 days and the number of colonies in the least dense wells were recorded. Selected wells of each cloning were tested by ELISA as above. In the final cloning, positive wells containing single colonies were expanded in RPMI with 11 % FBS. Three antibodies were determined to be reactive for the peptide raised against the amino terminus of CKIαHu (80 G10H11D, 80 H12F12B, and 80 J9E10C), and three antibodies were reactive with the peptide raised against the internal fragment of CKIα3Hu (75 D3G10A, 75 C10H1D, and 75 C2G11F). Clones 75D3G, 75C10H, 75C2G, and 80G10H were isotyped to be IgGl, clone 80H4F IgG3, and 80J9E IgG2a.

B. CKIHu/Thioredoxin Fusion Proteins

Expression plasmids were constructed in order to express the CKIHu isoforms as fusion proteins with thioredoxin. Specifically, the coding sequence for each isoform was amplified by PCR with primers which created a 5 'Xbal restriction site and a 3 'BamHl site. The primer used to create the Xbal site for the CKIαHu isoforms is set out in SEQ ID NO: 43 with the Xbal site underlined.

5'-T ACA TCT AGA ATT ATG GCG AGTAGC AGC GGC-3' (SEQ ID NO: 43) The primer used to create the 3 'BamHl site in the CKIαlHu coding sequence is set out in SEQ ID NO: 44, with SαmHI site underlined.

5 '-AAT GGA TCC TTA GAA ACC TGT GGG GGT-3 ' (SEQ ID NO: 44) The primer used to create the BamHl site in the CKIα2Hu and CKIα3Hu coding sequences is set out in SEQ ID NO: 45, with the BamHl site underlined.

5 '-AAT GGA TCC TTA GAA ACC TTT CAT GTT ACT CTT GGT-3 ' (SEQ ID NO: 45)

The Xbal and BαmHI sites were created in the CKIδHu coding sequences with primers set out in SEQ ID NOS: 46 and 47, respectively.

5'-TACATCT AGAATT ATG GAG CTGAGA GTC GGG-5' (SEQID NO: 46)

5'-GGATCC TCA TCG GTG CAC GAC AGA CTG-3' (SEQIDNO:47)

The primers used to create the Xbal and JSαmHI sites in the coding regions of the CKI7HU isoforms are set out in SEQ ID NO: 48 and 49.

5'T ACA TCT AGA ATT ATG GCA CGA CCT AGT GGT CGATCG-3' (SEQID NO: 48)

5'-G GGG ATC CTA CTT CAGTAG GGG CTG-3' (SEQID NO: 49)

Digestion of the resulting PCR products with Xbal and BamHl allowed the fragments to be directionly cloned in frame at the carboxy terminus of sequences encoding thioredoxin in plasmid pTRXFUS [LeVallie, et al, Nature/Biotechnology 77: 187-193 (1993)]. The resulting expression constructions contained the lag Iq gene, followed by the tacll promoter (from plasmid pMal-c2, New England Biolabs, Beverly, MA) which drives expression of the E.coli thioredoxin gene fused at the amino termini of the CKI catalytic domains.

E.coli XL-1 Blue cells (Stratagene) were transformed with the individual expression plasmids by standard methods and grown at 37 °C to mid-log phase. Samples were collected to serve as controls for uninduced cells and the remaining cells were induced for four hours with 0.25 mM IPTG at 37°C. Cells were then lysed and inclusion bodies in the insoluble extract from cleared lysate were used to inject mice.

C. Other CKI Peptides Monoclonal antibodies were also raised against other CKI peptides coupled to bovine gamma globulin as in section A of this example. Peptides derived from the amino termini of the CKLyHu isoforms are set out in SEQ ID NOS: 50 and 51; peptides derived from the amino termini of bovine CKIβ [Rowles, et al , supra] are set out in SEQ ID NOS: 52 and 53; peptides derived from the amino terminus and carboxy terminus of CKIδHu are set out in SEQ ID NOS: 54 and 55, respectively; a peptide derived from the carboxy termini of CKIα2Hu and CKIα3Hu is set out in SEQ ID NO: 56; and a peptide common to all CKIHu isoforms is set out in SEQ ID NO: 57. The common CKI sequence set out in SEQ ID NO: 57 was also injected into rabbits to produce polyclonal antisera.

NH₂-RSGHNTRGTGSS-COOH (SEQ ID NO: 50)

NH₂-RLGHNTRGTGSS-COOH (SEQ ID NO: 51)

NH₂-SSRPKTDVLVG-COOH (SEQ ID NO: 52)

NH₂-KSDNTKSEMKHS-COOH (SEQ ID NO: 53) NH₂-GTDIAAGE-COOH (SEQ ID NO: 54)

NH₂-ERRDREERLR-COOH (SEQ ID NO: 55)

NH₂-TGKQTDKTKSNMKGY-COOH (SEQ ID NO: 56)

NH₂-DLLGPSLEDLFGY-COOH (SEQ ID NO: 57) Mice were injected with 50 μg of the peptide/gamma globulin complex on a varying schedule over a period of eight months.

Subsequent to the filing of U.S. Patent Application Serial No. 07/728,783 on July 3, 1991, there have been numerous reports in the scientific literature of the isolation of DNAs encoding H7?R25-like proteins. For example, Rowles, et al, (Proc. Natl. Acad. Sci. USA, 88:9548-9592, 1991) reported the purification of a bovine thymus casein kinase I (CKI) enzyme. The sequencing of tryptic fragments reveled nearly 25 % of the primary sequence of the enzyme. PCR cloning resulted in development of partial clones coding for the CKI enzyme isolate and a homologue enzyme referred to as CKI-δ. Screening of bovine brain libraries with the partial clones yielded full length cDNAs for the CKI isolate (designated CKIα) and two additional homologues (CKI/3 and CKI7). The deduced sequence for bovine CKIα was noted by Rowles, et al, [supra] to be 60% homologous to HRR25 over its catalytic domain. As noted earlier, a comparison of the bovine CKIα sequence of Rowles, et al to human CKI l sequence set out in SEQ. ID. NO. 7 and 8 reveals 100% homology in the catalytic domain.

As another example, Robinson, et al. (Proc. Natl. Acad. Sci. USA, 89:28-32, 1992) describes the isolation of two Saccharomyces cerevisiae genes, YCKl and YCK2 which encode yeast casein kinase 1 homologues and also describes purification and partial sequencing of a rabbit casein kinase I from a rabbit reticulocyte lysate preparation. HRR25 was noted to be 50% homologous to YCKl and YCK2 and 60% homologous to the partial rabbit CKI sequence. As a further example, Wang, et al. (Molecular Biology of the Cell, 3:275-286, 1992) describes the isolation of a 54 kDa CKI from S. cerevisiae and the use of amino acid sequence information therefrom for cloning two yeast cDNAs encoding homologous casein kinase I proteins, CKII and CKI2. Comparison of the catalytic domains of the protein encoded by the CKII gene produced few alignments revealing greater than 20-25% homology. The closest matches were with HRR25 (50-56%) and with the three bovine isozymes of Rowles, et al. (51- 56%). The YCKl sequence of Robinson, et al. corresponds to the CKI2 sequence of Wang, et al; the YCK2 sequence corresponds to CKII. Brockman, et al. (Proc. Natl. Acad. Sci, USA, 89:9454-9458, 1992) reported the immunopurification and sequencing of a human erythroid casein kinase I and noted that it was 62% homologous to HRR25. As a final example, Graves, et al. (J.Biol Chem. 265:6394-6401, 1993) reported the cloning and characterization of a casein kinase I from rat testes. This CKI, designated CKIδ, shared 76% homology at the amino acid level with CKIα isolated from bovine brain and 65% homology with HRR25.

While the foregoing illustrative examples are specifically directed to isolation of "full length" polynucleotides encoding the H7?R25-like proteins HRR25, Ηhpl + , Hhp2+, CKIαlHu, CKIα2Hu, CKIα3Hu, CKIδHu, CKPylHu and CKI72HU, it will be readily understood that the present invention is not limited to those polynucleotides. Rather it embraces all polynucleotides which are comprehended within the class of genes encoding H7?/f25-like proteins characterized protein kinase activity and by homology of 35% or more with the HRR25 protein through the protein kinase catalytic domain. By way of example, employing information concerning the DNA sequence of HRR25, the procedures of Example 7 allowed the isolation partial cDNA clones of expected length from cDNA libraries derived from Arabidopsis thaliana, Drosophila melanogaster, Xenopus, chicken, mouse, rat, and human species. These partial cDNAs may, in turn, be employed in the manner of Examples 6 and 7 to isolate full length DNA clones encoding H_?R25-like proteins from these species. Each of these may be employed in the large scale production of the corresponding proteins by recombinant methods or for the generation of other useful polynucleotides such as antisense RNAs. Recombinant expression products of such HRR25-like DNAs may be employed for generation of antibodies and in screens for compounds which modulate the protein kinase and/or recombination/repair functions of these enzymes. Moreover, as suggested in the publication of Rowles, et al, Robinson, et al, and Wang, et al. , multiple H7y_25-like isozymes are expected to exist in a variety of eukaryotic species as both membrane bound and cytoplasmic proteins. It appears reasonable to expect that a number of genes and gene products exist in human species, all of which are functionally related as well as structurally related to each other and to HRR25.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

SUMMARY OF SEQUENCES

SEQ ID NO: 1 is the nucleic acid sequence and the deduced amino acid of a genomic fragment encoding a yeast-derived protein kinase, HRR25 of the present invention.

SEQ ID NO: 2 is the deduced amino acid sequence of a yeast- derived protein kinase HRR25 of the present invention.

SEQ ID NO: 3 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding Hhpl + of the present invention.

SEQ ID NO: 4 is the deduced amino acid sequence of Hhpl + of the present invention.

SEQ ID NO: 5 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding Hhp2+ of the present invention.

SEQ ID NO: 6 is the deduced amino acid sequence of Hhp2+ of the present invention.

SEQ ID NO: 7 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding CKlαlHu of the present invention.

SEQ ID NO: 8 is the deduced amino acid sequence of CKIαlHu of the present invention.

SEQ ID NO: 9 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding CKlα2Hu of the present invention. SEQ ID NO: 10 is the deduced amino acid sequence of CKlα2Hu of the present invention.

SEQ ID NO: 11 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding CKl 3Hu of the present invention.

SEQ ID NO: 12 is the deduced amino acid sequence of CKlα3Hu of the present invention.

SEQ ID NO: 13 is the primer, 4583, representing top strand DNA encoding residues 16-23 of HRR25.

SEQ ID NO: 14 is the primer, 4582, representing top strand DNA encoding residues 126-133 of HRR25.

SEQ ID NO: 15 is the primer, 4589, representing bottom strand DNA encoding residues 126-133 of HRR25.

SEQ ID NO: 16 is the primer, 4590, representing bottom strand DNA encoding residues 194-199 of HRR25.

SEQ ID NO: 17 is the primer JH21 , representing bovine top strand DNA bases 47-67.

SEQ ID NO: 18 is the primer JH22, representing bovine top strand DNA bases 223-240.

SEQ ID NO: 19 is the primer JH29, representing bovine top strand

DNA bases 604-623. SEQ ID NO: 20 is the primer JH30, representing bovine bottom strand DNA bases 623-604.

SEQ ID NO: 21 is the primer JH31, representing bovine bottom strand DNA bases 835-817.

SEQ ID NO: 22 is the mutated HRR25 kinase domain primer found on p. 33, Example 3.

SEQ ID NO: 23 is the nucleic acid sequence (and the deduced amino acid sequence) of a genomic fragment encoding NUFl of the present invention.

SEQ ID NO: 24 is the deduced amino acid sequence of NUFl of the present invention.

SEQ ID NOS: 25, 26 and 27 are the conserved motifs found on page 18.

SEQ ID NOS: 28 and 29 are redundant oligonucleotides, based on conserved regions of HRR25-like proteins, used to amplify a probe from a human cDNA library.

SEQ ID NO: 30 is the nucleotide sequence of the CKI7IHU gene.

SEQ ID NO: 31 is the deduced amino acid sequence of the CKI7IHU protein.

SEQ ID NO: 32 is the nucleotide sequence of the CKI72HU gene. SEQ ID NO: 33 is the deduced amino acid sequence of the CKI72HU protein.

SEQ ID NO: 34 is the nucleic acid sequence for CKIδHu.

SEQ ID NO: 35 is the deduced amino acid sequence for CKIδHu.

SEQ ID NO: 36 is the mutagenic oligonucleotide used to generate an Ncol restriction site in expression plasmid pRS305.

SEQ ID NO: 37 is the mutagenic oligonucleotide used to generate an Ncol restriction site in CKI7I.

SEQ ID NO: 38 is the mutagenic oligonucleotide used to create an Ncol^* restriction site in human CKIαa.

SEQ ID NO: 39 is the mutagenic oligonucleotide used to introduce a BgRl restriction site in CKIδ.

SEQ ID NO: 40 is the intervening nucleic acids sequence between the GALl promoter and initiating methionine codon in the CKIδ expression plasmid.

SEQ ID NOS: 41 and 42 are amino terminal and internal peptide fragments of CKIα isoforms to generate monoclonal antibodies.

SEQ ID NO: 43 is the primer used to create a Xbal restriction site in CKIαHu coding sequences.

SEQ ID NO: 44 is the primer used to create a BamHl restriction site in the CKI lHu coding sequence. SEQ ID NO: 45 is the primer used to create a BamHl restriction site in the CKIα2Hu and CKIα3Hu coding sequences.

SEQ ID NO: 46 is the primer used to create a Xbal restriction site in the CKIδHu coding sequence.

SEQ ID NO: 47 is the primer used to create a BamHl restriction site in the CKIδHu coding sequence.

SEQ ID NO: 48 is the primer used to create a Xbal restriction site in the CKI7IHU and CKI

coding sequences.

SEQ ID NO: 49 is the primer used to create a BamHl restriction site in the CKI7IHU and CKI 2Hu coding sequences.

SEQ ID NO: 50 is an amino terminal peptide fragment of CKI7HU coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.

SEQ ID NO: 51 is an amino terminal peptide fragment of CKI7HU coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.

SEQ ID NO: 52 is an amino terminal peptide fragment of bovine CKI/3 coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.

SEQ ID NO: 53 is an amino terminal peptide fragment of bovine

CKIjδ coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice. SEQ ID NO: 54 is an amino terminal peptide fragment of CKIδHu coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.

SEQ ID NO: 55 is a carboxy terminal peptide fragment of CKIδHu coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.

SEQ ID NO: 56 is an carboxy terminal peptide fragment of CKIα2Hu and CKIα3Hu coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.

SEQ ID NO: 57 is an internal terminal peptide fragment common to all human CKI isoforms coupled to bovine gamma globulin and used to generate monoclonal antibodies in mice.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: The Salk Institute For Biological Studies (ii) TITLE OF INVENTION: Protein Kinases (iii) NUMBER OF SEQUENCES: 57

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Marshall, O'Toole, Gerstein, Murray & Borun

(B) STREET: 233 South Wacker Drive, 6300 Sears Tower

(C) CITY: Chicago

(D) STATE: Illinois

(E) COUNTRY: USA

(F) ZIP: 60606-6402

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/008,001

(B) FILING DATE: 21-JAN-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/728,783

(B) FILING DATE: 03-JUL-1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Noland, Greta E.

(B) REGISTRATION NUMBER: 35,302

(C) REFERENCE/DOCKET NUMBER: 27866/31853

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 312-474-6300

(B) TELEFAX: 312-474-0448

(C) TELEX: 25-3856

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3098 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 879..2360

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

GTCGACTCGC CAATCACCAA GTTCTTATCC CACATCCGAC CAGTGTCTGA GTCATGGTTT 60

ACCACCACCA TACCATCGCT GGTCATTTGT AAATCCGTTT CTATTACATC AGCACCTGCT 120

GCATAAGCCT TCTCAAATGC TAGTAGCGTA TTTTCAGGAT ATCTTGCTTT AAAAGCTCTG 180

TGGCCCACAA TTTCAACCAT CCTCGTGTCC TTGTTGTTAT CTTACACTTC TTATTTATCA 240

ATAACACTAG TAACATCAAC AACACCAATT TTATATCTCC CTTAATTGTA TACTAAAAGA 300

TCTAAACCAA TTCGGTATTG TCCTCGATAC GGCATGCGTA TAAAGAGATA TAATTAAAAG 360

AGGTTATAGT CACGTGATGC AGATTACCCG CAACAGTACC ACAAAATGGA TACCATCTAA 420

TTGCTATAAA AGGCTCCTAT ATACGAATAA CTACCACTGG ATCGACGATT^" ATTTCGTGGC 480

AATCATATAC CACTGTGAAG AGTTACTGCA ACTCTCGCTT TGTTTCAACG CTTCTTCCCG 540

TCTGTGTATT TACTACTAAT AGGCAGCCCA CGTTTGAATT TCTTTTTTTC TGGAGAATTT 600

TTGGTGCAAC GAGGAAAAGG AGACGAAGAA AAAAAGTTGA AACACGACCA CATATATGGA 660

ACGTGGTTGA AATACAAAGA GAAGAAAGGT TCGACACTCG AGGAAAGCAT TTGGTGGTGA 720

AAACACATCT TAGTAGCATC TTTAAACCTC TGTTGGGTAC TTAGAAAAAT ATTTCCAGAC 780

TTCAAGGATA AAAAAAGTCG AAAAGTTACG ACATATTCGA CCAAAAAAAA AAACCAAAAA 840

GAAAAGATAT ATTTATAGAA AGGATACATT AAAAAGAG ATG GAC TTA AGA GTA 893

Met Asp Leu Arg Val 1 5

GGA AGG AAA TTT CGT ATT GGC AGG AAG ATT GGG AGT GGT TCC TTT GGT 941 Gly Arg Lys Phe Arg lie Gly Arg Lys lie Gly Ser Gly Ser Phe Gly 10 15 20

GAC ATT TAC CAC GGC ACG AAC TTA ATT AGT GGT GAA GAA GTA GCC ATC 989 Asp lie Tyr His Gly Thr Asn Leu lie Ser Gly Glu Glu Val Ala lie 25 30 35

AAG CTG GAA TCG ATC AGG TCC AGA CAT CCT CAA TTG GAC TAT GAG TCC 1037 Lys Leu Glu Ser lie Arg Ser Arg His Pro Gin Leu Asp Tyr Glu Ser 40 45 50

CGC GTC TAC AGA TAC TTA AGC GGT GGT GTG GGA ATC CCG TTC ATC AGA 1085 Arg Val Tyr Arg Tyr Leu Ser Gly Gly Val Gly lie Pro Phe lie Arg 55 60 65

TGG TTT GGC AGA GAG GGT GAA TAT AAT GCT ATG GTC ATC GAT CTT CTA 1133 Trp Phe Gly Arg Glu Gly Glu Tyr Asn Ala Met Val lie Asp Leu Leu 70 75 80 85

GGC CCA TCT TTG GAA GAT TTA TTC AAC TAC TGT CAC AGA AGG TTC TCC 1181 Gly Pro Ser Leu Glu Asp Leu Phe Asn Tyr Cys His Arg Arg Phe Ser 90 95 100

TTT AAG ACG GTT ATC ATG CTG GCT TTG CAA ATG TTT TGC CGT ATT CAG 1229 Phe Lys Thr Val lie Met Leu Ala Leu Gin Met Phe Cys Arg lie Gin 105 110 115 TAT ATA CAT GGA AGG TCG TTC ATT CAT AGA GAT ATC AAA CCA GAC AAC 1 77 Tyr lie His Gly Arg Ser Phe lie His Arg Asp lie Lys Pro Asp Asn 120 125 130

TTT TTA ATG GGG GTA GGA CGC CGT GGT AGC ACC GTT CAT GTT ATT GAT 1325 Phe Leu Met Gly Val Gly Arg Arg Gly Ser Thr Val His Val He Asp 135 140 145

TTC GGT CTA TCA AAG AAA TAC CGA GAT TTC AAC ACA CAT CGT CAT ATT 1373 Phe Gly Leu Ser Lys Lys Tyr Arg Asp Phe Asn Thr His Arg His He 150 155 160 165

CCT TAC AGG GAG AAC AAG TCC TTG ACA GGT ACA GCT CGT TAT GCA AGT 1421 Pro Tyr Arg Glu Asn Lys Ser Leu Thr Gly Thr Ala Arg Tyr Ala Ser 170 175 180

GTC AAT ACG CAT CTT GGA ATA GAG CAA AGT AGA AGA GAT GAC TTA GAA 1469 Val Asn Thr His Leu Gly He Glu Gin Ser Arg Arg Asp Asp Leu Glu 185 190 195

TCA CTA GGT TAT GTC TTG ATC TAT TTT TGT AAG GGT TCT TTG CCA TGG 1517 Ser Leu Gly Tyr Val Leu He Tyr Phe Cys Lys Gly Ser Leu Pro Trp 200 205 210

CAG GGT TTG AAA GCA ACC ACC AAG AAA CAA AAG TAT GAT CGT ATC ATG 1565 Gin Gly Leu Lys Ala Thr Thr Lys Lys Gin Lys Tyr Asp Arg He Met 215 220 225

GAA AAG AAA TTA AAC GTT AGC GTG GAA ACT CTA TGT TCA GGT TTA CCA 1613 Glu Lys Lys Leu Asn Val Ser Val Glu Thr Leu Cys Ser Gly Leu Pro 230 235 240 245

TTA GAG TTT CAA GAA TAT ATG GCT TAC TGT AAG AAT TTG AAA TTC GAT 1661 Leu Glu Phe Gin Glu Tyr Met Ala Tyr Cys Lys Asn Leu Lys Phe Asp 250 255 260

GAG AAG CCA GAT TAT TTG TTC TTG GCA AGG CTG TTT AAA GAT CTG AGT 1709 Glu Lys Pro Asp Tyr Leu Phe Leu Ala Arg Leu Phe Lys Asp Leu Ser 265 270 275

ATT AAA CTA GAG TAT CAC AAC GAC CAC TTG TTC GAT TGG ACA ATG TTG 1757 He Lys Leu Glu Tyr His Asn Asp His Leu Phe Asp Trp Thr Met Leu 280 285 290

CGT TAC ACA AAG GCG ATG GTG GAG AAG CAA AGG GAC CTC CTC ATC GAA 1805 Arg Tyr Thr Lys Ala Met Val Glu Lys Gin Arg Asp Leu Leu He Glu 295 300 305

AAA GGT GAT TTG AAC GCA AAT AGC AAT GCA GCA AGT GCA AGT AAC AGC 1853 Lys Gly Asp Leu Asn Ala Asn Ser Asn Ala Ala Ser Ala Ser Asn Ser 310 315 320 325

ACA GAC AAC AAG TCT GAA ACT TTC AAC AAG ATT AAA CTG TTA GCC ATG 1901 Thr Asp Asn Lys Ser Glu Thr Phe Asn Lys He Lys Leu Leu Ala Met 330 335 340

AAG AAA TTC CCC ACC CAT TTC CAC TAT TAC AAG AAT GAA GAC AAA CAT 1949 Lys Lys Phe Pro Thr His Phe His Tyr Tyr Lys Asn Glu Asp Lys His 345 350 355

AAT CCT TCA CCA GAA GAG ATC AAA CAA CAA* ACT ATC TTG AAT AAT AAT 1997 Asn Pro Ser Pro Glu Glu He Lys Gin Gin Thr He Leu Asn Asn Asn 360 365 370

GCA GCC TCT TCT TTA CCA GAG GAA TTA TTG AAC GCA CTA GAT AAA GGT 2045 Ala Ala Ser Ser Leu Pro Glu Glu Leu Leu Asn Ala Leu Asp Lys Gly 375 380 385 ATG GAA AAC TTG AGA CAA CAG CAG CCG CAG CAG CAG GTC CAA AGT TCG 2093 Met Glu Asn Leu Arg Gin Gin Gin Pro Gin Gin Gin Val Gin Ser Ser 390 395 400 405

CAG CCA CAA CCA CAG CCC CAA CAG CTA CAG CAG CAA CCA AAT GGC CAA 2141 Gin Pro Gin Pro Gin Pro Gin Gin Leu Gin Gin Gin Pro Asn Gly Gin 410 415 420

AGA CCA AAT TAT TAT CCT GAA CCG TTA CTA CAG CAG CAA CAA AGA GAT 2189 Arg Pro Asn Tyr Tyr Pro Glu Pro Leu Leu Gin Gin Gin Gin Arg Asp 425 430 435

TCT CAG GAG CAA CAG CAG CAA GTT CCG ATG GCT ACA ACC AGG GCT ACT 2237 Ser Gin Glu Gin Gin Gin Gin Val Pro Met Ala Thr Thr Arg Ala Thr 440 445 450

CAG TAT CCC CCA CAA ATA AAC AGC AAT AAT TTT AAT ACT AAT CAA GCA 2285 Gin Tyr Pro Pro Gin He Asn Ser Asn Asn Phe Asn Thr Asn Gin Ala 455 460 465

TCT GTA CCT CCA CAA ATG AGA TCT AAT CCA CAA CAG CCG CCT CAA GAT 2333 Ser Val Pro Pro Gin Met Arg Ser Asn Pro Gin Gin Pro Pro Gin Asp 470 475 480 485

AAA CCA GCT GGC CAG TCA ATT TGG TTG TAAGCAACAT ATATTGCTCA 2380

Lys Pro Ala Gly Gin Ser He Trp Leu 490

AAACGCACAA AAATAAACAT ATGTATATAT AGACATACAC ACACACATAT ATATATATAT 2440

ATTATTATTA TTATTTACAT ATACGTACAC ACAATTCCAT ATCGAGTTAA TATATACAAT 2500

TCTGGCCTTC TTACCTAAAA AGATGATAGC TAAAAGAACC ACTTTTTTTA TGCATTTTTT 2560

TCTTCGGGAA GGAAATTAAG GGGGAGCGGA GCACCTCTTG GCCAATTTGT TTTTTTTTTA 2620

TGTAATAAAG GGCTAACGAT CGAAGATCAA TCACGAATAT TGGACGGTTT TAAAGGAGGG 2680

CCTCTGAGAA GACAGCATCA ATTCGTATTT TCGATAATTA ACTTGCCTTA TAGTGTCTGA 2740

TTAGGAAACA ATCACGAGAC GATAACGACG GAATACCAAG GAAGTTTGTG CAAATATACA 2800

GCCGGCACAA ACAGCAGCTT CACTCAGGTT AACTCACATA CTGTTGAAAA TTGTCGGTAT 2860

GGAATTCGTT GCAGAAAGGG CTCAGCCAGT TGGTCAAACA ATCCAGCAGC AAAATGTTAA 2920

TACTTACGGG CAAGGCGTCC TACAACCGCA TCATGATTTA CAGCAGCGAC AACAACAACA 2980

ACAGCAGCGT CAGCATCAAC AACTGCTGAC GTCTCAGTTG CCCCAGAAAT CTCTCGTATC 3040

CAAAGGCAAA TATACACTAC ATGACTTCCA GATTATGAGA ACGCTTGGTA CTGGATCC 3098

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 494 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Asp Leu Arg Val Gly Arg Lys Phe Arg He Gly Arg Lys He Gly 1 5 10 15 Ser Gly Ser Phe Gly Asp He Tyr His Gly Thr Asn Leu He Ser Gly 20 25 30

Glu Glu Val Ala He Lys Leu Glu Ser He Arg Ser Arg His Pro Gin 35 40 45

Leu Asp Tyr Glu Ser Arg Val Tyr Arg Tyr Leu Ser Gly Gly Val Gly 50 55 60

He Pro Phe He Arg Trp Phe Gly Arg Glu Gly Glu Tyr Asn Ala Met 65 70 75 80

Val He Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asn Tyr Cys 85 90 95

His Arg Arg Phe Ser Phe Lys Thr Val He Met Leu Ala Leu Gin Met 100 105 110

Phe Cys Arg He Gin Tyr He His Gly Arg Ser Phe He His Arg Asp 115 120 125

He Lys Pro Asp Asn Phe Leu Met Gly Val Gly Arg Arg Gly Ser Thr 130 135 140

Val His Val He Asp Phe Gly Leu Ser Lys Lys Tyr Arg Asp Phe Asp 145 150 155 160

Thr His Arg His He Pro Tyr Arg Glu Asn Lys Ser Leu Thr Gly Thr 165 170 175

Ala Arg Tyr Ala Ser Val Asn Thr His Leu Gly He Glu Gin Ser Arg 180 185 190

Arg Asp Asp Leu Glu Ser Leu Gly Tyr Val Leu He Tyr Phe Cys Lys 195 200 205

Gly Ser Leu Pro Trp Gin Gly Leu Lys Ala Thr Thr Lys Lys Gin Lys 210 215 220

Tyr Asp Arg He Met Glu Lys Lys Leu Asn Val Ser Val Glu Thr Leu 225 230 235 240

Cys Ser Gly Leu Pro Leu Glu Phe Gin Glu Tyr Met Ala Tyr Cys Lys 245 250 255

Asn Leu Lys Phe Asp Glu Lys Pro Asp Tyr Leu Phe Leu Ala Arg Leu 260 265 270

Phe Lys Asp Leu Ser He Lys Leu Glu Tyr His Asn Asp His Leu Phe 275 280 285

Asp Trp Thr Met Leu Arg Tyr Thr Lys Ala Met Val Glu Lys Gin Arg 290 295 300

Asp Leu Leu He Glu Lys Gly Asp Leu Asn Ala Asn Ser Asn Ala Ala 305 310 315 320

Ser Ala Ser Asn Ser Thr Asp Asn Lys Ser Glu Thr Phe Asn Lys He 325 330 335

Lys Leu Leu Ala Met Lys Lys Phe Pro Thr His Phe His Tyr Tyr Lys 340 345 350

Asn Glu Asp Lys His Asn Pro Ser Pro Glu Glu He Lys Gin Gin Thr 355 360 365 He Leu Asn Asn Asn Ala Ala Ser Ser Leu Pro Glu Glu Leu Leu Asn 370 375 380

Ala Leu Asp Lys Gly Met Glu Asn Leu Arg Gin Gin Gin Pro Gin Gin 385 390 395 400

Gin Val Gin Ser Ser Gin Pro Gin Pro Gin Pro Gin Gin Leu Gin Gin 405 410 415

Gin Pro Asn Gly Gin Arg Pro Asn Tyr Tyr Pro Glu Pro Leu Leu Gin 420 425 430

Gin Gin Gin Arg Asp Ser Gin Glu Gin Gin Gin Gin Val Pro Met Ala 435 440 445

Thr Thr Arg Ala Thr Gin Tyr Pro Pro Gin He Asn Ser Asn Asn Phe 450 455 460

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

Gin Pro Pro Gin Asp Lys Pro Ala Gly Gin Ser He Trp Leu 485 490

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2469 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 113..1207

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

AATATTTCAA GCTATACCAA GCATACAATC AACTCCAAGC TTCGAGCGGC CGCCAGTGTG 60

CTCTAAAGGA AAAAGCGAGT GCCTTTAGCC TTAAAAGCGT TATAATATTA TT ATG 115

Met 1

GCT TTG GAC CTC CGG ATT GGG AAC AAG TAT CGC ATT GGT CGT AAA ATT 163 Ala Leu Asp Leu Arg He Gly Asn Lys Tyr Arg He Gly Arg Lys He 5 10 15

GGC AGT GGA TCT TTC GGA GAC ATT TAT CTT GGG ACT AAT GTC GTT TCT 211 Gly Ser Gly Ser Phe Gly Asp He Tyr Leu Gly Thr Asn Val Val Ser 20 25 30

GGT GAA GAG GTC GCT ATC AAG CTA GAA TCA ACT CGT GCT AAA CAC CCT 259 Gly Glu Glu Val Ala He Lys Leu Glu Ser Thr Arg Ala Lys His Pro 35 40 45

CAA TTG GAG TAT GAA TAC AGA GTT TAT CGC ATT TTG TCA GGA GGG GTC 307 Gin Leu Glu Tyr Glu Tyr Arg Val Tyr Arg He Leu Ser Gly Gly Val 50 55 60 65 GGA ATC CCG TTT GTT CGT TGG TTC GGT GTA GAA TGT GAT TAC AAC GCT 355 Gly He Pro Phe Val Arg Trp Phe Gly Val Glu Cys Asp Tyr Asn Ala 70 75 80

ATG GTG ATG GAT TTA TTG GGT CCT TCG TTG GAA GAC TTG TTT AAT TTT 403 Met Val Met Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asn Phe 85 90 95

TGC AAT CGA AAG TTT TCT TTG AAA ACA GTT CTT CTC CTT GCG GAC CAG 451 Cys Asn Arg Lys Phe Ser Leu Lys Thr Val Leu Leu Leu Ala Asp Gin 100 105 110

CTC ATT TCT CGA ATT GAA TTC ATT CAT TCA AAA TCT TTT CTT CAT CGT 499 Leu He Ser Arg He Glu Phe He His Ser Lys Ser Phe Leu His Arg 115 120 125

GAT ATT AAG CCT GAT AAC TTT TTA ATG GGA ATA GGT AAA AGA GGA AAT 547 Asp He Lys Pro Asp Asn Phe Leu Met Gly He Gly Lys Arg Gly Asn 130 135 140 145

CAA GTT AAC ATA ATT GAT TTC GGA TTG GCT AAG AAG TAT CGT GAT CAC 595 Gin Val Asn He He Asp Phe Gly Leu Ala Lys Lys Tyr Arg Asp His 150 155 160

AAA ACT CAC CTG CAC ATT CCT TAT CGC GAG AAC AAG AAT CTT ACA GGT 643 Lys Thr His Leu His He Pro Tyr Arg Glu Asn Lys Asn Leu Thr Gly 165 170 175

ACT GCA CGC TAT GCT AGC ATC AAT ACT CAT TTA GGT ATT GAA CAA TCC 691 Thr Ala Arg Tyr Ala Ser He Asn Thr His Leu Gly He Glu Gin Ser 180 185 190

CGC CGT GAT GAC CTC GAA TCT TTA GGT TAT GTG CTC GTC TAC TTT TGT 739 Arg Arg Asp Asp Leu Glu Ser Leu Gly Tyr Val Leu Val Tyr Phe Cys 195 200 205

CGT GGT AGC CTG CCT TGG CAG GGA TTG AAG GCT ACC ACG AAA AAG CAA 787 Arg Gly Ser Leu Pro Trp Gin Gly Leu Lys Ala Thr Thr Lys Lys Gin 210 215 220 225

AAG TAT GAA AAG ATT ATG GAG AAG AAG ATC TCT ACG CCT ACA GAG GTC 835 Lys Tyr Glu Lys He Met Glu Lys Lys He Ser Thr Pro Thr Glu Val 230 235 240

TTA TGT CGG GGA TTC CCT CAG GAG TTC TCA ATT TAT CTC AAT TAC ACG 883 Leu Cys Arg Gly Phe Pro Gin Glu Phe Ser He Tyr Leu Asn Tyr Thr 245 250 255

AGA TCT TTA CGT TTC GAT GAC AAA CCT GAT TAC GCC TAC CTT CGC AAG 931 Arg Ser Leu Arg Phe Asp Asp Lys Pro Asp Tyr Ala Tyr Leu Arg Lys 260 265 270

CTT TTC CGA GAT CTT TTT TGT CGG CAA TCT TAT GAG TTT GAC TAT ATG 979 Leu Phe Arg Asp Leu Phe Cys Arg Gin Ser Tyr Glu Phe Asp Tyr Met 275 280 285

TTT GAT TGG ACC TTG AAG AGA AAG ACT CAA CAA GAC CAA CAA CAT CAG 1027 Phe Asp Trp Thr Leu Lys Arg Lys Thr Gin Gin Asp Gin Gin His Gin 290 295 300 305

CAG CAA TTA CAG CAA CAA CTG TCT GCA ACT CCT CAA GCT ATT AAT CCG 1075 Gin Gin Leu Gin Gin Gin Leu Ser Ala Thr Pro Gin Ala He Asn Pro 310 315 320

CCG CCA GAG AGG TCT TCA TTT AGA AAT TAT CAA AAA CAA AAC TTT GAT 1123 Pro Pro Glu Arg Ser Ser Phe Arg Asn Tyr Gin Lys Gin Asn Phe Asp 325 330 335 GAA AAA GGC GGA GAC ATT AAT ACA ACC GTT CCT GTT ATA AAT GAT CCA 1171 Glu Lys Gly Gly Asp He Asn Thr Thr Val Pro Val He Asn Asp Pro 340 345 350

TCT GCA ACC GGA GCT CAA TAT ATC AAC AGA CCT AAT TGATTAGCCT 1217

Ser Ala Thr Gly Ala Gin Tyr He Asn Arg Pro Asn 355 360 365

TTCATATTAT TATTATATAG CATGGGCACA TTATTTTTAT ATTTTCTTCT CATCTGGAGT 1277

CTTCCAATAC TTGCCTTTTA TCCTCCAGAC GTCCTTTAAT TTTGTTGATA GCGCAGGGCT 1337

TTTTCCTTGG GATGGCGAAA GTTACTTTGC TTATAGTTTA TTGAGGGTTC ATAGCTTATT 1397

TGGCTGAAGA TCTTGTGTTG ACTTAAATTC TATGCTAACC TCATGATCAT ATCCTCATTA 1457

TGGCAAGTTT TGGTGAAAAA TTTTTTAATA TTAGTACATT TGCTAATAAT ACATTTGGTA 1517

TTTGTTTTTA CTACCTGTGA ATCTATTCAT ACATTATCAT ATATGTTTCG AGCCAGGAAC 1577

AGAAAAAAGT GAGAGAATTT TCTGCAGAAA TGATCATAAT TTTATCTTCG CTTAACACGA 1637

ATCCTGGTGA CAGATTATCG TGGTTTAAAG CCTTTTTTTT ACGACGCCAT AAGCAAATTG 1697

GTTACTTTTT TATGTGTGAT GAGCCTTGGG GTTTAATCTA ATTAGAAGGC ATTGCATTCA 1757

TATACTTTTA ATAATATATT ATCAGCTATT TGCTGCTTTT CTTTATAGAT ACCGTCTTTT 1817

CCAAGCTGAA CTCATTTAAT CAGCGTCGTT TAACCTTAGG ATGCTTAAGA TGCGTTTAAA 1877

TTCAATGACT TAATGCTCGA GGGATGAATG GTTTGTTTTA GTTCGTGTTC TGGGTGCATG 1937

ATCTCGTGCT TGACTGTTTT ATTGAAGCGT TCATTTCATG AAGTGTCTTT CGATGTTGTT 1997

CACACTTCTG TTTGCTAAAT ATAATAAATA TTTTGCTTTT CACTTTAGAG CACACTGGCG 2057

GCCGCTCGAA GCTTTGGACT TCTTCGCCAT TGGTCAAGTC TCCAATCAAG GTTGTCGGCT 2117

TGTCTACCTT GCCAGAAATT TACGAAAAGA TGGAAAAGGG ATCCAAATCG TTGGTAGATA 2177

CTTGTTGACA CTTCTAAATA AGCGAATTTC TTATGATTTA TGATTTTTAT TATTAAATAA 2237

GTTATAAAAA AAATAAGGTA TACAAATTTT AAAGTGACTC TTAGGTTTTA AAACGAAAAT 2297

TCTTATTCTT GAGTAACTCT TTCCTGTAGG TCAGGTTGCT TTCTCAGGTA TAGCATGAGG 2357

TCGCTCTTAT TGACCACACC TCTACCGGCA TGCCGAGCAA ATGCCTGCAA ATCGCTCCCC 2417

ATTTCACCCA ATTGTAGATA TGCTAACTCC AGCAATGAGC CGATGAATCT CC 2469

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 365 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ala Leu Asp Leu Arg He Gly Asn Lys Tyr Arg He Gly Arg Lys 1 5 10 15

He Gly Ser Gly Ser Phe Gly Asp He Tyr Leu Gly Thr Asn Val Val 20 25 30 Ser Gly Glu Glu Val Ala He Lys Leu Glu Ser Thr Arg Ala Lys His 35 40 45

Pro Gin Leu Glu Tyr Glu Tyr Arg Val Tyr Arg He Leu Ser Gly Gly 50 55 60

Val Gly He Pro Phe Val Arg Trp Phe Gly Val Glu Cys Asp Tyr Asn 65 70 75 80

Ala Met Val Met Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asn 85 90 95

Phe Cys Asn Arg Lys Phe Ser Leu Lys Thr Val Leu Leu Leu Ala Asp 100 105 110

Gin Leu He Ser Arg He Glu Phe He His Ser Lys Ser Phe Leu His 115 120 125

Arg Asp He Lys Pro Asp Asn Phe Leu Met Gly He Gly Lys Arg Gly 130 135 140

Asn Gin Val Asn He He Asp Phe Gly Leu Ala Lys Lys Tyr Arg Asp 145 150 155 160

His Lys Thr His Leu His He Pro Tyr Arg Glu Asn Lys Asn Leu Thr 165 170 175

Gly Thr Ala Arg Tyr Ala Ser He Asn Thr His Leu Gly He Glu Gin 180 185 190

Ser Arg Arg Asp Asp Leu Glu Ser Leu Gly Tyr Val Leu Val Tyr Phe 195 200 205

Cys Arg Gly Ser Leu Pro Trp Gin Gly Leu Lys Ala Thr Thr Lys Lys 210 215 220

Gin Lys Tyr Glu Lys He Met Glu Lys Lys He Ser Thr Pro Thr Glu 225 230 235 240

Val Leu Cys Arg Gly Phe Pro Gin Glu Phe Ser He Tyr Leu Asn Tyr 245 250 255

Thr Arg Ser Leu Arg Phe Asp Asp Lys Pro Asp Tyr Ala Tyr Leu Arg 260 265 270

Lys Leu Phe Arg Asp Leu Phe Cys Arg Gin Ser Tyr Glu Phe Asp Tyr 275 280 285

Met Phe Asp Trp Thr Leu Lys Arg Lys Thr Gin Gin Asp Gin Gin His 290 295 300

Gin Gin Gin Leu Gin Gin Gin Leu Ser Ala Thr Pro Gin Ala He Asn 305 310 315 320

Pro Pro Pro Glu Arg Ser Ser Phe Arg Asn Tyr Gin Lys Gin Asn Phe 325 330 335

Asp Glu Lys Gly Gly Asp He Asn Thr Thr Val Pro Val He Asn Asp 340 345 350

Pro Ser Ala Thr Gly Ala Gin Tyr He Asn Arg Pro Asn 355 360 365 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1989 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 50..1249

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CCGCCAGTGT GCTCTAAAGG TCATCTCTGT GAATTAGAAT CTTAGCAAA ATG ACG 55

Met Thr

1

GTT GTT GAC ATT AAG ATT GGT AAT AAA TAT CGT ATA GGT AGA AAA ATT 103 Val Val Asp He Lys He Gly Asn Lys Tyr Arg He Gly Arg Lys He 5 10 15

GGT TCT GGC TCC TTT GGT CAA ATT TAC CTG GGA TTA AAT ACG GTA AAT 151 Gly Ser Gly Ser Phe Gly Gin He Tyr Leu Gly Leu Asn Thr Val Asn 20 25 30

GGA GAA CAA GTT GCT GTG AAA TTG GAG CCT TTA AAG GCT CGT CAT CAT 199 Gly Glu Gin Val Ala Val Lys Leu Glu Pro Leu Lys Ala Arg His His 35 40 45 50

CAG TTA GAA TAT GAG TTT CGT GTG TAT AAT ATT CTT AAA GGA AAT ATT 247 Gin Leu Glu Tyr Glu Phe Arg Val Tyr Asn He Leu Lys Gly Asn He 55 60 65

GGC ATA CCC ACA ATT CGC TGG TTC GGT GTA ACC AAT AGT TAT AAT GCT 295 Gly He Pro Thr He Arg Trp Phe Gly Val Thr Asn Ser Tyr Asn Ala 70 75 80

ATG GTC ATG GAT TTA TTA GGC CCT TCT CTG GAA GAT TTA TTC TGC TAT 343 Met Val Met Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Cys Tyr 85 90 95

TGT GGA AGA AAG TTT ACT CTT AAA ACG GTT CTT TTA CTT GCT GAT CAA 391 Cys Gly Arg Lys Phe Thr Leu Lys Thr Val Leu Leu Leu Ala Asp Gin 100 105 110

CTC ATC AGT CGC ATT GAA TAT GTT CAC TCC AAG TCA TTC TTA CAT CGA 439 Leu He Ser Arg He Glu Tyr Val His Ser Lys Ser Phe Leu His Arg 115 120 125 130

GAC ATT AAG CCT GAT AAT TTT TTA ATG AAG AAG CAC AGC AAT GTT GTT 487 Asp He Lys Pro Asp Asn Phe Leu Met Lys Lys His Ser Asn Val Val 135 140 145

ACG ATG ATT GAC TTC GGA TTG GCG AAA AAA TAC AGG GAT TTT AAA ACT 535 Thr Met He Asp Phe Gly Leu Ala Lys Lys Tyr Arg Asp Phe Lys Thr 150 155 160 CAT GTT CAT ATT CCA TAT CGA GAT AAT AAG AAT CTT ACG GGA ACG GCT 583 His Val His He Pro Tyr Arg Asp Asn Lys Asn Leu Thr Gly Thr Ala 165 170 175

CGA TAT GCT AGT ATT AAC ACC CAT ATT GGT ATT GAA CAA TCT CGC CGT 631 Arg Tyr Ala Ser He Asn Thr His He Gly He Glu Gin Ser Arg Arg 180 185 190

GAT GAC CTC GAA TCG TTA GGT TAT GTT TTA CTT TAT TTT TGT CGC GGC 679 Asp Asp Leu Glu Ser Leu Gly Tyr Val Leu Leu Tyr Phe Cys Arg Gly 195 200 205 210

AGT TTG CCC TGG CAA GGC TTA CAA GCT GAT ACA AAG GAG CAA AAG TAT 727 Ser Leu Pro Trp Gin Gly Leu Gin Ala Asp Thr Lys Glu Gin Lys Tyr 215 220 225

CAA CGG ATA CGT GAT ACC AAG ATT GGC ACT CCT TTG GAA GTC CTT TGC 775 Gin Arg He Arg Asp Thr Lys He Gly Thr Pro Leu Glu Val Leu Cys 230 235 240

AAA GGT CTT CCC GAA GAG TTT ATC ACT TAC ATG TGT TAC ACT CGT CAG 823 Lys Gly Leu Pro Glu Glu Phe He Thr Tyr Met Cys Tyr Thr Arg Gin 245 250 255

CTT TCG TTT ACC GAG AAG CCA AAC TAT GCT TAT TTG AGA AAG CTG TTT 871 Leu Ser Phe Thr Glu Lys Pro Asn Tyr Ala Tyr Leu Arg Lys Leu Phe 260 265 270

CGT GAT TTA CTT ATT CGT AAA GGA TAC CAG TAT GAC TAT GTT TTT GAC 919 Arg Asp Leu Leu He Arg Lys Gly Tyr Gin Tyr Asp Tyr Val Phe Asp 275 280 285 290

TGG ATG ATA TTA AAA TAC CAA AAG CGA GCT GCT GCT GCT GCC GCC GCT 967 Trp Met He Leu Lys Tyr Gin Lys Arg Ala Ala Ala Ala Ala Ala Ala 295 300 305

TCT GCT ACA GCA CCT CCA CAG GTT ACA TCT CCT ATG GTG TCA CAA ACT 1015 Ser Ala Thr Ala Pro Pro Gin Val Thr Ser Pro Met Val Ser Gin Thr ^'310 315 320

CAA CCG GTT AAT CCC ATT ACT CCT AAT TAT TCA TCC ATT CCC TTA CCT 1063 Gin Pro Val Asn Pro He Thr Pro Asn Tyr Ser Ser He Pro Leu Pro 325 330 335

GCT GAG CGG AAT CCA AAG ACT CCA CAA TCT TTC TCC ACT AAT ATT GTT 1111 Ala Glu Arg Asn Pro Lys Thr Pro Gin Ser Phe Ser Thr Asn He Val 340 345 350

CAA TGT GCT TCT CCC TCA CCT CTT CCT CTC TCC TTT CGT TCT CCT GTT 1159 Gin Cys Ala Ser Pro Ser Pro Leu Pro Leu Ser Phe Arg Ser Pro Val 355 360 365 370

CCC AAC AAA GAT TAT GAA TAC ATT CCA TCT TCG TTG CAA CCT CAA TAC 1207 Pro Asn Lys Asp Tyr Glu Tyr He Pro Ser Ser Leu Gin Pro Gin Tyr 375 380 385

AGT GCT CAA CTG AGG CGT GTT TTA GAT GAA GAA CCA GCT CCT 1249

Ser Ala Gin Leu Arg Arg Val Leu Asp Glu Glu Pro Ala Pro

390 395 400

TGATTTTTTG ACTTTACTTT TCATCAATTC CTCTCTTACA CTACGTCTTT TAGTCTTAAA 1309 TTCCAAACCA TCTGTTGACG TTTTAAAGTT CCACAAATAT CTTTAATAAT TCCTGGCTTT 1369 CTTTTTTGTC TATGGATGGC CGGATTGCTA CACTAATACA CTTTGAGGTT TAGCTATTGT 1429 TTTGAGCTAT TCCATTTTGC CTAGAAGTTG AGTTTTAATG CCTTCTTTTT AAATAGACAT 1489 ATTGTGTAAA CCTCATACAT GCTTTACTGA AAAGACATAA TTAGAGGACA AAATTTAAAT 1549

CGTGCTGTTT GTTTATATTC AGCTCGTTCC GGTCAAGTTC TTGCCAAAGA ATTGAGTCAG 1609

TCGTGCTATT CATTTCTAAA TTTCTTCTTC CCAGAATTTT ATTTTATTGT TTTCGTTCCC 1669

CATTGGTTCT TACATTCCGT TTTTATTCAA AACTGAAAAG TTTGTACCTC CATTGCTAGA 1729

AGTAATATAC ACAAGGAGCA TGTTTCTTTT TTTACACTAT CATTTGCGTG GCTCTAAACC 1789

AGTCTTTATT GCCTACCTTT GCAATAAAAG ATATAATATC AATTGCATAA GAAATAATTC 1849

ATTAATAAAT GATAAATTTC ATCGATTAAA TAAAAAAAAA AAACTTTAGA GCTTTAGAGC 1909

ACAACTGGCG GCCGCTCGAA GCTTTGGACT TCTTCGCCAT TGGTCAAGTC TCAATCAAGG 1969

TTGTCGGCTT GTCTACCTTC 1989

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 400 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Thr Val Val Asp He Lys He Gly Asn Lys Tyr Arg He Gly Arg 1 5 10 15

Lys He Gly Ser Gly Ser Phe Gly Gin He Tyr Leu Gly Leu Asn Thr 20 25 30

Val Asn Gly Glu Gin Val Ala Val Lys Leu Glu Pro Leu Lys Ala Arg 35 40 45

His His Gin Leu Glu Tyr Glu Phe Arg Val Tyr Asn He Leu Lys Gly 50 55 60

Asn He Gly He Pro Thr He Arg Trp Phe Gly Val Thr Asn Ser Tyr 65 70 75 80

Asn Ala Met Val Met Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe 85 90 95

Cys Tyr Cys Gly Arg Lys Phe Thr Leu Lys Thr Val Leu Leu Leu Ala 100 105 110

Asp Gin Leu He Ser Arg He Glu Tyr Val His Ser Lys Ser Phe Leu 115 120 125

His Arg Asp He Lys Pro Asp Asn Phe Leu Met Lys Lys His Ser Asn 130 135 140

Val Val Thr Met He Asp Phe Gly Leu Ala Lys Lys Tyr Arg Asp Phe 145 150 155 160

Lys Thr His Val His He Pro Tyr Arg Asp Asn Lys Asn Leu Thr Gly 165 170 175

Thr Ala Arg Tyr Ala Ser He Asn Thr His He Gly He Glu Gin Ser 180 185 190 Arg Arg Asp Asp Leu Glu Ser Leu Gly Tyr Val Leu Leu Tyr Phe Cys 195 200 205

Arg Gly Ser Leu Pro Trp Gin Gly Leu Gin Ala Asp Thr Lys Glu Gin 210 215 220

Lys Tyr Gin Arg He Arg Asp Thr Lys He Gly Thr Pro Leu Glu Val 225 230 235 240

Leu Cys Lys Gly Leu Pro Glu Glu Phe He Thr Tyr Met Cys Tyr Thr 245 250 255

Arg Gin Leu Ser Phe Thr Glu Lys Pro Asn Tyr Ala Tyr Leu Arg Lys 260 265 270

Leu Phe Arg Asp Leu Leu He Arg Lys Gly Tyr Gin Tyr Asp Tyr Val 275 280 285

Phe Asp Trp Met He Leu Lys Tyr Gin Lys Arg Ala Ala Ala Ala Ala 290 295 300

Ala Ala Ser Ala Thr Ala Pro Pro Gin Val Thr Ser Pro Met Val Ser 305 310 315 320

Gin Thr Gin Pro Val Asn Pro He Thr Pro Asn Tyr Ser Ser He Pro 325 330 335

Leu Pro Ala Glu Arg Asn Pro Lys Thr Pro Gin Ser Phe Ser Thr Asn 340 345 350

He Val Gin Cys Ala Ser Pro Ser Pro Leu Pro Leu Ser Phe Arg Ser 355 360 365

Pro Val Pro Asn Lys Asp Tyr Glu Tyr He Pro Ser Ser Leu Gin Pro 370 375 380

Gin Tyr Ser Ala Gin Leu Arg Arg Val Leu Asp Glu Glu Pro Ala Pro 385 390 395 400

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1210 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 173..1147

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGCGGTGATC AGTTCCCCTC TGCTGATTCT GGGCCCGAAC CCGGTAAAGG CCTCCGTGTT 60 CCGTTTCCTG CCGCCCTCCT CCGTAGCCTT GCCTAGTGTA GGAGCCCCGA GGCCTCCGTC 120 CTCTTCCCAG AGGTGTCGGG GCTTGCCCCA GCCTCCATCT TCGTCTCTCA GG ATG 175

Met

1

GCG AGT AGC AGC GGC TCC AAG GCT GAA TTC ATT GTC GGA GGG AAA TAT 223 Ala Ser Ser Ser Gly Ser Lys Ala Glu Phe He Val Gly Gly Lys Tyr 5 10 15

AAA CTG GTA CGG AAG ATC GGG TCT GGC TCC TTC GGG GAC ATC TAT TTG 271 Lys Leu Val Arg Lys He Gly Ser Gly Ser Phe Gly Asp He Tyr Leu 20 25 30

GCG ATC AAC ATC ACC AAC GGC GAG GAA GTG GCA GTG AAG CTA GAA TCT 319 Ala He Asn He Thr Asn Gly Glu Glu Val Ala Val Lys Leu Glu Ser 35 40 45

CAG AAG GCC AGG CAT CCC CAG TTG CTG TAC GAG AGC AAG CTC TAT AAG 367 Gin Lys Ala Arg His Pro Gin Leu Leu Tyr Glu Ser Lys Leu Tyr Lys 50 55 60 65

ATT CTT CAA GGT GGG GTT GGC ATC CCC CAC ATA CGG TGG TAT GGT CAG 415 He Leu Gin Gly Gly Val Gly He Pro His He Arg Trp Tyr Gly Gin 70 75 80

GAA AAA GAC TAC AAT GTA CTA GTC ATG GAT CTT CTG GGA CCT AGC CTC 463 Glu Lys Asp Tyr Asn Val Leu Val Met Asp Leu Leu Gly Pro Ser Leu 85 90 95

GAA GAC CTC TTC AAT TTC TGT TCA AGA AGG TTC ACA ATG AAA ACT GTA 511 Glu Asp Leu Phe Asn Phe Cys Ser Arg Arg Phe Thr Met Lys Thr Val 100 105 110

CTT ATG TTA GCT GAC CAG ATG ATC AGT AGA ATT GAA TAT GTG CAT ACA 559 Leu Met Leu Ala Asp Gin Met He Ser Arg He Glu Tyr Val His Thr 115 120 125

AAG AAT TTT ATA CAC AGA GAC ATT AAA CCA GAT AAC TTC CTA ATG GGT 607 Lys Asn Phe He His Arg Asp He Lys Pro Asp Asn Phe Leu Met Gly 130 135 140 145

ATT GGG CGT CAC TGT AAT AAG TTA TTC CTT ATT GAT TTT GGT TTG GCC 655 He Gly Arg His Cys Asn Lys Leu Phe Leu He Asp Phe Gly Leu Ala 150 155 160

AAA AAG TAC AGA GAC AAC AGG ACA AGG CAA CAC ATA CCA TAC AGA GAA 703 Lys Lys Tyr Arg Asp Asn Arg Thr Arg Gin His He Pro Tyr Arg Glu 165 170 175

GAT AAA AAC CTC ACT GGC ACT GCC CGA TAT GCT AGC ATC AAT GCA CAT 751 Asp Lys Asn Leu Thr Gly Thr Ala Arg Tyr Ala Ser He Asn Ala His 180 185 190

CTT GGT ATT GAG CAG AGT CGC CGA GAT GAC ATG GAA TCA TTA GGA TAT 799 Leu Gly He Glu Gin Ser Arg Arg Asp Asp Met Glu Ser Leu Gly Tyr 195 200 205

GTT TTG ATG TAT TTT AAT AGA ACC AGC CTG CCA TGG CAA GGG CTA AAG 847 Val Leu Met Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gin Gly Leu Lys 210 215 220 225

GCT GCA ACA AAG AAA CAA AAA TAT GAA AAG ATT AGT GAA AAG AAG ATG 895 Ala Ala Thr Lys Lys Gin Lys Tyr Glu Lys He Ser Glu Lys Lys Met 230 235 240

TCC ACG CCT GTT GAA GTT TTA TGT AAG GGG TTT CCT GCA GAA TTT GCG 943 Ser Thr Pro Val Glu Val Leu Cys Lys Gly Phe Pro Ala Glu Phe Ala 245 250 255 ATG TAC TTA AAC TAT TGT CGT GGG CTA CGC TTT GAG GAA GCC CCA GAT 991 Met Tyr Leu Asn Tyr Cys Arg Gly Leu Arg Phe Glu Glu Ala Pro Asp 260 265 270

TAC ATG TAT CTG AGG CAG CTA TTC CGC ATT CTT TTC AGG ACC CTG AAC 1039 Tyr Met Tyr Leu Arg Gin Leu Phe Arg He Leu Phe Arg Thr Leu Asn 275 280 285

CAT CAA TAT GAC TAC ACA TTT GAT TGG ACA ATG TTA AAG CAG AAA GCA 1087 His Gin Tyr Asp Tyr Thr Phe Asp Trp Thr Met Leu Lys Gin Lys Ala 290 295 300 305

GCA CAG CAG GCA GCC TCT TCC AGT GGG CAG GGT CAG CAG GCC CAA ACC 1135 Ala Gin Gin Ala Ala Ser Ser Ser Gly Gin Gly Gin Gin Ala Gin Thr 310 315 320

CCC ACA GGT TTC TAAGCATGAA TTGAGGAACA GAAGAAGCAG AGCAGATGAT 1187

Pro Thr Gly Phe 325

CGAGCAGCAT TTGTTTCTCC CAA 1210

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 325 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Ala Ser Ser Ser Gly Ser Lys Ala Glu Phe He Val Gly Gly Lys 1 5 10 15

Tyr Lys Leu Val Arg Lys He Gly Ser Gly Ser Phe Gly Asp He Tyr 20 25 30

Leu Ala He Asn He Thr Asn Gly Glu Glu Val Ala Val Lys Leu Glu 35 40 45

Ser Gin Lys Ala Arg His Pro Gin Leu Leu Tyr Glu Ser Lys Leu Tyr 50 55 60

Lys He Leu Gin Gly Gly Val Gly He Pro His He Arg Trp Tyr Gly 65 70 75 80

Gin Glu Lys Asp Tyr Asn Val Leu Val Met Asp Leu Leu Gly Pro Ser 85 90 95

Leu Glu Asp Leu Phe Asn Phe Cys Ser Arg Arg Phe Thr Met Lys Thr 100 105 110

Val Leu Met Leu Ala Asp Gin Met He Ser Arg He Glu Tyr Val His 115 120 125

Thr Lys Asn Phe He His Arg Asp He Lys Pro Asp Asn Phe Leu Met 130 135 140

Gly He Gly Arg His Cys Asn Lys Leu Phe Leu He Asp Phe Gly Leu 145 150 155 160

Ala Lys Lys Tyr Arg Asp Asn Arg Thr Arg Gin His He Pro Tyr Arg 165 170 175 Glu Asp Lys Asn Leu* Thr Gly Thr Ala Arg Tyr Ala Ser He Asn Ala 180 185 190

His Leu Gly He Glu Gin Ser Arg Arg Asp Asp Met Glu Ser Leu Gly 195 200 205

Tyr Val Leu Met Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gin Gly Leu 210 215 220

Lys Ala Ala Thr Lys Lys Gin Lys Tyr Glu Lys He Ser Glu Lys Lys 225 230 235 240

Met Ser Thr Pro Val Glu Val Leu Cys Lys Gly Phe Pro Ala Glu Phe 245 250 255

Ala Met Tyr Leu Asn Tyr Cys Arg Gly Leu Arg Phe Glu Glu Ala Pro 260 265 270

Asp Tyr Met Tyr Leu Arg Gin Leu Phe Arg He Leu Phe Arg Thr Leu 275 280 285

Asn His Gin Tyr Asp Tyr Thr Phe Asp Trp Thr Met Leu Lys Gin Lys 290 295 300

Ala Ala Gin Gin Ala Ala Ser Ser Ser Gly Gin Gly Gin Gin Ala Gin 305 310 315 320

Thr Pro Thr Gly Phe 325

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2385 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 297..1388

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

GAATTCCGAT AGTATTATGT GGAGTTCCAT TTTTATGTAT TTTTTGTATG AAATATTCTA 60

GTATAAGTAA ATATTTTATC AGAAGTATTT ACATATCTTT TTTTTTTTTA GTTTGAGAGC 120

GGCGGTGATC AGGTTCCCCT CTGCTGATTC TGGGCCCCGA ACCCCGGTAA AGGCCTCCGT 180

GTTCCGTTTC CTGCCGCCCT CCTCCGTAGC CTTGCCTAGT GTAGGAGCCC CGAGGCCTCC 240

GTCCTCTTCC CAGAGGTGTC GGGGCTTGGC CCCAGCCTCC ATCTTCGTCT CTCAGG 296

ATG GCG AGT AGC AGC GGC TCC AAG GCT GAA TTC ATT GTC GGA GGG AAA 344 Met Ala Ser Ser Ser Gly Ser Lys Ala Glu Phe He Val Gly Gly Lys 1 5 10 15

TAT AAA CTG GTA CGG AAG ATC GGG TCT GGC TCC TTC GGG GAC ATC TAT 392 Tyr Lys Leu Val Arg Lys He Gly Ser Gly Ser Phe Gly Asp He Tyr 20 25 30 TTG GCG ATC AAC ATC ACC AAC GGC GAG GAA GTG GCA GTG AAG CTA GAA 440 Leu Ala He Asn He Thr Asn Gly Glu Glu Val Ala Val Lys Leu Glu 35 40 45

TCT CAG AAG GCC AGG CAT CCC CAG TTG CTG TAC GAG AGC AAG CTC TAT 488 Ser Gin Lys Ala Arg His Pro Gin Leu Leu Tyr Glu Ser Lys Leu Tyr 50 55 60

AAG ATT CTT CAA GGT GGG GTT GGC ATC CCC CAC ATA CGG TGG TAT GGT 536 Lys He Leu Gin Gly Gly Val Gly He Pro His He Arg Trp Tyr Gly 65 70 75 80

CAG GAA AAA GAC TAC AAT GTA CTA GTC ATG GAT CTT CTG GGA CCT AGC 584 Gin Glu Lys Asp Tyr Asn Val Leu Val Met Asp Leu Leu Gly Pro Ser 85 90 95

CTC GAA GAC CTC TTC AAT TTC TGT TCA AGA AGG TTC ACA ATG AAA ACT 632 Leu Glu Asp Leu Phe Asn Phe Cys Ser Arg Arg Phe Thr Met Lys Thr 100 105 110

GTA CTT ATG TTA GCT GAC CAG ATG ATC AGT AGA ATT GAA TAT GTG CAT 680 Val Leu Met Leu Ala Asp Gin Met He Ser Arg He Glu Tyr Val His 115 120 125

ACA AAG AAT TTT ATA CAC AGA GAC ATT AAA CCA GAT AAC TTC CTA ATG 728 Thr Lys Asn Phe He His Arg Asp He Lys Pro Asp Asn Phe Leu Met 130 135 140

GGT ATT GGG CGT CAC TGT AAT AAG TGT TTA GAA TCT CCA GTG GGG AAG 776 Gly He Gly Arg His Cys Asn Lys Cys Leu Glu Ser Pro Val Gly Lys 145 150 155 160

AGG AAA AGA AGC ATG ACT GTT AGT ACT TCT CAG GAC CCA TCT TTC TCA 824 Arg Lys Arg Ser Met Thr Val Ser Thr Ser Gin Asp Pro Ser Phe Ser 165 170 175

GGA TTA AAC CAG TTA TTC CTT ATT GAT TTT GGT TTG GCC AAA AAG TAC 872 Gly Leu Asn Gin Leu Phe Leu He Asp Phe Gly Leu Ala Lys Lys Tyr 180 185 190

AGA GAC AAC AGG ACA AGG CAA CAC ATA CCA TAC AGA GAA GAT AAA AAC 920 Arg Asp Asn Arg Thr Arg Gin His He Pro Tyr Arg Glu Asp Lys Asn 195 200 205

CTC ACT GGC ACT GCC CGA TAT GCT AGC ATC AAT GCA CAT CTT GGT ATT 968 Leu Thr Gly Thr Ala Arg Tyr Ala Ser He Asn Ala His Leu Gly He 210 215 220

GAG CAG AGT CGC CGA GAT GAC ATG GAA TCA TTA GGA TAT GTT TTG ATG 1016 Glu Gin Ser Arg Arg Asp Asp Met Glu Ser Leu Gly Tyr Val Leu Met 225 230 235 240

TAT TTT AAT AGA ACC AGC CTG CCA TGG CAA GGG CTA AAG GCT GCA ACA 1064 Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gin Gly Leu Lys Ala Ala Thr 245 250 255

AAG AAA CAA AAA TAT GAA AAG ATT AGT GAA AAG AAG ATG TCC ACG CCT 1112 Lys Lys Gin Lys Tyr Glu Lys He Ser Glu Lys Lys Met Ser Thr Pro 260 265 270

GTT GAA GTT TTA TGT AAG GGG TTT CCT GCA GAA TTT GCG ATG TAC TTA 1160 Val Glu Val Leu Cys Lys Gly Phe Pro Ala Glu Phe Ala Met Tyr Leu 275 280 285

AAC TAT TGT CGT GGG CTA CGC TTT GAG GAA GCC CCA GAT TAC ATG TAT 1208 Asn Tyr Cys Arg Gly Leu Arg Phe Glu Glu Ala Pro Asp Tyr Met Tyr 290 295 300 CTG AGG CAG CTA TTC CGC ATT CTT TTC AGG ACC CTG AAC CAT CAA TAT 1256 Leu Arg Gin Leu Phe Arg He Leu Phe Arg Thr Leu Asn His Gin Tyr 305 310 315 320

GAC TAC ACA TTT GAT TGG ACA ATG TTA AAG CAG AAA GCA GCA CAG CAG 1304 Asp Tyr Thr Phe Asp Trp Thr Met Leu Lys Gin Lys Ala Ala Gin Gin 325 330 335

GCA GCC TCT TCC AGT GGG CAG GGT CAG CAG GCC CAA ACC CCC ACA GGC 1352 Ala Ala Ser Ser Ser Gly Gin Gly Gin Gin Ala Gin Thr Pro Thr Gly 340 345 350

AAG CAA ACT GAC AAA ACC AAG AGT AAC ATG AAA GGT TAGTAGCCAA 1398

Lys Gin Thr Asp Lys Thr Lys Ser Asn Met Lys Gly 355 360

GAACCAAGTG ACGTTACAGG GAAAAAATTG AATACAAAAT TGGGTAATTC ATTTCTAACA 1458

GTGTTAGATC AAGGAGGTGG TTTTAAAATA CATAAAAATT TGGCTCTGCG TTAAAAAAAA 1518

AAAAGACGTC CTTGGAAAAT TTGACTACTA ACTTTAAACC CAAATGTCCT TGTTCATATA 1578

TATGTATATG TATTTGTATA TACATATATG TGTGTAT TT TATATCATTT CTCTTGGGAT 1638

TTTGGGTCAT TTTTTTAACA ACTGCATCTT TTTTACTCAT TCATTAACCC CCTTTCCAAA 1698

AATTTGGTGT TGGGAATATA ATATAATCAA TCAATCCAAA ATCCTAGACC TAACACTTGT 1758

TGATTTCTAA TAATGAATTT GGTTAGCCAT ATTTTGACTT TATTTCAGAC TAACAATGTT 1818

AAGATTTTTT ATTTTGCATG TTAATGCTTT AGCATTTAAA ATGGAAAATT GTGAACATGT 1878

TGTAATTTCA AGAGGTGAGT TTGGCATTAC CCCCAAAGTG TCTATCTTCT CAGTTGCAGA 1938

GCATCTCATT TTCTCTCTTA AATGCTCAAA TAAATGCAAA GCTCAGCACA TCTTTTCTAG 1998

TCACAAAAAT AATTCTTTTA TTTGCAGTTT ACGTATGATC TTAATTTCAA AACGATTTCT 2058

TTGTTTTTGG CTTGATTTTT CACAATGTTG CAAATATCAG GCTCCCAGGG TTTAATGTGG 2118

AATTGAAGTC TGCAGCCAGG CCTTGCAAAT TGAAGGTAAC TGGGGCAAAT GCCATTGAAA 2178

CCGCTAGTCT TATTTCCTTT CTACTTTTCT TTGGCACTCT TACTGCCTGT AAGGAGTAGA 2238

ACTGTTAAGG CACACTGTTG CTATACAGTT AACTCCCATT TTCATGTTTT GTCTTTCTTT 2298

TCCCATTTCT GGGGCTTACC TCCTGATACC TGCTTACTTT CTGGAAGTAG TGGGCAAGTA 2358

AGATTTGGCT CTTGGTTTCT GGAATTC 2385

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 364 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Ala Ser Ser Ser Gly Ser Lys Ala Glu Phe He Val Gly Gly Lys 1 5 10 15

Tyr Lys Leu Val Arg Lys He Gly Ser Gly Ser Phe Gly Asp He Tyr 20 25 30 Leu Ala He Asn He Thr Asn Gly Glu Glu Val Ala Val Lys Leu Glu 35 40 45

Ser Gin Lys Ala Arg His Pro Gin Leu Leu Tyr Glu Ser Lys Leu Tyr 50 55 60

Lys He Leu Gin Gly Gly Val Gly He Pro His He Arg Trp Tyr Gly 65 70 75 80

Gin Glu Lys Asp Tyr Asn Val Leu Val Met Asp Leu Leu Gly Pro Ser 85 90 95

Leu Glu Asp Leu Phe Asn Phe Cys Ser Arg Arg Phe Thr Met Lys Thr 100 105 110

Val Leu Met Leu Ala Asp Gin Met He Ser Arg He Glu Tyr Val His 115 120 125

Thr Lys Asn Phe He His Arg Asp He Lys Pro Asp Asn Phe Leu Met 130 135 140

Gly He Gly Arg His Cys Asn Lys Cys Leu Glu Ser Pro Val Gly Lys 145 150 155 160

Arg Lys Arg Ser Met Thr Val Ser Thr Ser Gin Asp Pro Ser Phe Ser 165 170 175

Gly Leu Asn Gin Leu Phe Leu He Asp Phe Gly Leu Ala Lys Lys Tyr 180 185 190

Arg Asp Asn Arg Thr Arg Gin His He Pro Tyr Arg Glu Asp Lys Asn 195 200 205

Leu Thr Gly Thr Ala Arg Tyr Ala Ser He Asn Ala His Leu Gly He 210 215 220

Glu Gin Ser Arg Arg Asp Asp Met Glu Ser Leu Gly Tyr Val Leu Met 225 230 235 240

Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gin Gly Leu Lys Ala Ala Thr 245 250 255

Lys Lys Gin Lys Tyr Glu Lys He Ser Glu Lys Lys Met Ser Thr Pro 260 265 270

Val Glu Val Leu Cys Lys Gly Phe Pro Ala Glu Phe Ala Met Tyr Leu 275 280 285

Asn Tyr Cys Arg Gly Leu Arg Phe Glu Glu Ala Pro Asp Tyr Met Tyr 290 295 300

Leu Arg Gin Leu Phe Arg He Leu Phe Arg Thr Leu Asn His Gin Tyr 305 310 315 320

Asp Tyr Thr Phe Asp Trp Thr Met Leu Lys Gin Lys Ala Ala Gin Gin 325 330 335

Ala Ala Ser Ser Ser Gly Gin Gly Gin Gin Ala Gin Thr Pro Thr Gly 340 345 350

Lys Gin Thr Asp Lys Thr Lys Ser Asn Met Lys Gly 355 360 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2914 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 265..1275

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

GAATTCCCGA GAAACAAGTG GCCCAGCCTG GTAACCGCCG AGAAGCCCTT CACAAACTGC 60

GGCCTGGCAA AAAGAAACCT GACTGAGCGG CGGTGATCAG GTTCCCCTCT GCTGATTCTG 120

GGCCCCGAAC CCCGGTAAAG GCCTCCGTGT TCCGTTTCCT GCCGCCCTCC TCCGTAGCCT 180

TGCCTAGTGT AGGAGCCCCG AGGCCTCCGT CCTCTTCCCA GAGGTGTCGG GGCTTGGCCC 240

CAGCCTCCAT CTTCGTCTCT CAGG ATG GCG AGT AGC AGC GGC TCC AAG GCT 291

Met Ala Ser Ser Ser Gly Ser Lys Ala

1 5

GAA TTC ATT GTC GGA GGG AAA TAT AAA CTG GTA CGG AAG ATC GGG TCT 339 Glu Phe He Val Gly Gly Lys Tyr Lys Leu Val Arg Lys He Gly Ser 10 15 20 25

GGC TCC TTC GGG GAC ATC TAT TTG GCG ATC AAC ATC ACC AAC GGC GAG 387 Gly Ser Phe Gly Asp He Tyr Leu Ala He Asn He Thr Asn Gly Glu 30 35 40

_*

GAA GTG GCA GTG AAG CTA GAA TCT CAG AAG GCC AGG CAT CCC CAG TTG 435 Glu Val Ala Val Lys Leu Glu Ser Gin Lys Ala Arg His Pro Gin Leu 45 50 55

CTG TAC GAG AGC AAG CTC TAT AAG ATT CTT CAA GGT GGG GTT GGC ATC 483 Leu Tyr Glu Ser Lys Leu Tyr Lys He Leu Gin Gly Gly Val Gly He 60 65 70

CCC CAC ATA CGG TGG TAT GGT CAG GAA AAA GAC TAC AAT GTA CTA GTC 531 Pro His He Arg Trp Tyr Gly Gin Glu Lys Asp Tyr Asn Val Leu Val 75 80 85

ATG GAT CTT CTG GGA CCT AGC CTC GAA GAC CTC TTC AAT TTC TGT TCA 579 Met Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asn Phe Cys Ser 90 95 100 105

AGA AGG TTC ACA ATG AAA ACT GTA CTT ATG TTA GCT GAC CAG ATG ATC 627 Arg Arg Phe Thr Met Lys Thr Val Leu Met Leu Ala Asp Gin Met He 110 115 120

AGT AGA ATT GAA TAT GTG CAT ACA AAG AAT TTT ATA CAC AGA GAC ATT 675 Ser Arg He Glu Tyr Val His Thr Lys Asn Phe He His Arg Asp He 125 130 135

AAA CCA GAT AAC TTC CTA ATG GGT ATT GGG CGT CAC TGT AAT AAG TTA 723 Lys Pro Asp Asn Phe Leu Met Gly He Gly Arg His Cys Asn Lys Leu 140 145 150 TTC CTT ATT GAT TTT GGT TTG GCC AAA AAG TAC AGA GAC AAC AGG ACA 771 Phe Leu He Asp Phe Gly Leu Ala Lys Lys Tyr Arg Asp Asn Arg Thr 155 160 165

AGG CAA CAC ATA CCA TAC AGA GAA GAT AAA AAC CTC ACT GGC ACT GCC 819 Arg Gin His He Pro Tyr Arg Glu Asp Lys Asn Leu Thr Gly Thr Ala 170 175 180 185

CGA TAT GCT AGC ATC AAT GCA CAT CTT GGT ATT GAG CAG AGT CGC CGA 867 Arg Tyr Ala Ser He Asn Ala His Leu Gly He Glu Gin Ser Arg Arg 190 195 200

GAT GAC ATG GAA TCA TTA GGA TAT GTT TTG ATG TAT TTT AAT AGA ACC 915 Asp Asp Met Glu Ser Leu Gly Tyr Val Leu Met Tyr Phe Asn Arg Thr 205 210 215

AGC CTG CCA TGG CAA GGG CTA AAG GCT GCA ACA AAG AAA CAA AAA TAT 963 Ser Leu Pro Trp Gin Gly Leu Lys Ala Ala Thr Lys Lys Gin Lys Tyr 220 225 230

GAA AAG ATT AGT GAA AAG AAG ATG TCC ACG CCT GTT GAA GTT TTA TGT 1011 Glu Lys He Ser Glu Lys Lys Met Ser Thr Pro Val Glu Val Leu Cys 235 240 245

AAG GGG TTT CCT GCA GAA TTT GCG ATG TAC TTA AAC TAT TGT CGT GGG 1059 Lys Gly Phe Pro Ala Glu Phe Ala Met Tyr Leu Asn Tyr Cys Arg Gly 250 255 260 265

CTA CGC TTT GAG GAA GCC CCA GAT TAC ATG TAT CTG AGG CAG CTA TTC 1107 Leu Arg Phe Glu Glu Ala Pro Asp Tyr Met Tyr Leu Arg Gin Leu Phe 270 275 280

CGC ATT CTT TTC AGG ACC CTG AAC CAT CAA TAT GAC TAC ACA TTT GAT 1155 Arg He Leu Phe Arg Thr Leu Asn His Gin Tyr Asp Tyr Thr Phe Asp 285 290 295

TGG ACA ATG TTA AAG CAG AAA GCA GCA CAG CAG GCA GCC TCT TCC AGT 1203 Trp Thr Met Leu Lys Gin Lys Ala Ala Gin Gin Ala Ala Ser Ser Ser 300 305 310

GGG CAG GGT CAG CAG GCC CAA ACC CCC ACA GGC AAG CAA ACT GAC AAA 1251 Gly Gin Gly Gin Gin Ala Gin Thr Pro Thr Gly Lys Gin Thr Asp Lys 315 320 325

ACC AAG AGT AAC ATG AAA GGT TTC TAAGCATGAA TTGAGGAACA GAAGAAGCAG 1305 Thr Lys Ser Asn Met Lys Gly Phe 330 335

AGCAGATGAT CGGAGCAGCA TTTGTTTCTC CCCAAATCTA GAAATTTTAG TTCATATGTA 1365

CACTAGCCAG TGGTTGTGGA CAACCATTTA CTTGGTGTAA AGAACTTAAT TTCAGTATAA 1425

ACTGACTCTG GGCAGCATTG GTGATGCTGT ATCCTGAGTT GTAGCCTCTG TAATTGTGAA 1485

TATTAACTGA GATAGTGAAA CATGGTGTCC GGTTTTCTAT TGCATTTTTT CAAGTGGAAA 1545

AGTTAACTAA ATGGTTGACA CACAAAAATT GGTGGAGAAA TTGTGCATAT GCCAATTTTT 1605

TGTTAAAACC TTTTGTTTTG AACTATACTG CTTTGAGATC TCATTTCAGA AGAACGGCAT 1665

GAACAGTCTT CAGCCACAGT TGTGATGGTT GTTAAATGCT CACAATTGTG CATTCTTAGG 1725

GTTTTTCCAT CCCTGGGGTT TGCAAGTTGT TCACTTAAAA CATTCTTAAA ATGGTTGGCT 1785

TCTTGTCTGC AAGCCAGCTG ATATGGTAGC AACCAAAGAT TCCAGTGTTT GAGCATATGA 1845

AAGACTCTGC CTGCTTAATT GTGCTAGAAA TAACAGCATC TAAAGTGAAG ACTTAAGAAA 1905 AACTTAGTGA CTACTAGATT ATCCTTAGGA CTCTGCATTA ACTCTATAAT GTTCTTGGTA 1965

TTAAAAAAAA AGCATATTTG TCACAGAAAT TTAGTTAACA TCTTACAACT GAACATGTAT 2025

GTATGTTGCT TAGATAAATG TAATCACTGT AAACATCTAT ATGATCTGGG ATTTTGTTTT 2085

TATTTTGAAA TGGGAGCTTT TTTGTTTACA AGTTCATTAA AAACTAAAAA CTGTTTCTGT 2145

AAGGAAATGA GATTTTTTTT AAACAACAAA AAATGCCTTG CTGACTCACT ATTAAATAAA 2205

AATCTCCCCA ATTTTTTGAT AGACTACTTC AAGCCATTTG TTACATGGTA TTCCTTTGCA 2265

AGTCAATTTA GGTTTCGTGT TATAACTTTT CCTCTTTTTT TAAGAAAAAT GAAAAAAGTA 2325

ATTCTTTTGT CTGAAGGGGA AAGGCATTCT TTCATTTTTT TCTTTTTTTT TTTTTTTTTT 2385

TTATGACTTG CAGGCACAAT ATCTAGTACT GCAACTGCCA GAACTTGGTA TTGTAGCTGC 2445

TGCCCGCTGA CTAGCAGCTG GACTGATTTT GAATAAAAAT GAAAGCAGTA CTGGGATTAC 2505

AGGTGAGCCA CAGTGCCTGG CCCTTTTTTG TTTTTATTGT CTGTCTCCCC ACTAGAAGGT 2565

ACGCTCTACA AGGGCAGGGA TTTGTGCATC TTATTCATAG TGTTTCCCAC GTGGCAGATG 2625

CTCACTAAAG ATTTCAAAGG AGAAACTGTG ATGGACTCGT TCTGTAGATG AGAGAACAGA 2685

GGCACAGAGA CCTGTCCATG GTCCCCTGGC AGAAGGAGGT GGGGTCTGGA TTCCACCCCA 2745

GGGCTGCGTG GCTGCAGGAC CTCAGTGCTT GACTCCACAC TGCTGAGGGC TGTGAGTCCC 2805

TGGCCAGCCC AGACACAGTC CTGCAGCCCA GGCTGAGCAT TCTCAGACCT TCATGGAGAT 2865

GCCCACTCTC CTGTGAGCCT CCTGCTTCCT TTGCCCAGGG CCGGAATTC 2914

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 337 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Ala Ser Ser Ser Gly Ser Lys Ala Glu Phe He Val Gly Gly Lys 1 5 10 15

Tyr Lys Leu Val Arg Lys He Gly Ser Gly Ser Phe Gly Asp He Tyr 20 25 30

Leu Ala He Asn He Thr Asn Gly Glu Glu Val Ala Val Lys Leu Glu 35 40 45

Ser Gin Lys Ala Arg His Pro Gin Leu Leu Tyr Glu Ser Lys Leu Tyr 50 55 60

Lys He Leu Gin Gly Gly Val Gly He Pro His He Arg Trp Tyr Gly 65 70 75 80

Gin Glu Lys Asp Tyr Asn Val Leu Val Met Asp Leu Leu Gly Pro Ser 85 90 95

Leu Glu Asp Leu Phe Asn Phe Cys Ser Arg Arg Phe Thr Met Lys Thr 100 105 110 Val Leu Met Leu Ala Asp Gin Met He Ser Arg He Glu Tyr Val His 115 120 125

Thr Lys Asn Phe He His Arg Asp He Lys Pro Asp Asn Phe Leu Met 130 135 140

Gly He Gly Arg His Cys Asn Lys Leu Phe Leu He Asp Phe Gly Leu 145 150 155 160

Ala Lys Lys Tyr Arg Asp Asn Arg Thr Arg Gin His He Pro Tyr Arg 165 170 175

Glu Asp Lys Asn Leu Thr Gly Thr Ala Arg Tyr Ala Ser He Asn Ala 180 185 190

His Leu Gly He Glu Gin Ser Arg Arg Asp Asp Met Glu Ser Leu Gly 195 200 205

Tyr Val Leu Met Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gin Gly Leu 210 215 220

Lys Ala Ala Thr Lys Lys Gin Lys Tyr Glu Lys He Ser Glu Lys Lys 225 230 235 240

Met Ser Thr Pro Val Glu Val Leu Cys Lys Gly Phe Pro Ala Glu Phe 245 250 255

Ala Met Tyr Leu Asn Tyr Cys Arg Gly Leu Arg Phe Glu Glu Ala Pro 260 265 270

Asp Tyr Met Tyr Leu Arg Gin Leu Phe Arg He Leu Phe Arg Thr Leu 275 280 285

Asn His Gin Tyr Asp Tyr Thr Phe Asp Trp Thr Met Leu Lys Gin Lys 290 295 300

Ala Ala Gin Gin Ala Ala Ser Ser Ser Gly Gin Gly Gin Gin Ala Gin 305 310 315 320

Thr Pro Thr Gly Lys Gin Thr Asp Lys Thr Lys Ser Asn Met Lys Gly 325 330 335

Phe

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..23

(D) OTHER INFORMATION: /note= "Bases designated N at positions 3, 6, 9, 12 and 18 are Inosine." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGNWSNGGNW SNTTYGGNGA YAT 23

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..23

(D) OTHER INFORMATION: /note= "Bases designated N at positions 6, 12 and 18 are Inosine."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CAYGMNGAYA TNAARCCNGA YAA 23

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..24

(D) OTHER INFORMATION: /note= "Bases designated N at positions 7, 13 and 19 are Inosine."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: RTTRTCNGGY TTNATRTCNC KRTG 24

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 1..18

(D) OTHER INFORMATION: /note= "Bases designated N at positions 1, 4, 7 and 13 are Inosine."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: NCCNARNSWY TCNARRTC 18

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATATAAACTG GTACGGAAGA 20

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..17

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACATACGGTG GTATGGT 17

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..19

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

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..19

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CCTAATGATT CCATGTCAT 19

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TCAGGTACAT GTAATCCG 18

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..39

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCTGATCGAT TCCAGCCTGA TCGCTACTTC TTCACCACT 39

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3627 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(i ) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1633..3204

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

GATCAGATGA TATAGCTTTT TGTGTGCCGT ACCTTTCCGC GATTCTGCCC GTATATCTTG 60

GTCCCTGAGC TATTTTCTGA GATTCTTTTT GTTGCTTTGC CAAATCATTG GCGTCATTCA 120

TGGTCATACC AAATCCCAAT TTGGCAAACT TGGGTGTTAA AGTATCTTGC TGTTCTTTTC 180

TAGTTGTGTC GAAGCTGTTT GAAGTGTCAT TTAAAAAATC ATTGAATTCA TCAGGCTGGG 240

TATTAATATC ATCTATACTG TTATTATTGT TGCCTTTACT GTTATTCATA AATTGGGAAT 300

CGTAATCATT TGTCTAATTT TGGTGCTAGA AGACGAATTA GTGAACTCGT CCTCCTTTTC 360

TTGTTGAGCC TCTTTTTTAA ATTGATCAAA CAAGTCTTCT GCCTGTGATT TGTCGACTTT 420

CTTTGCGGTT AGTCTAGTGG GCTTTCTTGA CGAAGACAAA ATTGAATGTT TCTTTTTATC 480

TTGCGAGTTT AATACCGGTT TCTTTCTGCA TGCCGTTAAG ATGGAACTCT CGTTTTAGTG 540

ACAGTGGTCT TGGGTGTGCT GCCTGTGGTG TTGTTTTTTG GGGCGAGAGA GCCTGTATTT 600

ACATTGAGTT TAGAACTGGA ATTGGAGCTT GGTTTTTGCC AATTAGAGAA AAAATCGTCA 660

ACACTATTTT CTTTGGAAGT CGACCTGGAA GCGTCTGAAT CGGTGTCCAA CGGTGAGTCC 720

GAAGAATCTT GACCGTTCAA GACTAATTCT GATGGGTATA ACTCCATATC CTTTTGAACC 780

TTCTTGTCGA GATGTATCTT ATATTTCTTA GCAACAGGGC TCGTATATTT TGTTTTCGCG 840

TCAACATTTG CTGTATTTAG TAGCTGTTTC CCATTGTTCT TTAAGAAAAA ATCACGAGCC 900

TTATGGTTCC CACCCAACTT AAACCTTCTT AAATTGTTAA TTGTCCATTT ATCTAATGTA 960

GAAGACTTTA CAAAGGTGAT ATGAACACCC ATGTTTCTAT GCACAGCAGA GCATTGAATA 1020 CACAGCATCA CACCAAAAGG TACCGAAGTC CAGTAGGATT CTTGTTACCA CAATCAAAAC 1080

AAACTCGATT TTCCATGTTG CTACCTAGCT TCTGAAAAAC TTGTTGAGTA GTCTGTTCCG 1140

TGGCAAATGT TTCTCCTTCA TCGTTACTCA TTGTCGCTAT GTGTATACTA AATTGCTCAA 1200

GAAGACCGGA TCAACAAGTA CTTAACAAAT ACCCTTTCTT TGCTATCGCC TTGATCTCCT 1260

TTTATAAAAT GCCAGCTAAA TCGTGTTTAC GAAGAATAGT TGTTTTCTTT TTTTTTTTTT 1320

TTTTTCGAAA CTTTACCGTG TCGTCGAAAA TGACCAAACG ATGTTACTTT TCCTTTTGTG 1380

TCATAGATAA TACCAATATT GAAAGTAAAA TTTTAAACAT TCTATAGGTG AATTGAAAAG 1440

GGCAGCTTAG AGAGTAACAG GGGAACAGCA TTCGTAACAT CTAGGTACTG GTATTATTTG 1500

CTGTTTTTTA AAAAAGAAGG AAATCCGTTT TGCAAGAATT GTCTGCTATT TAAGGGTATA 1560

CGTGCTACGG TCCACTAATC AAAAGTGGTA TCTCATTCTG AAGAAAAAGT GTAAAAAGGA 1620

CGATAAGGAA AG ATG TCC CAA CGA TCT TCA CAA CAC ATT GTA GGT ATT 1668 Met Ser Gin Arg Ser Ser Gin His He Val Gly He

1 5 10

CAT TAT GCT GTA GGA CCT AAG ATT GGC GAA GGG TCT TTC GGA GTA A.TA 1716 His Tyr Ala Val Gly Pro Lys He Gly Glu Gly Ser Phe Gly Val He 15 20 25

TTT GAG GGA GAG AAC ATT CTT CAT TCT TGT CAA GCG CAG ACC GGT AGC 1764 Phe Glu Gly Glu Asn He Leu His Ser Cys Gin Ala Gin Thr Gly Ser 30 35 40

AAG AGG GAC TCT AGT ATA ATA ATG GCG AAC GAG CCA GTC GCA ATT AAA 1812 Lys Arg Asp Ser Ser He He Met Ala Asn Glu Pro Val Ala He Lys 45 50 55 60

TTC GAA CCG CGA CAT TCG GAC GCA CCC CAG TTG CGT GAC GAA TTT AGA 1860 Phe Glu Pro Arg His Ser Asp Ala Pro Gin Leu Arg Asp Glu Phe Arg 65 70 75

GCC TAT AGG ATA TTG AAT GGC TGC GTT GGA ATT CCC CAT GCT TAT TAT 1908 Ala Tyr Arg He Leu Asn Gly Cys Val Gly He Pro His Ala Tyr Tyr 80 85 90

TTT GGT CAA GAA GGT ATG CAC AAC ATC TTG ATT ATC GAT TTA CTA GGG 1956 Phe Gly Gin Glu Gly Met His Asn He Leu He He Asp Leu Leu Gly 95 100 105

CCA TCA TTG GAA GAT CTC TTT GAG TGG TGT GGT AGA AAA TTT TCA GTG 2004 Pro Ser Leu Glu Asp Leu Phe Glu Trp Cys Gly Arg Lys Phe Ser Val 110 115 120

AAA ACA ACC TGT ATG GTT GCC AAG CAA ATG ATT GAT AGA GTT AGA GCA 2052 Lys Thr Thr Cys Met Val Ala Lys Gin Met He Asp Arg Val Arg Ala 125 130 135 140

ATT CAT GAT CAC GAC TTA ATC TAT CGC GAT ATT AAA CCC GAT AAC TTT 2100 He His Asp His Asp Leu He Tyr Arg Asp He Lys Pro Asp Asn Phe 145 150 155

TTA ATT TCT CAA TAT CAA AGA ATT TCA CCT GAA GGA AAA GTC ATT AAA 2148 Leu He Ser Gin Tyr Gin Arg He Ser Pro Glu Gly Lys Val He Lys 160 165 170

TCA TGT GCC TCC TCT TCT AAT AAT GAT CCC AAT TTA ATA TAC ATG GTT 2196 Ser Cys Ala Ser Ser Ser Asn Asn Asp Pro Asn Leu He Tyr Met Val 175 180 185 GAC TTT GGT ATG GCA AAA CAA TAT AGA GAT CCA AGA ACG AAA CAA CAT 2244 Asp Phe Gly Met Ala Lys Gin Tyr Arg Asp Pro Arg Thr Lys Gin His 190 195 200

ATA CCA TAC CGT GAA CGA AAA TCA TTG AGC GGT ACC GCC AGA TAT ATG 2292 He Pro Tyr Arg Glu Arg Lys Ser Leu Ser Gly Thr Ala Arg Tyr Met 205 210 215 220

TCT ATT AAT ACT CAT TTT GGA AGA GAA CAG TCA CGT AGG GAT GAT TTA 2340 Ser He Asn Thr His Phe Gly Arg Glu Gin Ser Arg Arg Asp Asp Leu 225 230 235

GAA TCG CTA GGT CAC GTT TTT TTT TAT TTC TTG AGG GGA TCC TTG CCA 2388 Glu Ser Leu Gly His Val Phe Phe Tyr Phe Leu Arg Gly Ser Leu Pro 240 245 250

TGG CAA GGT TTG AAA GCA CCA AAC AAC AAA CTG AAG TAT GAA AAG ATT 2436 Trp Gin Gly Leu Lys Ala Pro Asn Asn Lys Leu Lys Tyr Glu Lys He 255 260 265

GGT ATG ACT AAA CAG AAA TTG AAT CCT GAT GAT CTT TTA TTG AAT AAT 2484 Gly Met Thr Lys Gin Lys Leu Asn Pro Asp Asp Leu Leu Leu Asn Asn 270 275 280

GCT ATT CCT TAT CAG TTT GCC ACA TAT TTA AAA TAT GCA CGT TCC TTG 2532 Ala He Pro Tyr Gin Phe Ala Thr Tyr Leu Lys Tyr Ala Arg Ser Leu 285 290 295 300

AAG TTC GAC GAA GAT CCG GAT TAT GAC TAT TTA ATC TCG TTA ATG GAT 2580 Lys Phe Asp Glu Asp Pro Asp Tyr Asp Tyr Leu He Ser Leu Met Asp 305 310 315

GAC GCT TTG AGA TTA AAC GAC TTA AAG GAT GAT GGA CAC TAT GAC TGG 2628 Asp Ala Leu Arg Leu Asn Asp Leu Lys Asp Asp Gly His Tyr Asp Trp 320 325 330

ATG GAT TTG AAT GGT GGT AAA GGC TGG AAT ATC AAG ATT AAT AGA AGA 2676 Met Asp Leu Asn Gly Gly Lys Gly Trp Asn He Lys He Asn Arg Arg 335 340 345

GCT AAC TTG CAT GGT TAC GGA AAT CCA AAT CCA AGA GTC AAT GGC AAT 2724 Ala Asn Leu His Gly Tyr Gly Asn Pro Asn Pro Arg Val Asn Gly Asn 350 355 360

ACT GCA AGA AAC AAT GTG AAT ACG AAT TCA AAG ACA CGA AAT ACA ACG 2772 Thr Ala Arg Asn Asn Val Asn Thr Asn Ser Lys Thr Arg Asn Thr Thr 365 370 375 380

CCA GTT GCG ACA CCT AAG CAA CAA GCT CAA AAC AGT TAT AAC AAG GAC 2820 Pro Val Ala Thr Pro Lys Gin Gin Ala Gin Asn Ser Tyr Asn Lys Asp 385 390 395

AAT TCG AAA TCC AGA ATT TCT TCG AAC CCG CAG AGC TTT ACT AAA CAA 2868 Asn Ser Lys Ser Arg He Ser Ser Asn Pro Gin Ser Phe Thr Lys Gin 400 405 410

CAA CAC GTC TTG AAA AAA ATC GAA CCC AAT AGT AAA TAT ATT CCT GAA 2916 Gin His Val Leu Lys Lys He Glu Pro Asn Ser Lys Tyr He Pro Glu 415 420 425

ACA CAT TCA AAT CTT CAA CGG CCA ATT AAA AGT CAA AGT CAA ACG TAC 2964 Thr His Ser Asn Leu Gin Arg Pro He Lys Ser Gin Ser Gin Thr Tyr 430 435 440

GAC TCC ATC AGT CAT ACA CAA AAT TCA CCA TTT GTA CCA TAT TCA AGT 3012 Asp Ser He Ser His Thr Gin Asn Ser Pro Phe Val Pro Tyr Ser Ser 445 450 455 460 TCT AAA GCT AAC CCT AAA AGA AGT AAT AAT GAG CAC AAC TTA CCA AAC 3060 Ser Lys Ala Asn Pro Lys Arg Ser Asn Asn Glu His Asn Leu Pro Asn 465 470 475

CAC TAC ACA AAC CTT GCA AAT AAG AAT ATC AAT TAT CAA AGT CAA CGA 3108 His Tyr Thr Asn Leu Ala Asn Lys Asn He Asn Tyr Gin Ser Gin Arg 480 485 490

AAT TAC GAA CAA GAA AAT GAT GCT TAT TCT GAT GAC GAG AAT GAT ACA 3156 Asn Tyr Glu Gin Glu Asn Asp Ala Tyr Ser Asp Asp Glu Asn Asp Thr 495 500 505

TTT TGT TCT AAA ATA TAC AAA TAT TGT TGT TGC TGT TTT TGT TGC TGT 3204 Phe Cys Ser Lys He Tyr Lys Tyr Cys Cys Cys Cys Phe Cys Cys Cys 510 515 520

TGATAAAGCG ATTTTTATAC TTTTCTCTTT TTCCTTTTTT TTTTTGATTG GCTGTTTCCT 3264

TATGCCGCTC TTTCCCAATT TATGACTTTC CAATAATGTA TTATTTTGTT TCTCTTTCTC 3324

TCTGTTACCC TTTATTTTAT CATCTACAAT AATTGAATTC CGGAGAGGGT AAAGAAACAG 3384

GAAAAAGAAG AAAATGAGAC ATAGTCAGCA TCGTAATCGT TTTCCTTCTG TATATTCCTT 3444

TATCAAAAGA CTACACGCAC ATATATATTA ATCCCGGTAT GTTTTTGGTG TGCTAAATCT 3504

ATCTTCAAGC ACTATTATAG CATTTTTTTA AGAATATCCA AAATAATATG TAATTTATGA 3564

TTAATCAAGG TTCAAGAATT GGAGAAACCG TGAGCGACTT CTTTGATACT TGGATGTAAG 3624

CTT 3627

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 524 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Ser Gin Arg Ser Ser Gin His He Val Gly He His Tyr Ala Val 1 5 10 15

Gly Pro Lys He Gly Glu Gly Ser Phe Gly Val He Phe Glu Gly Glu 20 25 30

Asn He Leu His Ser Cys Gin Ala Gin Thr Gly Ser Lys Arg Asp Ser 35 40 45

Ser He He Met Ala Asn Glu Pro Val Ala He Lys Phe Glu Pro Arg 50 55 60

His Ser Asp Ala Pro Gin Leu Arg Asp Glu Phe Arg Ala Tyr Arg He 65 70 75 80

Leu Asn Gly Cys Val Gly He Pro His Ala Tyr Tyr Phe Gly Gin Glu 85 90 95

Gly Met His Asn He Leu He He Asp Leu Leu Gly Pro Ser Leu Glu 100 105 110

Asp Leu Phe Glu Trp Cys Gly Arg Lys Phe Ser Val Lys Thr Thr Cys

115 120 125 Met Val Ala Lys Gin Met He Asp Arg Val Arg Ala He His Asp His 130 135 140

Asp Leu He Tyr Arg Asp He Lys Pro Asp Asn Phe Leu He Ser Gin 145 150 155 160

Tyr Gin Arg He Ser Pro Glu Gly Lys Val He Lys Ser Cys Ala Ser 165 170 175

Ser Ser Asn Asn Asp Pro Asn Leu He Tyr Met Val Asp Phe Gly Met 180 185 190

Ala Lys Gin Tyr Arg Asp Pro Arg Thr Lys Gin His He Pro Tyr Arg 195 200 205

Glu Arg Lys Ser Leu Ser Gly Thr Ala Arg Tyr Met Ser He Asn Thr 210 215 220

His Phe Gly Arg Glu Gin Ser Arg Arg Asp Asp Leu Glu Ser Leu Gly 225 230 235 240

His Val Phe Phe Tyr Phe Leu Arg Gly Ser Leu Pro Trp Gin Gly Leu 245 250 255

Lys Ala Pro Asn Asn Lys Leu Lys Tyr Glu Lys He Gly Met Thr Lys 260 265 270

Gin Lys Leu Asn Pro Asp Asp Leu Leu Leu Asn Asn Ala He Pro Tyr 275 280 285

Gin Phe Ala Thr Tyr Leu Lys Tyr Ala Arg Ser Leu Lys Phe Asp Glu 290 295 300

Asp Pro Asp Tyr Asp Tyr Leu He Ser Leu Met Asp Asp Ala Leu Arg 305 310 315 320

Leu Asn Asp Leu Lys Asp Asp Gly His Tyr Asp Trp Met Asp Leu Asn 325 330 335

Gly Gly Lys Gly Trp Asn He Lys He Asn Arg Arg Ala Asn Leu His 340 345 350

Gly Tyr Gly Asn Pro Asn Pro Arg Val Asn Gly Asn Thr Ala Arg Asn 355 360 365

Asn Val Asn Thr Asn Ser Lys Thr Arg Asn Thr Thr Pro Val Ala Thr 370 375 380

Pro Lys Gin Gin Ala Gin Asn Ser Tyr Asn Lys Asp Asn Ser Lys Ser 385 390 395 400

Arg He Ser Ser Asn Pro Gin Ser Phe Thr Lys Gin Gin His Val Leu 405 410 415

Lys Lys He Glu Pro Asn Ser Lys Tyr He Pro Glu Thr His Ser Asn 420 425 430

Leu Gin Arg Pro He Lys Ser Gin Ser Gin Thr Tyr Asp Ser He Ser 435 440 445

His Thr Gin Asn Ser Pro Phe Val Pro Tyr' Ser Ser Ser Lys Ala Asn 450 455 460

Pro Lys Arg Ser Asn Asn Glu His Asn Leu Pro Asn His Tyr Thr Asn 465 470 475 480 Leu Ala Asn Lys Asn He Asn Tyr Gin Ser Gin Arg Asn Tyr Glu Gin 485 490 495

Glu Asn Asp Ala Tyr Ser Asp Asp Glu Asn Asp Thr Phe Cys Ser Lys 500 505 510

He Tyr Lys Tyr Cys Cys Cys Cys Phe Cys Cys Cys 515 520

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..6

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

Gly Pro Ser Leu Glu Asp

1 5

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..9

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

Arg Asp He Lys Pro Asp Asn Phe Leu

1 5

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (vii) IMMEDIATE SOURCE:

(B) CLONE: Protein Kinase

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..6

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

His He Pro Tyr Arg Glu 1 5

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine. "

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

GARYTNMGNY TNGGNAAYYT N 21

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA ( ix ) FEATURE :

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine. "

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(C) OTHER INFORMATION: /note= "The nucleotide at this position is inosine."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GTYTTRTTNC CNGGNCKNCC NAT 23

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2405 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 67..1197

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

AAAGTGGAGT ACCGCAAACT TGATATGGAA AATAAAAAGA AAGACAAGGA CAAATCAGAT 60

GATAGA ATG GCA CGA CCT AGT GGT CGA TCG GGA CAC AAC ACT CGA GGA 108 Met Ala Arg Pro Ser Gly Arg Ser Gly His Asn Thr Arg Gly 1 5 10 ACT GGG TCT TCA TCG TCT GGA GTT TTA ATG GTT GGA CCT AAC TTT AGA 156 Thr Gly Ser Ser Ser Ser Gly Val Leu Met Val Gly Pro Asn Phe Arg 15 20 25 30

GTT GGA AAA AAA ATT GGA TGT GGC AAT TTT GGA GAA TTA CGA TTA GGG 204 Val Gly Lys Lys He Gly Cys Gly Asn Phe Gly Glu Leu Arg Leu Gly 35 40 45

AAA AAT TTA TAC ACA AAT GAA TAT GTG GCA ATT AAG TTG GAG CCC ATG 252 Lys Asn Leu Tyr Thr Asn Glu Tyr Val Ala He Lys Leu Glu Pro Met 50 55 60

AAA TCA AGA GCA CCA CAG CTA CAT TTG GAA TAC AGA TTC TAT AAG CAG 300 Lys Ser Arg Ala Pro Gin Leu His Leu Glu Tyr Arg Phe Tyr Lys Gin 65 70 75

TTA GGA TCT GGA GAT GGT ATA CCT CAA GTT TAC TAT TTC GGC CCC TGT 348 Leu Gly Ser Gly Asp Gly He Pro Gin Val Tyr Tyr Phe Gly Pro Cys 80 85 90

GGT AAA TAC AAT GCT ATG GTG CTG GAA CTG CTG GGA CCT AGT TTG GAA 396 Gly Lys Tyr Asn Ala Met Val Leu Glu Leu Leu Gly Pro Ser Leu Glu 95 100 105 110

GAC TTG TTT GAC TTG TGT GAC AGA ACA TTT TCT CTT AAA ACA GTT CTC 444 Asp Leu Phe Asp Leu Cys Asp Arg Thr Phe Ser Leu Lys Thr Val Leu 115 120 125

ATG ATA GCT ATA CAA CTG ATT TCT CGC ATG GAA TAT GTC CAT TCA AAG 492 Met He Ala He Gin Leu He Ser Arg Met Glu Tyr Val His Ser Lys 130 135 140

AAC TTG ATA TAC AGA GAT GTA AAA CCT GAG AAC TTC TTA ATA GGA CGA 540 Asn Leu He Tyr Arg Asp Val Lys Pro Glu Asn Phe Leu He Gly Arg 145 150 155

CCA GGA AAC AAA ACC CAG CAA GTT ATT CAC ATT ATA GAT TTT GGT TTG 588 Pro Gly Asn Lys Thr Gin Gin Val He His He He Asp Phe Gly Leu 160 165 170

GCA AAG GAA TAT ATT GAT CCG GAG ACA AAG AAA CAC ATA CCA TAC AGA 636 Ala Lys Glu Tyr He Asp Pro Glu Thr Lys Lys His He Pro Tyr Arg 175 180 185 190

GAA CAC AAG AGC CTT ACA GGA ACA GCT AGA TAT ATG AGC ATA AAC ACA 684 Glu His Lys Ser Leu Thr Gly Thr Ala Arg Tyr Met Ser He Asn Thr 195 200 205

CAT TTA GGA AAA GAA CAA AGT AGA AGA GAC GAT TTA GAA GCT TTA GGT 732 His Leu Gly Lys Glu Gin Ser Arg Arg Asp Asp Leu Glu Ala Leu Gly 210 215 220

CAT ATG TTC ATG TAT TTT CTG AGA GGC AGT CTT CCT TGG CAA GGC TTA 780 His Met Phe Met Tyr Phe Leu Arg Gly Ser Leu Pro Trp Gin Gly Leu 225 230 235

AAG GCT GAC ACA TTA AAG GAG AGG TAT CAG AAA ATT GGA GAT ACA AAA 828 Lys Ala Asp Thr Leu Lys Glu Arg Tyr Gin Lys He Gly Asp Thr Lys 240 245 250

CGG GCT ACA CCA ATA GAA GTG TTA TGT GAA AAT TTT CCA GAA GAA ATG 876 Arg Ala Thr Pro He Glu Val Leu Cys Glu Asn Phe Pro Glu Glu Met 255 260 265 270

GCA ACA TAT CTT CGT TAT GTA AGA AGG CTA GAT TTT TTT GAA AAA CCA 924 Ala Thr Tyr Leu Arg Tyr Val Arg Arg Leu Asp Phe Phe Glu Lys Pro 275 280 285 GAC TAT GAC TAC TTA AGA AAG CTT TTT ACT GAC TTG TTT GAT CGA AAA 972 Asp Tyr Asp Tyr Leu Arg Lys Leu Phe Thr Asp Leu Phe Asp Arg Lys 290 295 300

GGA TAT ATG TTT GAT TAT GAA TAT GAC TGG ATT GGT AAA CAG TTG CCT 1020 Gly Tyr Met Phe Asp Tyr Glu Tyr Asp Trp He Gly Lys Gin Leu Pro 305 310 315

ACT CCA GTG GGT GCA GTT CAG CAA GAT CCT GCT CTG TCA TCA AAC AGA 1068 Thr Pro Val Gly Ala Val Gin Gin Asp Pro Ala Leu Ser Ser Asn Arg 320 325 330

GAA GCA CAT CAA CAC AGA GAT AAG ATG CAA CAA TCC AAA AAC CAG GTT 1116 Glu Ala His Gin His Arg Asp Lys Met Gin Gin Ser Lys Asn Gin Val 335 340 345 350

GTA AGT TCT ACA AAT GGA GAG TTA AAC ACA GAT GAC CCC ACC GCA GAC 1164 Val Ser Ser Thr Asn Gly Glu Leu Asn Thr Asp Asp Pro Thr Ala Asp 355 360 365

GTT CAA ATG CAC CCA TCA CAG CCC CTA CTG AAG TAGAAGTGAT GGATGAAACC 1217 Val Gin Met His Pro Ser Gin Pro Leu Leu Lys 370 375

AACTGCCAGA AAGTGTTGAA CATGTGGTGC TGCTGTTTTT TCAAACGAAG GAAAAGGAAA 1277

ACCATACAGC GCCACAAATG ACTCTGGACA CAGACAGATC CTGGGGAGTT ACTTACATGT 1337

TCATCTGCTG TCTTGTGATT AAAATCATCT CTGTAGTGAC CACGTATATT TTCAAGGACT 1397

CACTCTTAGA AACAAAAATG TCATACTTTC ATACTTCATT TTGTGGTTGT CTTACATTCT 1457

TTTTCTTTTT TTTTTTCTCT AATTTAACCT TTATGGAAGC TTTAAAGTTT TGTCAAAAAC 1517

ATGAGTGCTT TTGCCCCATC AGTGAATGGA ATGGACCAAT GAGGTGGTAT CAATGAATAT 1577

AGTTCCATAG AACATTTCCA GAAGTTCTTC TGTTGTAGAA AGCAGTACAG TATCTTAAGT 1637

GTCAACCAGT TATATACCTA ATCTGGTTTT TTATAACTTC TGTAAGAGCA TAATCAAACA 1697

GGAATTTTCT TTTCTCAGTG GATAATACAA CAGAGAAAAC AGAGTTGCCC AAATATTTAA 1757

AAGAAGTTAT TCCTTGAGAA GTTCATATTT TGTGACATCT GCATTGATTT CAGTATTACT 1817

GATGGTACTG TTATTCATAA GTCATATTAA CATTCTCTCC GTGAAATCAT GGTACAGTCG 1877

CTGCCCAGAG GTACTGAGGA AAAAGCAATA TGGGTTCGGC AGATGGTGGT GGTAAAATGA 1937

ATCTTAAGGA GTGTGGTAAA TATGCGTCCG CTTTTGTTGC ATCACTATGT GAAGTACTGT 1997

GTTGCAGAAG TGGCAAAAGC GCTTATTTTT AAAAATGCAA AATATTTGTA CAATGTAACT 2057

TTATGCTTCC AAATAATAAT GTATGTTAGA CAGCAAGAAA TGAATACTTT AAAAAGTGAT 2117

GTATGTTGGA GTTATAAAGA AATACACTAA GGAGAGGTAG TAAATGTGAA CCTTGTTGCA 2177

GTGTATAAGG TGGAAGCCTA AAGAAATCTC ACCGAAACTT ACTGCTGAAT GATTACATTC 2237

TCCCTTAAGC AGAAAACTTT GGATGTGCCA TGCAATGGTG TCTGTGTAAT TATTTTGCTC 2297

TTTGATTAAA AAAAAGACCC CCAGCAATAA AAAGTGGGTC ACTCTAAAAA AAAAAAAAAA 2357

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA ACGACAGCAA CGGAATTC 2405 (2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 377 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Met Ala Arg Pro Ser Gly Arg Ser Gly His Asn Thr Arg Gly Thr Gly 1 5 10 15

Ser Ser Ser Ser Gly Val Leu Met Val Gly Pro Asn Phe Arg Val Gly 20 25 30

Lys Lys He Gly Cys Gly Asn Phe Gly Glu Leu Arg Leu Gly Lys Asn 35 40 45

Leu Tyr Thr Asn Glu Tyr Val Ala He Lys Leu Glu Pro Met Lys Ser 50 55 60

Arg Ala Pro Gin Leu His Leu Glu Tyr Arg Phe Tyr Lys Gin Leu Gly 65 70 75 80

Ser Gly Asp Gly He Pro Gin Val Tyr Tyr Phe Gly Pro Cys Gly Lys 85 90 95

Tyr Asn Ala Met Val Leu Glu Leu Leu Gly Pro Ser Leu Glu Asp Leu 100 105 110

Phe Asp Leu Cys Asp Arg Thr Phe Ser Leu Lys Thr Val Leu Met He

115 120 125

Ala He Gin Leu He Ser Arg Met Glu Tyr Val His Ser Lys Asn Leu 130 135 140

He Tyr Arg Asp Val Lys Pro Glu Asn Phe Leu He Gly Arg Pro Gly 145 150 155 160

Asn Lys Thr Gin Gin Val He His He He Asp Phe Gly Leu Ala Lys 165 170 175

Glu Tyr He Asp Pro Glu Thr Lys Lys His He Pro Tyr Arg Glu His 180 185 190

Lys Ser Leu Thr Gly Thr Ala Arg Tyr Met Ser He Asn Thr His Leu 195 200 205

Gly Lys Glu Gin Ser Arg Arg Asp Asp Leu Glu Ala Leu Gly His Met 210 215 220

Phe Met Tyr Phe Leu Arg Gly Ser Leu Pro Trp Gin Gly Leu Lys Ala 225 230 235 240

Asp Thr Leu Lys Glu Arg Tyr Gin Lys He Gly Asp Thr Lys Arg Ala 245 250 255

Thr Pro He Glu Val Leu Cys Glu Asn Phe Pro Glu Glu Met Ala Thr 260 265 270

Tyr Leu Arg Tyr Val Arg Arg Leu Asp Phe Phe Glu Lys Pro Asp Tyr 275 280 285

Asp Tyr Leu Arg Lys Leu Phe Thr Asp Leu Phe Asp Arg Lys Gly Tyr 290 295 300 Met Phe Asp Tyr Glu Tyr Asp Trp He Gly Lys Gin Leu Pro Thr Pro 305 310 315 320

Val Gly Ala Val Gin Gin Asp Pro Ala Leu Ser Ser Asn Arg Glu Ala 325 330 335

His Gin His Arg Asp Lys Met Gin Gin Ser Lys Asn Gin Val Val Ser 340 345 350

Ser Thr Asn Gly Glu Leu Asn Thr Asp Asp Pro Thr Ala Asp Val Gin 355 360 365

Met His Pro Ser Gin Pro Leu Leu Lys 370 375

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1233 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..1041

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

AGA GTT GGA AAA AAA ATT GGA TGT GGC AAT TTT GGA GAA TTA CGA TTA 48 Arg Val Gly Lys Lys He Gly Cys Gly Asn Phe Gly Glu Leu Arg Leu 1 5 10 15

GGG AAA AAT TTA TAC ACA AAT GAA TAT GTG GCA ATT AAG TTG GAG CCC 96 Gly Lys Asn Leu Tyr Thr Asn Glu Tyr Val Ala He Lys Leu Glu Pro 20 25 30

ATG AAA TCA AGA GCA CCA CAG CTA CAT TTG GAA TAC AGA TTC TAT AAG 144 Met Lys Ser Arg Ala Pro Gin Leu His Leu Glu Tyr Arg Phe Tyr Lys 35 40 45

CAG TTA GGA TCT GGA GAT GGT ATA CCT CAA GTT TAC TAT TTC GGC CCT 192 Gin Leu Gly Ser Gly Asp Gly He Pro Gin Val Tyr Tyr Phe Gly Pro 50 55 60

TGT GGT AAA TAC AAT GCT ATG GTG CTG GAA CTG CTG GGA CCT AGT TTG 240 Cys Gly Lys Tyr Asn Ala Met Val Leu Glu Leu Leu Gly Pro Ser Leu 65 70 75 80

GAA GAC TTG TTT GAC TTG TGT GAC AGA ACA TTT TCT CTT AAA ACA GTT 288 Glu Asp Leu Phe Asp Leu Cys Asp Arg Thr Phe Ser Leu Lys Thr Val 85 90 95

CTC ATG ATA GCT ATA CAA CTG ATT TCT CGC ATG GAA TAT GTC CAT TCA 336 Leu Met He Ala He Gin Leu He Ser Arg Met Glu Tyr Val His Ser 100 105 110

AAG AAC TTG ATA TAC AGA GAT GTA AAA CCT GAG AAC TTC TTA ATA GGA 384 Lys Asn Leu He Tyr Arg Asp Val Lys Pro Glu Asn Phe Leu He Gly

115 120 125 CGA CCA GGA AAC AAA ACC CAG CAA GTT ATT CAC ATT ATA GAT TTT GGT 432 Arg Pro Gly Asn Lys Thr Gin Gin Val He His He He Asp Phe Gly 130 135 140

TTG GCA AAG GAA TAT ATT GAT CCG GAG ACA AAG AAA CAC ATA CCA TAC 480 Leu Ala Lys Glu Tyr He Asp Pro Glu Thr Lys Lys His He Pro Tyr 145 150 155 160

AGA GAA CAC AAG AGC CTT ACA GGA ACA GCT AGA TAT ATG AGC ATA AAC 528 Arg Glu His Lys Ser Leu Thr Gly Thr Ala Arg Tyr Met Ser He Asn 165 170 175

ACA CAT TTA GGA AAA GAA CAA AGT AGA AGA GAC GAT TTA GAA GCT TTA 576 Thr His Leu Gly Lys Glu Gin Ser Arg Arg Asp Asp Leu Glu Ala Leu 180 185 190

GGT CAT ATG TTC ATG TAT TTT CTG AGA GGC AGT CTT CCT TGG CAA GGC 624 Gly His Met Phe Met Tyr Phe Leu Arg Gly Ser Leu Pro Trp Gin Gly 195 200 205

TTA AAG GTT GAC ACA TTA AAG GAG AGG TAT CAG AAA ATT GGA GAT ACA 672 Leu Lys Val Asp Thr Leu Lys Glu Arg Tyr Gin Lys He Gly Asp Thr 210 215 220

AAA CGG GCT ACA CCA ATA GAA GTG TTA TGT GAA AAT TTT CCA GAA ATG 720 Lys Arg Ala Thr Pro He Glu Val Leu Cys Glu Asn Phe Pro Glu Met 225 230 235 240

GCA ACA TAT CTT CGT TAT GTA AGA AGG CTA GAT TTT TTT GAA AAA CCA 768 Ala Thr Tyr Leu Arg Tyr Val Arg Arg Leu Asp Phe Phe Glu Lys Pro 245 250 255

GAC TAT GAC TAC TTA AGA AAG CTT TTT ACT GAC TTG TTT GAT CGA AAA 816 Asp Tyr Asp Tyr Leu Arg Lys Leu Phe Thr Asp Leu Phe Asp Arg Lys 260 265 270

GGA TAT ATG TTT GAT TAT GAA TAT GAC TGG ATT GGT AAA CAG TTG CCT 864 Gly Tyr Met Phe Asp Tyr Glu Tyr Asp Trp He Gly Lys Gin Leu Pro 275 280 285

ACT CCA GTG GGT GCA GTT CAG CAA GAT CCT GCT CTG TCA TCA AAC AGA 912 Thr Pro Val Gly Ala Val Gin Gin Asp Pro Ala Leu Ser Ser Asn Arg 290 295 300

GAA GCA CAT CAA CAC AGA GAT AAG ATG CAA CAA TCC AAA AAC CAG GTT 960 Glu Ala His Gin His Arg Asp Lys Met Gin Gin Ser Lys Asn Gin Val 305 310 315 320

GTA AGT TCT ACA AAT GGA GAG TTA AAC ACA GAT GAC CCC ACC GCA GAC 1008 Val Ser Ser Thr Asn Gly Glu Leu Asn Thr Asp Asp Pro Thr Ala Asp 325 330 335

GTT CAA ATG CAC CCA TCA CAG CCC CTA CTG AAG TAGAAGTGAT GGATGAAACC 1061 Val Gin Met His Pro Ser Gin Pro Leu Leu Lys 340 345

AACTGCCAGA AAGTGTTGAA CATGTGGTGC TGCTGTTTTT TCAAACGAAG GAAAAGGAAA 1121

ACCATACAGC GCCACAAATG ACTCTGGACA CAGACAGATC CTGGGGAGTT ACTTACATGT 1181

TCATCTGCTG TCTTGTGATT AAATCATCTC TGTAGTGACC ACGTATATTT TC 1233 (2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 347 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Arg Val Gly Lys Lys He Gly Cys Gly Asn Phe Gly Glu Leu Arg Leu 1 5 10 15

Gly Lys Asn Leu Tyr Thr Asn Glu Tyr Val Ala He Lys Leu Glu Pro 20 25 30

Met Lys Ser Arg Ala Pro Gin Leu His Leu Glu Tyr Arg Phe Tyr Lys 35 40 45

Gin Leu Gly Ser Gly Asp Gly He Pro Gin Val Tyr Tyr Phe Gly Pro 50 55 60

Cys Gly Lys Tyr Asn Ala Met Val Leu Glu Leu Leu Gly Pro Ser Leu 65 70 75 80

Glu Asp Leu Phe Asp Leu Cys Asp Arg Thr Phe Ser Leu Lys Thr Val 85 90 95

Leu Met He Ala He Gin Leu He Ser Arg Met Glu Tyr Val His Ser 100 105 110

Lys Asn Leu He Tyr Arg Asp Val Lys Pro Glu Asn Phe Leu He Gly 115 120 125

Arg Pro Gly Asn Lys Thr Gin Gin Val He His He He Asp Phe Gly 130 135 140

Leu Ala Lys Glu Tyr He Asp Pro Glu Thr Lys Lys His He Pro Tyr 145 150 155 160

Arg Glu His Lys Ser Leu Thr Gly Thr Ala Arg Tyr Met Ser He Asn 165 170 175

Thr His Leu Gly Lys Glu Gin Ser Arg Arg Asp Asp Leu Glu Ala Leu 180 185 190

Gly His Met Phe Met Tyr Phe Leu Arg Gly Ser Leu Pro Trp Gin Gly 195 200 205

Leu Lys Val Asp Thr Leu Lys Glu Arg Tyr Gin Lys He Gly Asp Thr 210 215 220

Lys Arg Ala Thr Pro He Glu Val Leu Cys Glu Asn Phe Pro Glu Met 225 230 235 240

Ala Thr Tyr Leu Arg Tyr Val Arg Arg Leu Asp Phe Phe Glu Lys Pro 245 250 255

Asp Tyr Asp Tyr Leu Arg Lys Leu Phe Thr Asp Leu Phe Asp Arg Lys 260 265 270

Gly Tyr Met Phe Asp Tyr Glu Tyr Asp Trp He Gly Lys Gin Leu Pro 275 280 285

Thr Pro Val Gly Ala Val Gin Gin Asp Pro Ala Leu Ser Ser Asn Arg 290 295 300 Glu Ala His Gin His Arg Asp Lys Met Gin Gin Ser Lys Asn Gin Val 305 310 315 320

Val Ser Ser Thr Asn Gly Glu Leu Asn Thr Asp Asp Pro Thr Ala Asp 325 330 335

Val Gin Met His Pro Ser Gin Pro Leu Leu Lys 340 345

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3505 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(i ) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 154..1398

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

GAATTCCGAC AGGAAAGCGA TGGTGAAAGC GGGGCCGTGA GGGGGGCGGA GCCGGGAGCC 60

GGACCCGCAG TAGCGGCAGC AGCGGCGCCG CCTCCCGGAG TTCAGACCCA GGAAGCGGCC 120

GGGAGGGCAG GAGCGAATCG GGCCGCCGCC GCC ATG GAG CTG AGA GTC GGG AAC 174

Met Glu Leu Arg Val Gly Asn

1 5

AGG TAC CGG CTG GGC CGG AAG ATC GGC AGC GGC TCC TTC GGA GAC ATC 222 Arg Tyr Arg Leu Gly Arg Lys He Gly Ser Gly Ser Phe Gly Asp He 10 15 20

TAT CTC GGT ACG GAC ATT GCT GCA GGA GAA GAG GTT GCC ATC AAG CTT 270 Tyr Leu Gly Thr Asp He Ala Ala Gly Glu Glu Val Ala He Lys Leu 25 30 35

GAA TGT GTC AAA ACC AAA CAC CCT CAG CTC CAC ATT GAG AGC AAA ATC 318 Glu Cys Val Lys Thr Lys His Pro Gin Leu His He Glu Ser Lys He 40 45 50 55

TAC AAG ATG ATG CAG GGA GGA GTG GGC ATC CCC ACC ATC AGA TGG TGC 366 Tyr Lys Met Met Gin Gly Gly Val Gly He Pro Thr He Arg Trp Cys 60 65 70

GGG GCA GAG GGG GAC TAC AAC GTC ATG GTG ATG GAG CTG CTG GGG CCA 414 Gly Ala Glu Gly Asp Tyr Asn Val Met Val Met Glu Leu Leu Gly Pro 75 80 85

AGC CTG GAG GAC CTC TTC AAC TTC TGC TCC AGG AAA TTC AGC CTC AAA 462 Ser Leu Glu Asp Leu Phe Asn Phe Cys Ser Arg Lys Phe Ser Leu Lys 90 95 100

ACC GTC CTG CTG CTT GCT GAC CAA ATG ATC AGT CGC ATC GAA TAC ATT 510 Thr Val Leu Leu Leu Ala Asp Gin Met He Ser Arg He Glu Tyr He 105 110 115

CAT TCA AAG AAC TTC ATC CAC CGG GAT GTG AAG CCA GAC AAC TTC CTC 558 His Ser Lys Asn Phe He His Arg Asp Val Lys Pro Asp Asn Phe Leu 120 125 130 135 ATG GGC CTG GGG AAG AAG GGC AAC CTG GTG TAC ATC ATC GAC TTC GGG 606 Met Gly Leu Gly Lys Lys Gly Asn Leu Val Tyr He He Asp Phe Gly 140 145 150

CTG GCC AAG AAG TAC CGG GAT GCA CGC ACC CAC CAG CAC ATC CCC TAT 654 Leu Ala Lys Lys Tyr Arg Asp Ala Arg Thr His Gin His He Pro Tyr 155 160 165

CGT GAG AAC AAG AAC CTC ACG GGG ACG GCG CGG TAC GCC TCC ATC AAC 702 Arg Glu Asn Lys Asn Leu Thr Gly Thr Ala Arg Tyr Ala Ser He Asn 170 175 180

ACG CAC CTT GGA ATT GAA CAA TCC CGA AGA GAT GAC TTG GAG TCT CTG 750 Thr His Leu Gly He Glu Gin Ser Arg Arg Asp Asp Leu Glu Ser Leu 185 190 195

GGC TAC GTG CTA ATG TAC TTC AAC CTG GGC TCT CTC CCC TGG CAG GGG 798 Gly Tyr Val Leu Met Tyr Phe Asn Leu Gly Ser Leu Pro Trp Gin Gly 200 205 210 215

CTG AAG GCT GCC ACC AAG AGA CAG AAA TAC GAA AGG ATT AGC GAG AAG 846 Leu Lys Ala Ala Thr Lys Arg Gin Lys Tyr Glu Arg He Ser Glu Lys 220 225 230

AAA ATG TCC ACC CCC ATC GAA GTG TTG TGT AAA GGC TAC CCT TCC GAA 894 Lys Met Ser Thr Pro He Glu Val Leu Cys Lys Gly Tyr Pro Ser Glu 235 240 245

TTT GCC ACA TAC CTG AAT TTC TGC CGT TCC TTG CGT TTT GAC GAC AAG 942 Phe Ala Thr Tyr Leu Asn Phe Cys Arg Ser Leu Arg Phe Asp Asp Lys 250 255 260

CCT GAC TAC TCG TAC CTG CGG CAG CTT TTC CGG AAT CTG TTC CAT CGC 990 Pro Asp Tyr Ser Tyr Leu Arg Gin Leu Phe Arg Asn Leu Phe His Arg 265 270 275

CAG GGC TTC TCC TAT GAC TAC GTG TTC GAC TGG AAC ATG CTC AAA TTT 1038 Gin Gly Phe Ser Tyr Asp Tyr Val Phe Asp Trp Asn Met Leu Lys Phe 280 285 290 295

GGT GCC AGC CGG GCC GCC GAT GAC GCC GAG CGG GAG CGC AGG GAC CGA 1086 Gly Ala Ser Arg Ala Ala Asp Asp Ala Glu Arg Glu Arg Arg Asp Arg 300 305 310

GAG GAG CGG CTG AGA CAC TCG CGG AAC CCG GCT ACC CGC GGC CTC CCT 1134 Glu Glu Arg Leu Arg His Ser Arg Asn Pro Ala Thr Arg Gly Leu Pro 315 320 325

TCC ACA GCC TCC GGC CGC CTG CGG GGG ACG CAG GAA GTG GCT CCC CCC 1182 Ser Thr Ala Ser Gly Arg Leu Arg Gly Thr Gin Glu Val Ala Pro Pro 330 335 340

ACA CCC CTC ACC CCT ACC TCA CAC ACG GCT AAC ACC TCC CCC CGG CCC 1230 Thr Pro Leu Thr Pro Thr Ser His Thr Ala Asn Thr Ser Pro Arg Pro 345 350 355

GTC TCC GGC ATG GAG AGA GAG CGG AAA GTG AGT ATG CGG CTG CAC CGC 1278 Val Ser Gly Met Glu Arg Glu Arg Lys Val Ser Met Arg Leu His Arg 360 365 370 375

GGG GCC CCC GTC AAC ATC TCC TCG TCC GAC CTC ACA GGC CGA CAA GAT 1326 Gly Ala Pro Val Asn He Ser Ser Ser Asp Leu Thr Gly Arg Gin Asp 380 385 390

ACC TCT CGC ATG TCC ACC TCA CAG ATT CCT GGT CGG GTG GCT TCC AGT 1374 Thr Ser Arg Met Ser Thr Ser Gin He Pro Gly Arg Val Ala Ser Ser 395 400 405 GGT CTT CAG TCT GTC GTG CAC CGA TGAGAACTCT CCTTATTGCT GTGAAGGGCA 1428 Gly Leu Gin Ser Val Val His Arg 410 415

GACAATGCAT GGCTGATCTA CTCTGTTACC AATGGCTTTA CTAGTGACAC GTCCCCCGGT 1488

CTAGGATCGA AATGTTAACA CCGGGAGCTC TCCAGGCCAC TCACCCAGCG ACGCTCGTGG 1548

GGGAAACATA CTAAACGGAC AGACTCCAAG AGCTGCCACC GCTGGGGCTG CACTGCGGCC 1608

CCCCACGTGA ACTCGGTTGT AACGGGGCTG GGAAGAAAAG CAGAGAGAGA ATTGCAGAGA 1668

ATCAGACTCC TTTTCCAGGG CCTCAGCTCC CTCCAGTGGT GGCCGCCCTG TACTCCCTGA 1728

CGATTCCACT GTAACTACCA ATCTTCTACT TGGTTAAGAC AGTTTTGTAT CATTTTGCTA 1788

AAAATTATTG GCTTAAATCT GTGTAAAGAA AATCTGTCTT TTTATTGTTT CTTGTCTGTT 1848

TTTGCGGTCT TACAAAAAAA ATGTTGACTA AGGAATTCTG AGACAGGCTG GCTTGGAGTT 1908

AGTGTATGAG GTGGAGTCGG GCAGGGAGAA GGTGCAGGTG GATCTCAAGG GTGTGTGCTG 1968

TGTTTGTTTT GCAGTGTTTT ATTGTCCGCT TTGGAGAGGA GATTTCTCAT CAAAAGTCCG 2028

TGGTGTGTGT GTGTGCCCGT GTGTGGTGGG ACCTCTTCAA CCTGATTTTG GCGTCTCACC 2088

CTCCCTCCTC CCGTAATTGA CATGCCTGCT GTCAGGAACT CTTGAGGCCC TCGGAGAGCA 2148

GTTAGGGACC GCAGGCTGCC GCGGGGCAGG GGTGCAGTGG GTGTTACCAG GCAAAGCACT 2208

GCGCGCTTCT TCCCCAGGAG GTGGGCAGGC AGCTGAGAGC TTGGAAGCAG AGGCTTTGAG 2268

ACCCTAGCAG GACAATTGGG AGTCCCAGGA TTCAAGGTGG AAGATGCGTT TCTGGTCCCT 2328

TGGGAGAGGA CTGTGAACCG AGAGGTGGTT ACTGTAGTGT TTGTTGCCTT GCTGCCTTTG 2388

CACTCAGTCC ATTTTCTCAG CACTCAATGC TCCTGTGCGG ATTGGCACTC CGTCTGTATG 2448

AATGCCTGTG GTTAAAACCA GGAGCGGGGC TGTCCTTGCC ACGTGCCAAG ACTAGCTCAG 2508

AAAAGCCGGC AGGCCAGAAG GACCCACCCT GAGGTGCCAA GGAGCAGGTG ACTCTCCCAA 2568

CCGGACCCAG AACCTTCACG GCCAGAAAGT AGAGTCTGCG CTGTGACCTT CTGTTGGGCG 2628

CGTGTCTGTT GGTCAGAAGT GAAGCAGCGT GCGTGGGGCC GAGTCCCACC AGAAGGCAGG 2688

TGGCCTCCGT GAGCTGGTGC TGCCCCAGGC TCCATGCTGC TGTGCCCTGA GGTTCCCAGG 2748

ATGCCTTCTC GCCTCTCACT CCGCAGCACT TGGGCGGTAG CCAGTGGCCA TGTGCTCCCA 2808

ACCCCAATGC GCAGGGCAGT CTGTGTTCGT GGGCACTTCG GCTGGACCCC ATCACGATGG 2868

ACGATGTTCC CTTTGGACTC TAGGGCTTCG AAGGTGTGCA CCTTGGTTCT CCCTTCTCCT 2928

CCCCAGAGTT CCCCCGGATG CCATAACTGG CTGGCGTCCC AGAACACAGT TGTCAACCCC 2988

CCCACCAGCT GGCTGGCCGT CTGTCTGAGC CCATGGATGC TTTCTCAATC CTAGGCTGGT 3048

TACTGTGTAA GCGTGTTGGA GTACGGCGCC TTGAGCGGGT GGGAGCTGTG TGTTGAAGTA 3108

CAGAGGGAGG TTGGGGTGGG TCAGAGCCGA GTTAAGAGAT TTTCTTTGTT GCTGGACCCC 3168

TTCTTGAAGG TAGACGTCCC CCACCCGGAG AGACGTCGCG CTGTGGCCTG AAGTGGCGCA 3228

AGCTTGCTTT GTAAATATCT GTGGTCCCGA TGTAGTGCCC AGAACGTTTG TGCGAGGCAG 3288

CTCTGCGCCC GGGTTCCAGC CCGAGCCTCG CCGGGTCGCG TCTTCGGAGT GCTTGTGACA 3348 GTCCTTGCCC AGTATCTAGT CCCCGTCGCC CCGTGCAGGA GACGTAGGTA GGACGTCGTG 3408 TCAGCTGTGC ACTGACGGCC AGTCTCCGAG CTGTGCGTTT GTATCGCCAC TGTATTTGTG 3468 TACTTTAACA ATCGTGTAAA TAATAAATTC GGAATTC 3505

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 415 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Met Glu Leu Arg Val Gly Asn Arg Tyr Arg Leu Gly Arg Lys He Gly 1 5 10 15

Ser Gly Ser Phe Gly Asp He Tyr Leu Gly Thr Asp He Ala Ala Gly 20 25 30

Glu Glu Val Ala He Lys Leu Glu Cys Val Lys Thr Lys His Pro Gin 35 40 45

Leu His He Glu Ser Lys He Tyr Lys Met Met Gin Gly Gly Val Gly 50 55 60

He Pro Thr He Arg Trp Cys Gly Ala Glu Gly Asp Tyr Asn Val Met 65 70 75 80

Val Met Glu Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asn Phe Cys 85 90 95

Ser Arg Lys Phe Ser Leu Lys Thr Val Leu Leu Leu Ala Asp Gin Met 100 105 110

He Ser Arg He Glu Tyr He His Ser Lys Asn Phe He His Arg Asp 115 120 125

Val Lys Pro Asp Asn Phe Leu Met Gly Leu Gly Lys Lys Gly Asn Leu 130 135 140

Val Tyr He He Asp Phe Gly Leu Ala Lys Lys Tyr Arg Asp Ala Arg 145 150 155 160

Thr His Gin His He Pro Tyr Arg Glu Asn Lys Asn Leu Thr Gly Thr 165 170 175

Ala Arg Tyr Ala Ser He Asn Thr His Leu Gly He Glu Gin Ser Arg 180 185 190

Arg Asp Asp Leu Glu Ser Leu Gly Tyr Val Leu Met Tyr Phe Asn Leu 195 200 205

Gly Ser Leu Pro Trp Gin Gly Leu Lys Ala Ala Thr Lys Arg Gin Lys 210 215 220

Tyr Glu Arg He Ser Glu Lys Lys Met Ser Thr Pro He Glu Val Leu 225 230 235 240

Cys Lys Gly Tyr Pro Ser Glu Phe Ala Thr Tyr Leu Asn Phe Cys Arg 245 250 255 Ser Leu Arg Phe Asp Asp Lys Pro Asp Tyr Ser Tyr Leu Arg Gin Leu 260 265 270

Phe Arg Asn Leu Phe His Arg Gin Gly Phe Ser Tyr Asp Tyr Val Phe 275 280 285

Asp Trp Asn Met Leu Lys Phe Gly Ala Ser Arg Ala Ala Asp Asp Ala 290 295 300

Glu Arg Glu Arg Arg Asp Arg Glu Glu Arg Leu Arg His Ser Arg Asn 305 310 315 320

Pro Ala Thr Arg Gly Leu Pro Ser Thr Ala Ser Gly Arg Leu Arg Gly 325 330 335

Thr Gin Glu Val Ala Pro Pro Thr Pro Leu Thr Pro Thr Ser His Thr 340 345 350

Ala Asn Thr Ser Pro Arg Pro Val Ser Gly Met Glu Arg Glu Arg Lys 355 360 365

Val Ser Met Arg Leu His Arg Gly Ala Pro Val Asn He Ser Ser Ser 370 375 380

Asp Leu Thr Gly Arg Gin Asp Thr Ser Arg Met Ser Thr Ser Gin He 385 390 395 ' 400

Pro Gly Arg Val Ala Ser Ser Gly Leu Gin Ser Val Val His Arg 405 410 415

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CTAGATCTAG CTAGACCATG GTAGTTTTTT CTCCTTGACG 40

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: CATGCCATGG CACGACCTAG T 21 (2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: CTAGATCTAG CTAGACCATG GTAGTTTTTT CTCCTTGACG 40

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single '(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GAATCGGGCC GCCGAGATCT CATATGGAGC TGAGAGTC 38

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: CCCGGATCTA GCAGATCTCA T 21

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Ala Ser Ser Ser Gly Ser Lys Ala Glu Phe He Val Gly Gly Tyr

1 5 10 15 (2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

Arg Ser Met Thr Val Ser Thr Ser Gin Asp Pro Ser Phe Ser Gly Tyr

1 5 10 15

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: TACATCTAGA ATTATGGCGA GTAGCAGCGG C 31

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: AATGGATCCT TAGAAACCTG TGGGGGT 27

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: AATGGATCCT TAGAAACCTT TCATGTTACT CTTGGT 36 (2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TACATCTAGA ATTATGGAGC TGAGAGTCGG G 31

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GGATCCTCAT CGGTGCACGA CAGACTG 27

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: TACATCTAGA ATTATGGCAC GACCTAGTGG TCGATCG 37

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GGGGATCCTA CTTCAGTAGG GGCTG 25 (2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

Arg Ser Gly His Asn Thr Arg Gly Thr Gly Ser Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

Arg Leu Gly His Asn Thr Arg Gly Thr Gly Ser Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Ser Ser Arg Pro Lys Thr Asp Val Leu Val Gly

1 5 10

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

Lys Ser Asp Asn Thr Lys Ser Glu Met Lys His Ser

1 5 10 (2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

Gly Thr Asp He Ala Ala Gly Glu 1 5

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

Glu Arg Arg Asp Arg Glu Glu Arg Leu Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

Thr Gly Lys Gin Thr Asp Lys Thr Lys Ser Asn Met Lys Gly Tyr 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Gly Tyr

1 5 10

Claims

1. An isolated polynucleotide sequence encoding a polypeptide with an amino acid sequence having at least about 35% homology in the protein kinase domain with the polynucleotide encoding HRR25 protein kinase.

2. The polynucleotide of claim 1 wherein the encoded polypeptide possesses casein kinase activity.

3. The polynucleotide of claim 1 wherein the encoded polypeptide possesses protein-serine/threonine kinase activity.

4. The polynucleotide of claim 1 wherein the encoded polypeptide possesses protein-tyrosine kinase activity.

5. The polynucleotide of claim 1 wherein the encoded polypeptide possess protein-serine/threonine and protein-tyrosine kinase activity.

6. The polynucleotide of claim 1, wherein the polypeptide is characterized as: a) promoting normal meiotic recombination; and b) promoting the repair a DNA strand break which occurs at the cleavage site:

;

CAACAG GTTGTC . t

7. The polynucleotide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of GPSLED (amino acids 86 to 91 in SEQ ID NO: 2), RDIKPDNFL (amino acids 127 to 135 in SEQ ID NO: 2), HIPYRE (amino acids 164 to 169 in SEQ ID NO: 2), and SVN (amio acids 181 to 183 in SEQ ID NO: 2) and conservative variations thereof.

8. The polynucleotide of claim 1, selected from the group consisting of RNA, mRNA, genomic DNA and cDNA.

9. An antisense polynucleotide according to claim 1.

10. The polynucleotide of claim 1, selected from the group consisting of the DNA sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 23, 30, 31, and 34.

11. An isolated and purified polypeptide encoded by a DNA sequence of claim 10.

12. The polynucleotide of claim 1 wherein the polynucleotide is isolated from organisms selected from the group consisting of Saccharomyces, Schizosaccharomyces, human, bovine, porcine, murine, avian and Drosophila species.

13. An autonomously replicating DNA vector comprising a DNA according to claim 8.

14. A procaryotic or eukaryotic host cell stably transformed or transfected with a DNA according to claim 8.

15. A method for the production of a polypeptide possessing protein kinase and/or recombination/repair promoting activity comprising growing a host cell according to claim 14 in a suitable nutrient medium and isolating the desired polypeptide from said host cell or from the medium of its growth.

16. A polypeptide product of the method of claim 15.

17. An antibody substance specific for a polypeptide of claim 15.

18. A monoclonal antibody according to claim 17.

19. A method for identifying a composition which modulates the protein kinase and/or recombination/repair promoting activity of an HRR25-like protein comprising:

(a) incubating a system of components comprising the composition and the protein in the presence of a substrate for said protein wherein incubation is carried out under conditions sufficient to allow the components to interact; and

(b) measuring the change in activity of said protein on said substrate.

20. The method of claim 19 wherein the activity is promotion of repair of a DNA double strand break.

21. The method of claim 19 wherein the activity is protein kinase activity.

22. A method of treating a cell proliferative disorder associated with an HRR25-like protein comprising administering, to a subject with the disorder, a therapeutically effective amount of a composition which modulates the activity of the protein.