EUCARYOTIC CELLS TRANSFORMED WITH A MAMMALIAN PHOSPHOLIPID KINASE OR PROTEIN KINASE AND ASSAYS USING THEM

Abstract: A eukaryotic cell transformed with a DNA construct comprising a coding sequence encoding a polypeptide having the activity of a mammalian phospholipid kinase or a mammalian protein kinase activated by a phospholipid or its metabolite and regulatory elements to allow transcription of the coding sequence in the said cell wherein the regulatory elements include a repressible or inducible promoter and the expression of the said coding sequence is lethal or growth inhibitory to the cell. In a preferred embodiment the cells are (Schizosaccharomyce pombe). The cells are used as the basis of an assay for compounds involved in cell growth regulation. Such compounds can be used to treat cancers and the formation of blood vessel plaques.

Description: EUCARYOTIC CELLS TRANSFORMED WITH A Mf ftLIAN PHOSPHOLIPID KINASE OR PROTEIN KINASE AND ASSAYS USING THEM The present invention relates to assays for compounds involved in cell growth regulation, and more particularly to those involved in transducing signals from hormones, growth factors and oncogenes. Such compounds represent potential drugs or targets for drugs to treat cancers and to prevent the formation of plaques which cause heart disease. Phosphatidylinositol 3-OH kinase (Ptdlns 3-kinase) catalyses the phosphorylation of the 3-hydroxyl of inositol in Ptdlns, PtdIns-4-phosphate or in PtdIns-4,5-bisphosphate. This activity is involved in transducing signals from a number of hormones, growth factors and oncogenes. The standard assay for the activity of the Ptdlns 3-kinase involving lipid moieties does not readily lend itself to a screen for potential inhibitors (or activators) of catalytic function. Members of the protein kinase C family of enzymes are involved in transducing signals from a number of hormones, growth factors and oncogenes. The standard assay for protein kinase C does not lend itself to a screen for potential inhibitors (or activators) of catalytic function. Thus it has been desirable to investigate other means of searching for inhibitors. A first aspect of the invention provides a eukaryotic cell transformed with a DNA construct comprising a coding sequence encoding a polypeptide having the activity of a mammalian phospholipid kinase or a mammalian protein kinase activated by a phospholipid or its metabolite and regulatory elements to allow transcription of the coding sequence in the said cell wherein the regulatory elements include a repressible or inducible promoter and the expression of the said coding sequence is lethal or growth inhibitory to the cell. Polypeptides having the activity of a phospholipid kinase or a protein kinase activated by a phospholipid or its metabolite are involved in cell growth regulation. By "growth inhibitory" we mean that the growth rate of cells transformed with the said DNA construct is at least two to three fold lower than the same cells not transformed with the said DNA construct when grown in the same culture conditions. By "repressible" we mean that in the presence of a repressing agent the expression from the promoter is at least two-fold lower than expression from the promoter in the absence of the repressing agent. It is preferred if expression from the promoter in the presence of a repressing agent is at least five-fold lower, more preferably ten-fold lower or even more preferably 100-fold lower than expression from the promoter in the absence of the repressing agent. By "inducible" we mean that in the presence of an inducing agent the expression from the promoter is at least two-fold higher than expression from the promoter in the absence of the inducing agent. It is preferred if expression from the promoter in the presence of an inducing agent is at least five-fold higher, more preferably ten-fold higher or even more preferably 100-fold higher than expression from the promoter in the absence of the inducing agent. When an inducible promoter is used there is sufficiently low expression of the polypeptide in the uninduced state that the lethal or growth inhibitory phenotype is not observed whereas when the inducing agent is present the lethal or growth inhibitory phenotype is observed. When a repressible promoter is used there is sufficientiy low expression of the polypeptide in the repressed state that the lethal or growth inhibitory phenotype is not observed whereas when the repressing agent is absent the lethal or growth inhibitory phenotype is observed. Suitable eukaryotic cells include mammalian cells, such as COS cells and CHO cells, insect cells, slime mould such as Dictyosteliwn, and yeast. Suitable regulatable mammalian cell promoters include glucocorticoid-inducible promoters and the metallothionein promoter. It is preferred if the cell is a yeast cell. Exemplary genera of yeast contemplated to be useful in the practice of the present invention are Pichia, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Hansenula, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like. It is preferred if the yeast is a fission yeast. It is further preferred if the yeast is Schizosaccharomyces. Preferably, the said polypeptide has the activity of a phospholipid kinase, for example a catalytically effective portion of the said kinase. Phospholipid kinases include phosphatidyl inositol 3-kinase, phosphatidyl inositol 4-kinase and phosphatidyl inositol 5-kinase which phosphorylate the inositol ring on the 3', 4' or 5' hydroxyl, respectively. Suitably, the said polypeptide is a catalytically effective portion of a phosphatidylinositol 3-OH kinase. It is convenient to use the 110 kDa mammalian Ptdlns 3-kinase catalytic subunit. In further preference, the said polypeptide is a catalytically effective portion of a protein kinase C (PKC). Suitably, the protein kinase C is PKC-γ or PKC-δ A constitutive promoter such as adh may be used (disclosed in ref 1). Also, the SV40 promoter may be used. Thus, a further aspect of the invention provides a Schizosaccharomyces cell transformed with a DNA construct comprising a coding sequence encoding a polypeptide having the activity of a mammalian phospholipid kinase or a mammalian protein kinase activated by a phospholipid or its metabolite and regulatory elements to allow transcription of the coding sequence in the said cell wherein the regulatory elements include a constitutive promoter and the expression of the said coding sequence is lethal or growth inhibitory to the cell. Any gene that arrests growth or is lethal can be expressed only transiently for the purposes of subsequent inhibitor screening. In the case of a constitutive promoter in a plasmid carrying a marker, freshly transfected cells are diluted directly into medium using a combination of growth conditions to select for transfectants (for example, medium containing no leucine) and added potential inhibitors of the constitutively expressed mammalian gene to test for their efficacy. Mammalian genes whose expression can be controlled by growth conditions can be introduced into the yeast under conditions where expression is low (ie suppressed or not induced). It is preferred if the mammalian genes so introduced are stably maintained in the yeast. It is further preferred if the mammalian genes are stably integrated into the yeast genome. Expression is then increased following growth under de-repressing conditions (for example removal of thiamine) and potential inhibitors scored on their ability to permit growth under these conditions. The use of an integrant and a controllable promoter provides the most amenable procedure. The induction of cell arrest or cell death provides a powerful screen for a suppressor of such events. The present invention provides a screen for suppressors of regulatory proteins that control other mammalian functions either directly, for example protein kinases, or indirectly through the production of small regulatory molecules, for example an inositol lipid kinase. Thus, in a preferred embodiment, the S. pombe cells contain a coding sequence for the 110 kDa mammalian Ptdlns 3-kinase catalytic subunit under the regulatory control of the rant promoter and with other suitable regulatory elements, such as a transcription terminator, as is known in the art, for expression of the said catalytic subunit. In the presence of thiamine the promoter is inoperative and the cells carrying the Ptdlns 3-kinase catalytic subunit plasmid grow as the parental strain. (It will be appreciated by those skilled in the art that the parental strain may not be wild-type. For example mutant strains containing Ade " or Leu " or Ura " mutations may be used as the parental strain to allow selection of plasmid uptake). In the absence of thiamine the nmt promoter functions and the Ptdlns 3-kinase catalytic subunit is induced. This has been shown by demonstrating a substantial increase in Ptdlns 3-kinase activity under these conditions. However, following this induction the cells cease to divide; cultures plated in the absence of thiamine do not grow but die. Derivative of the nmt promoter that retain the thiamine-repressibility characteristics of the wild type promoter may also be used. As an alternative to the thiamine-repressible nmt promoter, the fbpl gene promoter from S. pombe can be used. The jbpl gene promoter is repressed in the presence of 8% glucose as disclosed by Hoffman & Winston (1990) Genetics ΩA, 807-816 incorporated herein by reference. Thus, in a further embodiment, the S. pombe cells contain a coding sequence for the 110 kDa mammalian Ptdlns 3-kinase catalytic subunit under the regulatory control of the fbpl promoter and with other suitable regulatory elements for expression of the said catalytic subunit. In the presence of 8% glucose the function of the promoter is repressed and the cells carrying the Ptdlns 3-kinase catalytic subunit plasmid grow on the parental strains. In the absence of glucose the fbpl promoter functions and the Ptdlns 3-kinase catalytic subunit is induced. The lethal phenotype of the S. pombe expressing mammalian Ptdlns 3-kinase provides a very powerful tool with which to screen for inhibitors of this activity. Cells plated in the absence of thiamine will survive and proliferate if the activity of the Ptdlns 3-kinase is suppressed. A direct demonstration that this is indeed the case, is afforded by the finding that a mammalian Ptdlns 3- kinase regulatory subunit (p85α.) when coexpressed with the Ptdlns 3-kinase catalytic subunit will rescue these cells and allow proliferation. Clearly, therefore, coexpression of (or generally the presence of) the p85α subunit should be avoided in the assay of this embodiment, as should, in other embodiments, other activity-suppressing compounds. In further embodiments the S. pombe cells contain a coding sequence for a mammalian protein kinase C under the regulatory control of the nmt promoter or the fbpl promoter. As an inhibitor screening process, a further advantage afforded by this approach is that general cytostatic and cytotoxic compounds will score negative; the screen will distinguish the action of the mammalian Ptdlns 3-kinase or protein kinase C against the background of a plethora of essential eukaryotic gene functions. Thus, a further aspect of the invention provides an assay kit comprising a eukaryotic cell according to the first aspect of the invention and culture medium such that the cell will divide and grow and such that the said coding sequence is expressed, the expressed polypeptide at least preventing cell division in the cell culture. Conveniently the kit comprises S. pombe as the eukaryotic cell. The invention also encompasses compounds identified as being useful in the assays of the invention. These compounds are useful in the treatment of disease and medical conditions where there is an undesirable function of a phospholipid kinase or a protein kinase activated by a phospholipid or its metabolite. Such diseases and conditions include cancer, inflammation, Alzheimer's disease, restenosis, atherosclerosis and wound healing. Suitable promoters and coding sequence can be incorporated into vectors in the correct orientation by methods known in the art, some of which are described in Sambrook et al (1989) Molecular Cloning, a practical approach (2nd Edition), Sambrook, J., Fritsch, E. & Maniatis, T., eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, -New York, incorporated herein by reference. A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules. Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities. The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment. Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA. A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art. Transformation of appropriate cell hosts is accomplished by well known methods that typically depend on the type of vector used and host cell. Transformation of Saccharomyces and related cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA. Schizosaccharomyces pombe may be transformed following LiCl treatment or by electroporation. Conveniently, a Bio-Rad Pulse Controller may be used for electroporation of 5. pombe cells. a) Grow up cells to OD 595 less than or equal to 0.5 in minimal medium. b) Centrifuge cells at 1500 g for 5 min, remove supernatant and resuspend in 20 ml ice-cold distilled water, centrifuge again, remove supernatant and resuspend in 20 ml ice-cold 1 M sorbitol, centrifuge again and remove supernatant. c) Resuspend cells in ice-cold 1 M sorbitol to a density of ~5xl0 9 cells/ml (concentrated 500 times when compared to original culture). d) Use 40-100 μl of cell suspension per transformation. Add DNA (up to 100 ng) in 1 μl in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to cells and incubate on ice 5 min. e) Transfer to pre-chilled cuvettes (0.2 cm gap) and apply pulse (1.5 KV, 25 μF, 200 Ω). f) Immediately add 900 μl of ice-cold 1 M sorbitol and transfer to a chilled tube on ice. g) Promptly spread 100-200 μl onto a selective minimal medium plate containing 1 M sorbitol and culture at 32°C until grown. The technique of electroporation of yeast is disclosed in Becker, D.M. and Guarente, L. (1990) Meth. Enzymol. 194, 182. Machines for electroporation are available from other manufacturers and can be used to transform yeast and mammalian cells according to their instructions. Successfully transformed cells, ie cells that contain a DNA construct, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies, for example by western blotting. The invention will now be described in detail with reference to the following Examples and Figures wherein: Figure 1 shows the nucleotide sequence (SEQ ID No 1) and deduced amino acid (SEQ ID No 2) of the sequence 110 kDa catalytic subunit of Ptdlns 3- kinase (PI 10). Figure 2 shows the nucleotide sequence of the nmt promoter region (SEQ ID No 3). Figure 3 shows the nucleotide sequence of PKC-e (SEQ ID No 4). Figure 4 shows the nucleotide sequence of PKC-γ (SEQ ID No 5). Figure 5 shows the nucleotide sequence of PKC-δ (SEQ ID No 6). Figure 6 shows the nucleotide sequence of PKC-77 (SEQ ID No 7). Figure 7 shows that the lethal effect of pi 10 expression in S. pombe is suppressed by p85 expression. Figure 8 shows the isotype-specific effects of PKC expression in S. pombe. Figure 9 shows the effect of PKC expression on growth rates in liquid culture. Figure 10 shows that PKC-δ-induced growth inhibition is the result of kinase activity. Example 1: Assay using catalytic subunit of Ptdlns 3-kinase and nmt promoter Isolation of Ptdlns 3-kinase catalytic subunit cDNA. The cDNA for the 110 kDa catalytic subunit can be isolated by a conventional cloning strategy. Purification of the bovine enzyme from brain tissue (Morgan, Smith et al 1990) has demonstrated that sufficient protein can be isolated for protein sequence determination. This is unequivocally established for the 85 kDa regulatory subunit which has been sequenced from this source and, as a consequence, cloned (Otsu, Hiles et al 1991). The Ptdlns 3-kinase from bovine brain (85- 110 dimer) is purified according to Morgan, Smith et al (1990) by sequential fractionation with ammonium sulphate and chromatography on DEAE-cellulose, phosphocellulose, Sephacryl S-200 and Mono Q. In order to remove contaminants and separate subunits, the protein is further purified by sodium dodecyl sulphate polyacrylamide gel electrophoresis according to Laemmli (1970), the 110 kDa protein visualised in ammonium chloride (4N), electroeluted and digested with trypsin as described in Katan, Kriz et al (1988). Tryptic peptides are then separated by standard procedures and subjected to amino acid sequence determination. Sequence established for the 110 kDa catalytic subunit is used to predict redundant oligonucleotide probes for screening a bovine brain cDNA library. Standard cloning procedures are then employed in the isolation of a cDNA encoding the complete open reading frame of the 110 kDa subunit (Sambrook et al 1989). The sequence of the cDNA is determined by commonly employed dideoxy-sequencing procedures. A specific example of using this strategy is described by Hiles et al (1992) Cell 70, 419- 429. Materials: Restriction enzymes and DNA modification enzymes were obtained from standard commercial sources and used according to the manufacturer's recommendations. Oligonucleotides were synthesised on an Applied Biosystems 380B DNA synthesiser and used directly in subsequent procedures. Protein Purification and Amino Acid Sequence Determination: The purification of the p85α. and pi 10 proteins by chromatography on a peptide affinity column corresponding to amino acids 742-758 of the kinase insert region of the human PDGF-0 receptor has been described (Otsu et al (1991) Cell 65, 91-104). Proteins were released from the affinity matrix using SDS- containing buffers, separated on a Prosieve agarose gel, and visualised by staining with Coomassie blue G250. The band corresponding to pi 10 was excised and protein was eluted by tube gel HPEC. Protein was precipitated from pl lO-containing fractions by treatment with trichloroacetic acid and then washed with acetone. The pi 10-containing pellet was resuspended and digested with lysylendopeptidase in the presence of SDS, and peptides were separated by tandem ion-exchange chromatography and reverse-phase HPLC. This procedure was carried out on three separate PI3 -kinase preparations. A fourth preparation was eluted from the matrix as before and boiled for 5 min. After cooling, the sample was diluted with 25 mM Tris-HCl (pH 8.8) and digested directly with lysylendopeptidase for 72 hr at 30° C. Peptides were separated as above. Peptide sequences were determined using a modified Applied Biosystems 477A automated pulse-liquid sequencer. mRNA Isolation and cDNA Cloning: Total RNA was isolated from SGBAF-1 cells by the method of Chirgwin et al (1979) Biochemistry 18, 294-299 and poly(A) mRNA was selected by chromatography on oligo-(dT)-cellulose (Maniatis et al (1982) Molecular Cloning: A laboratory manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). An oligo-dT primed cDNA library of 5 x 10 6 primary recombinants was constructed in lambda Uni-Zap (Stratagene) from 5 μg of this mRNA using the Stratagene Uni-Zap cDNA cloning system. The construction of the total bovine brain cDN A library in lambda Uni-Zap has been described previously (Otsu et al (1991) Cell 65, 91- 104). Library Screening and Hybridisations: The unamplified SGBAF-1 cDNA library (10 6 recombinants) was plated on E. coli K12 PLK-F (Stratagene) at a density of 10 5 plaques per 15 cm dish, and lifts were taken in duplicate onto nitrocellulose membranes (Millipore) . For screening, filters were prehybridised for at least 1 hr at 42°C in 6 x SSPE, 0.5% SDS, 10 x Denhardt's solution, and 100 μg/ml denatured sonicated herring sperm DNA (Sigma). Hybridisation was carried out in the same solution containing 10 ng/ml radiolabeled oligonucleotide. Oligonucleotides used were: peptide N, (MDWIFHT; SEQ ID No 8) 5'-AA(G/A)ATGGA(T/C)TGGAT(C/T/A)TT(T/C)CA(T/C)AC-3' (SEQ ID No 9); peptide J (DDGQLFHIDFGHF; SEQ ID No 10) 5'- GATGATGGCC-A(G/A)CTGTT(T/C)CA(T/C)AT(T/A)GA(T/C)- TTTGGCCA(T/C)TT (SEQ ID No 11). Oligonucleotides were labeled with 32 P at the 5' end in a 20 μl reaction containing 100 ng of oligonucleotide, 1 x kinase buffer (Promega), 0.1 mM spermidine, 5 mM dithiothreitol, 100 μCi of [7- 32 P]ATP (5000 Ci/mmol, Amersham), and 2 μl (20 U) of T4 polynucleotide kinase (Amersham). Filters were washed in 6 x SSC, 0.1 % SDS at room temperature and then subjected to autoradiography using Kodak XAR film. Hybridising clones were plaque purified and rescued as plasmids according to the manufacturer's instructions. Characterisation of cDNA Clones: Sequencing was carried out by the chain termination method using the Sequenase system (US Biochemicals). Clones for sequencing were obtained by directed cloning of restriction fragments into M13mpl8 and mpl9 vectors (Yanisch-Perron et al (1985) Gene 33, 103-119) and by making a series of exonuclease Ill-mediated deletions (Henikoff (1984) Gene 28, 351-359; Pharmacia Exonuclease III deletion kit). DNA sequences were analysed on a Micro-VAX computer using the Wisconsin sequence analysis package (UWGCG; Devereux et al (1984) Nucl. Acids Res. 12, 387- 395). RACE PCR: RACE PCR was carried out essentially as described previously (Frohman et al (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002; Harvey and Garlison (1991) Nucl. Acids Res. 19, 4002). In brief, first-strand cDNA primed with random hexamers (Amersham) was synthesised from 1 μg of SGBAF-1 cell mRNA using the Stratagene first-strand cDNA synthesis kit. First-strand cDNA was isolated by isopropanol precipitation and tailed with oligo-(dA) using terminal deoxynucleotidyl transferase (Bethesda Research Laboratories). PCR was performed using oligo 2224 (5'- AATTCACACACTGGCATGCCGAT; SEQ ID No 12) and adaptor dT (5'- GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT; SEQ ID No 13) as primers, using a Perkin-Elmer Cetus Taq polymerase PCR kit (conditions: 30 cycles of 94°C for 1 min, 35°C for 1 min, 72°C for 2 min). Products were fractionated on a 1.5 % low melting point agarose gel and visualised by staining with ethidium bromide. The gel was sliced into six bands (ranging from 150 bp to 2000 bp), and DNA was isolated from each gel slice. A further round of PCR was performed on this DNA using oligonucleotide 2280 (5'- TTTAAGCTTAGGCATTCTAAAGTCACTATCATCCC; SEQ ID No 14) and adaptor (5'-GACTCGAGTCGACATCGA; SEQ ID No 15) as primers (conditions: 35 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 2 min). Products were fractionated on an agarose gel and visualised by staining with ethidium bromide. A band 250 bp shorter than the size of the DNA in the gel slice used for the PCR was expected. An intensely staining band of 350 bp obtained from the ~600 bp gel slice was excised, digested with HmdIII and Sail, and ligated into Bluescript KS- digested with H/ndlll and Xhol to give plasmid pBS/race. Two independent inserts were completely sequenced. The sequence of pi 10, the 110-kD catalytic subunit of PI3-kinase is shown in Figure 1 and has the GenEMBL Accession No M93259 (SEQ ID No 1). Isolation of nmt promoter. The promoter has been isolated by Maundrell (2) and may be isolated by repeating the procedures reported in that reference. Moreover, the sequence of the gene, including the promoter, has been submitted to the GenBank™/EMBL database as Accession No J05493 and is shown in Figure 2 (SEQ ID No 3). Vectors containing the nmt promoter and derivatives of the nmt promoter suitable for use in the present invention are described by Basi et al (1992) Yeast 8, S597 (special issue) and Maundrell (1990). The upstream regulatory region and downstream polyadenylation site of nmtl have been incorporated into two types of S. pombe/E. coli shuttle vector: pREP extrachromosomally replicating plasmids and pRIP integrating plasmids. Using either of these constructs thiamine mediated transcriptional regulation can be transferred to heterologous coding sequences. The time course of induction and repression have been studied as a function of changes in the intracellular thiamine concentration. Addition of thiamine to cells growing in minimal medium results in a rapid rise in the internal thiamine from a basal level of around 10 pmoles/10 7 cells to up to 1000 fold this level and this is accompanied by repression of nmtl promoter activity. If cells are then washed and allowed to continue growth in minimal medium, the intracellular thiamine is progressively diluted as the cell mass doubles and transcription is reinitiated as the internal thiamine concentration falls below 50 pmoles/10 7 cells. The time taken to re-activate the nmtl promoter therefore depends on the internal thiamine concentration at the time when the cells are transferred to thiamine free medium. Quantitation of promoter strength was assessed using chloramphenicol acetyl transferase as a reporter gene. The fully induced nmtl promoter is about 6 fold more active than the S. pombe adh promoter and its activity is reduced about 80 fold when cells are grown in repressing conditions. These vectors are ideally suited to applications requiring maximal expression of a gene of interest. In addition, two modified versions of the promoter with reduced activity have been created following an analysis of the effects of TATA box mutations. Truncating the wild type TATA box, TATATAAA to ATAAA (the '4' series) or AT (the '8' series) down-regulates transcriptional activity of the nmtl promoter by approximately 1 and 2 orders of magnitude respectively (see Table). These mutations in the TATA box do not affect thiamine repressibility or the site of transcription initiation. The table below summarises the salient features of some of the vectors which have been constructed thus far: 10 15 activity is based on the quantitation of CAT assays. Data are expressed in arbitrary units relative to the wild type promoter cultured in the presence of thiamine. b the Ball site is replaced with an Xhol site allowing expression from the ATG. c in some of the vectors the complementation gene used for selection of plasmid uptake has been changed from the LEU2 gene to the sup 3.5 gene which complements the Ade 6.704 mutation or to the URA4 gene. The backbone of the plasmid is not altered (ie promoter and stop sequence from the nmtl gene, ARS1 and pUC119 backbone). Construction of an 5. pombe pllO expression system. A suitable restriction fragment containing the complete 110 kDa subunit open reading frame and flanking sequences is subcloned into the nmt promoter plasmid containing a suitable marker gene for selection creating an rønt-100 plasmid in order to allow expression of the 110 kDa protein under the control of the thiamine repressible nmt promoter. The nmt- 10 plasmid is grown in a suitable bacterial host and the plasmid purified by conventional techniques (Sambrook et al 1989). A 3.4 kb Bam l/Fspl fragment containing the cDNA of pi 10 was isolated and subcloned into the Bam l/Smal sites of pREP3X-pl l0 (nmt- 110). The nmt-\ 10 plasmid is then transfected by standard procedures (Moreno, Klar et al 1991) into a Schizosaccharomyces pombe strain that is auxotrophic for leucine cells are transformed using electroporation. Transfected cells are then plated in the presence of thiamine and in the absence of leucine. As an alternative Schizosaccharomyces pombe strains which are auxotrophic for adenine or uracil (that is Ade " or Ura " ) may be used; in this case the cells are plated in the presence of thiamine and absence of adenine, or the presence of thiamine and absence of uracil, respectively. Colonies growing up under these conditions are then analysed for the presence of the n t-l lO plasmid. The lethal phenotype caused by the expression of 110 kDa protein is checked by replating colonies in the presence or absence of thiamine; under the latter conditions colonies will arrest and/or die. For the purposes of setting up a screen for inhibitors, a stable transformant is isolated. This is carried out by standard procedures involving growth in the presence and absence of the selectable marker leucine (or adenine or uracil). Isolates obtained in this manner are checked for the stable insertion of the 110 kDa sequence into genomic DNA by Southern analysis or stable replication of a non-integrated plasmid. Expression of the pi 10 protein is also confirmed by western blot analysis of the transformants using antibodies reactive against pi 10, or by measuring the activity of the pi 10 subunit in the transformed cells. The inducible lethal phenotype is rechecked by growth of these isolates in the presence and absence of thiamine ( > 10 nM). It is preferred if 100 nM, or > 1 pM or > 1 μM is used. It is most preferred if 15 μM thiamine is used. Operating the screen. The screen for inhibitor activity is carried out on a 96- well microtitre plate format. An integrant colony is picked and put into liquid culture in minimal medium, 2% glucose, 15 μM thiamine and supplements appropriate for the strain (eg uracil 50 μg/ml would be included for a ura " strain if the integrated plasmid did not harbour a URA4-based selection marker). This culture is grown up and, after extensive washing, used to seed two 10 ml cultures, one containing thiamine as above, and one without. The cultures are expanded overnight and then diluted to an optical density (OD) at 595 nm of 0.01-0.10. For those cells requiring treatment for arrest of growth additions are made at this stage prior to plating. The diluted cultures are then aliquoted into wells of a sterile 96 well microtitre plate containing individual test compounds in the presence or absence of thiamine. The growth of the cells is monitored over time until the OD 595 reached is ~0.8 for control cultures. Control cultures are those cultured with thiamine. The OD 595 is assessed using a microtitre plate reader. The cells precultured in thiamine and retained in thiamine serve to indicate optimum growth rate. Cells precultured in the absence of thiamine and then put into wells containing thiamine provide a control for the rescue of growth. Cells precultured in the absence of thiamine and put into wells in the absence of thiamine or test compound provide a baseline for non-growth. Individual test compounds are assessed for their potency in permitting growth in the absence of thiamine in cells plated in the absence of thiamine. Accumulated experience in the operation of this screen for a particular gene product permits a less frequent monitoring of the growth curves and a single time point may be found to be sufficient. Similarly, cultures propagated throughout in the presence of thiamine may be found to be a non-essential control. These alterations to the procedure may provide some practical advantages in increasing the number of test compounds per 96 well plate and in reducing the time required for assessment of growth. The above procedures have been employed in creating an S. pombe strain harbouring a pi 10 cDNA under the control of the nmt promoter. Switching these cells from a medium containing thiamine (15 μM) to one in the absence of thiamine causes growth arrest. Evidence that the arrest is a consequence of the expression of the mammalian protein has come from a number of observations: 1. Transient transfection and subsequent expression has been observed on multiple occasions with the pi 10 cDNA and not with the vector alone. 2. On expression of the pi 10 protein, it is possible to detect the activity of the expressed mammalian protein in cell extracts, ie the catalytic activity is retained on expression in S. pombe. 3. On expression of the mammalian regulatory subunit of the kinase, p85α [4], increased expression of pi 10 no longer induces growth arrest. The use of this system as a viable tool for screening pi 10 inhibitors is evidenced by the ability of p85α, the regulatory subunit, to suppress the growth arrest phenotype. Biochemical evidence has already established that the p85α- pl lO complex is less active than the free pi 10 protein [9]. The lethal effect of pi 10 expression in S. pombe is suppressed by p85 expression as shown in Figure 7. Stable pi 10-expressing 5. pombe cells were transformed with the ρREP4 vector, or the pREP4-p85α or pREP4-p853 constructs and, after selection for plasmid uptake, were streaked onto selective minimal medium plates in the presence or absence of thiamine. Expression of pi 10 alone is lethal but this effect is rescued by co-expression of either p85α or p85/3. The p85α and p85/3 cDNAs can be obtained using the methods described by Otsu et al (1991) Cell 65, 91-104 incorporated herein by reference. Example 2: Isotype-specific effects of PKC expression in iS. pombe and the effect of PKC expression on growth rates in liquid culture S. pombe strains containing integrated plasmids for expression of mammalian PKC-γ, -δ, -e, -ζ or -η were streaked onto selective minimal medium plates in the absence of thiamine or the presence of thiamine or TPA as shown in Figure 8. Growth of control (vector) or PKC-f cells was similar under all three conditions. PKC-γ expression (Figure 8, plate B) marginally decreased growth and TPA addition to these cells totally supressed growth (Figure 8, plate C). PKC-δ, -e and -η expression alone was markedly growth inhibitory (Figure 8, plate B). Stable PKC-S. pombe strains were cultured in minimal medium in the absence of thiamine for 18 hours until an OD 595 of 0.2-0.5 was attained (see Figure 9). Strains were then (at time zero) diluted to an OD 595 of 0.02 in minimal medium and cultured in the presence of 1 μM thiamine (controls) (A), in the absence of thiamine (■) or in the absence of thiamine with 100 ng/ml TPA (O). At the indicated times, the cell density was calculated by measuring the OD 595 . PKC-f cells grew at a rate essentially indistinguishable from vector controls. PKC-δ, -e and -η expression markedly delayed growth when compared with vector controls (-thiamine). Growth of PKC-γ, -δ and -η expressing cells was essentially nil when cultured in the presence of TPA. Example 3: An inhibitor screen for protein kinase C-e Protein kinase C-e [10] cDNA (Figure 3; SEQ ID No 4) has been introduced into a plasmid under the control of the nmt promoter yielding nmt-PKC-e. A 2.7 kb Xhol fragment with the full coding sequence for PKC-e was isolated from ρMT2-PKC-e and subcloned into Sα/I-digested pREP3X. Then 300 bp of 5' non-coding sequence was removed by digesting with Xhol and Ncό , blunting the ends and religating to give pREP3X-PKC-e. The plasmid pMT2-PKC-e can be prepared by the methods described by Schaap et al (1989) FEBS Lett. 243, 351-357. Transfection of this construct into S. pombe employing selection for uptake of the LEU2 gene in the presence of thiamine, yields populations of cells that on switching to "no thiamine" conditions while retaining selection for LEU2, reduce growth rate. Growth inhibition is consistent with the expression of the mammalian PKC-e gene product since: 1. Growth inhibition correlates with an induction of the PKC-e protein as judged by Western analysis. 2. The induced phenotype also correlates with expression of PKC-e activity as determined in cell extracts. 3. Suppression of PKC-e expression by exposure to the phorbol ester TPA can rescue cells that are expressing low levels of PKC-e (cells expressing high levels of PKC-e are not rescued and the steady state level of PKC-e is not significantly depressed by TPA treatment). The expression of a functional PKC-e activity in S. pombe and its correlation with growth arrest under various growth conditions provides the basis for an inhibitor screen. The transformed cells are plated in the presence of thiamine (control) and the absence of thiamine (test) and the compound to be assayed is added to the "test" plates. Example 4: An inhibitor screen for protein kinase C-γ. A cDNA for PKC-γ (Figure 4; SEQ ID No 5) has been introduced into a plasmid under the control of the nmt promoter, producing wnt-PKC-γ. A 2.4 kb -δαmHI/blunt Hinάlll fragment with the full coding sequence of PKC-γ was isolated from pSP64-PKC-γ and subcloned into the BamHl/Smal sites of pREP3X to give pREP3X-PKC-γ. The plasmid pSP64-PKC-γ can be prepared as described by Patel & Stabel (1989) Cell. Signall. 1, 227-240.Transfection of S. pombe with nmt-PKC-y yields populations of cells that on switching to medium without thiamine induce PKC-γ protein as determined by Western blotting and the detection of PKC activity in cell extracts. These cells continue to grow on induction but if the PKC-γ is selectively activated by inclusion of the phorbol ester TPA in the growth medium, the cells will arrest. The dependence of growth arrest upon the inclusion of TPA provides direct evidence that the catalytic function of PKC-γ is responsible for the phenotype. No such arrest is observed on treatment of the original S. pombe strain. Other PKC activators, such as Mezerein, or other phorbol esters or diacylglycerols may be used in place of TPA. That activation of PKC-γ induces growth arrest provides a screen for inhibition of function of this mammalian gene product. Operating the screen. The screen for inhibitor activities is carried out on a 96-well microtitre plate format. For thiamine repressible genes, stable integrants are grown up overnight (12 h) in the absence of thiamine. The culture is then diluted in the absence of thiamine to an OD 595 = 0.01 to 0.10. The culture is then aliquoted into microtitre wells containing the potential inhibitors and, in the case of PKC-γ, also phorbol ester. The growth of cells monitored at 595 nm using a microtitre plate reader. Cells are allowed to grow until parallel wells/plates containing cells growing in the presence of thiamine (15 μM) have increased their OD 595 to 1.0 units. Cells from the test wells that have proliferated can be scored relative to both control wells (eg + thiamine) and no addition wells (-inhibitor, -thiamine). Thus, for PKC-γ there are the following possibilities: (i) control plates which are + thiamine or -thiamine or -thiamine + TPA and (ii) test plates which are + thiamine + compound or -thiamine + compound or -thiamine + TPA + compound. Example 5: An inhibitor screen for protein kinase C-δ. A cDNA for PKC-δ (Figure 5; SEQ ID No 6) has been introduced into a plasmid under the control of the nmt promoter, producing nmf-PKC-δ. A 2.4 kb blunt PfiMl/Ndel fragment containing the full coding sequence of PKC-δ was isolated from pBluescript-PKC-δ and subcloned into blunt Sα I-digested ρREP3X to give pREP3X-PKC-δ. The plasmid pBluescript-PKC-δ can be obtained using the methods described in Olivier & Parker (1991) Eur. J. Biochem. 200, 805-810 incorporated herein by reference. Transfection of S. pombe with nmt-PKC-δ yields populations of cells that on switching to medium without thiamine induce PKC-δ protein as determined by Western blotting and by activity measurements. There is marked growth inhibition by expression alone and if the PKC-δ is activated by inclusion of the phorbol ester TPA in the growth medium, the phenotype is strengthened. Experiments with PKC-δ also provide firm evidence that the phenotype is a result of the function of the kinase. Part of the kinase domain of PKC-δ was deleted thus rendering it enzymatically inactive. The product was expressed to a high level in S. pombe but there was no growth inhibition thus indicating that the phenotype is due to the functional kinase. That activation of PKC-δ induces growth inhibition provides a screen for inhibition of function of this mammalian gene product. Operating the screen. The screen for inhibitor activities is carried out on a 96-well microtitre plate format. For thiamine repressible genes, stable integrants are grown up overnight (12 h) in the absence of thiamine. The culture is then diluted in the absence of thiamine to an OD 595 = 0.01 to 0.10. The culture is then aliquoted into microtitre wells containing the potential inhibitors and, in the case of PKC-γ, also phorbol ester. The growth of cells monitored at 595 nm using a microtitre plate reader. Cells are allowed to grow until parallel wells/plates containing cells growing in the presence of thiamine (15 μM) have increased their OD 595 to 1.0 units. Cells from the test wells that have proliferated can be scored relative to both control wells (+ thiamine) and no addition wells (-inhibitor, -thiamine). Additionally, the test wells may contain or lack TPA. Figure 10 shows that the PKC-δ-induced growth inhibition is the result of kinase activity. S. pombe cells were transformed with a control vector or vectors to express the full length PKC-δ protein or a PKC-δ protein in which part of the catalytic domain has been deleted to render it functionally inactive as a protein kinase (PKC-δΔ). After selection for uptake of plasmid, a number of colonies were plated onto selective medium plates in the presence of thiamine, the absence of thiamine or the presence of TPA. PKC-δ expression markedly inhibits growth (-thiamine plate) and addition of TPA increases the effect. In contrast, expression of PKC-δΔ has no effect on growth under any condition. Example 6: An inhibitor screen for protein kinase C-rj. A cDNA for PKC-r? (Figure 6; SEQ ID No 7) has been introduced into a plasmid under the control of the nmt promoter, producing nmt-PKC-η. A 3.3 kb Xhol fragment containing the coding sequence for PKC-η was isolated from pBluescript-PKC-77 and subcloned into Sα/I-digested pREP3X to give pREP3X- PKC-7?. The plasmid pBluescript-PKC-η can be obtained using the methods described by Dekker et al (1992) FEBS Lett. 312, 195-199. Transfection of S. pombe with nmt-PKC-η yields populations of cells that on switching to medium without thiamine induce PKC-77 protein as determined by Western blotting and the detection of PKC activity in cell extracts. However, there is some expression even in the presence of thiamine which produces ~50% growth inhibition. There is an even more marked growth inhibition by derepressed expression alone and if the PKC-77 is selectively activated by inclusion of the phorbol ester TPA in the growth medium, there is no growth. That activation of PKC-77 induces growth inhibition provides a screen for inhibition of function of this mammalian gene product. Operating the screen. The screen for inhibitor activities is carried out on a 96-well microtitre plate format. For thiamine repressible genes, stable integrants are grown up overnight (12 h) in the absence of thiamine. The culture is then diluted in the absence of thiamine to an OD 595 = 0.01 to 0.10. The culture is then aliquoted into microtitre wells containing the potential inhibitors and, in the case of PKC-γ, also phorbol ester. The growth of cells monitored at 595 nm using a microtitre plate reader. Cells are allowed to grow until parallel wells/plates containing cells growing in the presence of thiamine (15 μM) have increased their OD 595 to 1.0 units. Cells from the test wells that have proliferated can be scored relative to both control wells (+ thiamine) and no addition wells (-inhibitor, -thiamine). Additionally, the test wells may contain or lack TPA. REFERENCES 1. Russell, P. & Nurse, P. (1986) Cell 45, 145-153. 2. Maundrell, K. (1990) J. Biol Chem. 265, 10857-10864. 3. Morgan, S.J. et al (1990) Eur. J. Biochem. 191, 761-767. 4. Otsu, M. et al (1991) Cell 65, 91-104. 5. Laemmli, U.K. (1970) Nature 227, 680-685. 6. Katan, M. et al (1988) Cell 54, 171-177. 7. Sambrook, J. et al (1989) "Molecular Cloning, a Laboratory Manual" C.S.H. Laboratory Press. 8. Moreno, S. et al (1991) "Molecular Genetic Analysis of fission yeast Schizosaccharomyces pombe ". In Methods in Enzymology. pp 795- 823. 9. Shibasaki, F. et al (1991) J. Biol. Chem. 266, 8108-8114. 10. Schaap, D. et al (1989) FEBS Lett. 243, 351-357. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Imperial Cancer Research Technology Ltd (B) STREET: Sardinia House, Sardinia Street (C) CITY: London (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): WC2A 3NL (G) TELEPHONE: 071 242 1136 (H) TELEFAX: 071 831 4991 (ii) TITLE OF INVENTION: Transformed cells and assays using -them (iii) NUMBER OF SEQUENCES: 15 (iv) 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 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3498 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..3204 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG CCT CCA AGA CCA TCA TCA GGT GAA CTG TGG GGC ATC CAC TTG ATG 48 Met Pro Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly lie His Leu Met 1 5 10 15 CCC CCA AGA ATC CTA GTA GAA TGT TTA CTA CCA AAT GGG ATG ATA GTG 96 Pro Pro Arg lie Leu Val Glu Cys Leu Leu Pro Asn Gly Met lie Val 20 25 30 ACT TTA GAA TGC CTC CGT GAG GCT ACG TTA ATA ACG ATA AAG CAT GAA 144 Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu lie Thr lie Lys His Glu 35 40 45 CTA TTT AAA GAA GCA AGA AAA TAC CCT CTC CAT CAA CTT CTT CAA GAT 192 Leu Phe Lys Glu Ala Arg Lys Tyr Pro Leu His Gin Leu Leu Gin Asp 50 55 60 GAA TCT TCT TAC ATT TTC GTA AGT GTT ACC CAA GAA GCA GAA AGG GAA 240 Glu Ser Ser Tyr lie Phe Val Ser Val Thr Gin Glu Ala Glu Arg Glu 65 70 75 80 GAA TTT TTT GAT GAA ACA AGA CGA CTT TGT GAC CTT CGG CTT TTT CAA 288 Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gin 85 90 95 CCC TTT TTA AAA GTA ATT GAA CCA GTA GGC AAC CGT GAA GAA AAG ATC 336 Pro Phe Leu Lys Val lie Glu Pro Val Gly Asn Arg Glu Glu Lys lie 100 105 110 CTC AAT CGA GAA ATT GGT TTT GCT ATC GGC ATG CCA GTG TGT GAA TTC 384 Leu Asn Arg Glu lie Gly Phe Ala lie Gly Met Pro Val Cys Glu Phe 115 120 125 GAT ATG GTT AAA GAT CCA GAA GTA CAG GAC TTC CGA AGA AAT ATT CTC 432 Asp Met Val Lys Asp Pro Glu Val Gin Asp Phe Arg Arg Asn lie Leu 130 135 140 AAT GTT TGT AAA GAA GCT GTG GAT CTT AGG GAT CTT AAT TCA CCT CAT 480 Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His 145 150 155 160 AGT AGA GCA ATG TAT GTT TAT CCT CCA AAT GTA GAA TCT TCA CCA GAA 528 Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175 CTG CCA AAG CAC ATA TAT AAT AAA TTG GAT AAA GGG CAA ATA ATA GTG 576 Leu Pro Lys His lie Tyr Asn Lys Leu Asp Lys Gly Gin lie lie Val 180 185 190 GTG ATT TGG GTA ATA GTT TCT CCA AAT AAT GAC AAA CAG AAG TAT ACT 624 Val lie Trp Val lie Val Ser Pro Asn Asn Asp Lys Gin Lys Tyr Thr 195 200 205 CTG AAA ATC AAC CAT GAC TGT GTG CCA GAA CAA GTA ATT GCT GAA GCA 672 Leu Lys lie Asn His Asp Cys Val Pro Glu Gin Val lie Ala Glu Ala 210 215 220 ATC AGG AAA AAA ACT CGA AGT ATG TTG CTA TCA TCT GAA CAA CTA AAA 720 lie Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gin Leu Lys 225 230 235 240 CTC TGT GTT TTA GAA TAT CAG GGC AAG TAT ATT TTA AAA GTG TGT GGA 768 Leu Cys Val Leu Glu Tyr Gin Gly Lys Tyr He Leu Lys Val Cys Gly 245 250 255 TGT GAT GAA TAC TTC CTA GAA AAA TAT CCT CTG AGT CAG TAT AAG TAT 816 Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gin Tyr Lys Tyr 260 265 270 ATA AGA AGC TGT ATA ATG CTT GGG AGG ATG CCC AAT TTG ATG CTG ATG 864 He Arg Ser Cys He Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285 GCT AAA GAA AGC CTC TAT TCT CAA CTG CCA ATG GAC TGT TTT ACA ATG 912 Ala Lys Glu Ser Leu Tyr Ser Gin Leu Pro Met Asp Cys Phe Thr Met 290 295 300 CCA TCA TAT TCC AGA CGC ATC TCC ACA GCT ACG CCA TAT ATG AAT GGA 960 Pro Ser Tyr Ser Arg Arg He Ser Thr Ala Thr Pro Tyr Met Asn Gly 305 310 315 320 GAA ACA TCT ACA AAA TCC CTT TGG GTT ATA AAT AGT GCA CTC AGA ATA 1008 Glu Thr Ser Thr Lys Ser Leu Trp Val He Asn Ser Ala Leu Arg He 325 330 335 AAA ATT CTT TGT GCA ACC TAT GTG AAT GTA AAT ATT CGA GAC ATT GAC 1056 Lys He Leu Cys Ala Thr Tyr Val Asn Val Asn He Arg Asp He Asp 340 345 350 AAG ATT TAT GTT CGA ACA GGT ATC TAC CAT GGA GGA GAA CCC TTA TGT 1104 Lys He Tyr Val Arg Thr Gly He Tyr His Gly Gly Glu Pro Leu Cys 355 360 365 GAT AAT GTG AAC ACT CAA AGA GTA CCT TGT TCC AAT CCC AGG TGG AAT 1152 Asp Asn Val Asn Thr Gin Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380 GAA TGG CTG AAT TAC GAT ATA TAC ATT CCT GAT CTT CCT CGT GCT GCT 1200 Glu Trp Leu Asn Tyr Asp He Tyr He Pro Asp Leu Pro Arg Ala Ala 385 390 395 400 CGA CTT TGC CTT TCC ATT TGT TCT GTT AAA GGC CGA AAG GGT GCT AAA 1248 Arg Leu Cys Leu Ser He Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415 GAG GAA CAC TGT CCA TTG GCC TGG GGA AAT ATA AAC TTG TTT GAT TAC 1296 Glu Glu His Cys Pro Leu Ala Trp Gly Asn He Asn Leu Phe Asp Tyr 420 425 430 ACA GAT ACT CTA GTA TCT GGA AAA ATG GCT TTG AAT CTT TGG CCA GTA 1344 Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445 CCT CAT GGA CTA GAA GAT TTG CTG AAC CCT ATT GGT GTT ACT GGA TCA 1392 Pro His Gly Leu Glu Asp Leu Leu Asn Pro He Gly Val Thr Gly Ser 450 455 460 AAT CCA AAT AAA GAA ACT CCA TGT TTA GAG TTG GAG TTT GAC TGG TTC 1440 Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe 465 470 475 480 AGC AGT GTG GTA AAG TTT CCA GAT ATG TCA GTG ATT GAA GAG CAT GCC 1488 Ser Ser Val Val Lys Phe Pro Asp Met Ser Val He Glu Glu His Ala 485 490 495 AAT TGG TCT GTA TCC CGT GAA GCA GGA TTT AGT TAT TCC CAT GCA GGA 1536 Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His Ala Gly 500 505 510 CTG AGT AAC AGA CTA GCT AGA GAC AAT GAA TTA AGA GAA AAT GAT AAA 1584 Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525 GAA CAG CTC CGA GCA ATT TGT ACA CGA GAT CCT CTA TCT GAA ATC ACT 1632 Glu Gin Leu Arg Ala He Cys Thr Arg Asp Pro Leu Ser Glu He Thr 530 535 540 GAG CAA GAG AAA GAT TTT CTG TGG AGC CAC AGA CAC TAT TGT GTA ACT 1680 Glu Gin Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr 545 550 555 560 ATC CCC GAA ATT CTA CCC AAA TTG CTT CTG TCT GTT AAA TGG AAC TCT 1728 He Pro Glu He Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575 AGA GAT GAA GTA GCT CAG ATG TAC TGC TTG GTA AAA GAT TGG CCT CCA 1776 Arg Asp Glu Val Ala Gin Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590 ATC AAG CCT GAA CAG GCT ATG GAG CTT CTG GAC TGC AAT TAC CCA GAT 1824 He Lys Pro Glu Gin Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605 CCT ATG GTT CGA GGT TTT GCT GTT CGG TGC TTA GAA AAA TAT TTA ACA 1872 Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620 GAT GAC AAA CTT TCT CAG TAC CTA ATT CAG CTA GTA CAG GTA CTA AAA 1920 Asp Asp Lys Leu Ser Gin Tyr Leu He Gin Leu Val Gin Val Leu Lys 625 630 635 640 TAT GAA CAG TAT TTG GAT AAC CTG CTT GTG AGA TTT TTA CTC AAA AAA 1968 Tyr Glu Gin Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655 GCG TTA ACT AAT CAA AGG ATC GGT CAC TTT TTC TTT TGG CAT TTA AAA 2016 Ala Leu Thr Asn Gin Arg He Gly His Phe Phe Phe Trp His Leu Lys 660 665 670 TCT GAG ATG CAC AAT AAA ACA GTT AGT CAG AGG TTT GGC CTG CTT TTG 2064 Ser Glu Met His Asn Lys Thr Val Ser Gin Arg Phe Gly Leu Leu Leu 675 680 685 GAG TCC TAT TGC CGT GCA TGT GGG ATG TAT CTG AAG CAC CTT AAT AGG 2112 Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700 CAA GTT GAG GCT ATG GAA AAG CTC ATT AAC TTG ACT GAC ATT CTC AAA 2160 Gin Val Glu Ala Met Glu Lys Leu He Asn Leu Thr Asp lie: Leu Lys 705 710 715 720 CAA GAG AAG AAG GAT GAA ACA CAA AAG GTA CAG ATG AAG TTT TTA GTT 2208 Gin Glu Lys Lys Asp Glu Thr Gin Lys Val Gin Met Lys Phe Leu Val 725 730 735 GAG CAA ATG CGG CGA CCA GAT TTC ATG GAT GCT CTC CAG GGC TTT CTG 2256 Glu Gin Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gin Gly Phe Leu 740 745 750 TCT CCT CTA AAC CCT GCT CAT CAG CTG GGA AAT CTC AGG CTT GAA GAG 2304 Ser Pro Leu Asn Pro Ala His Gin Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765 TGT CGA ATT ATG TCT TCT GCA AAA AGG CCA CTG TGG TTG AAT TGG GAG 2352 Cys Arg He Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780 AAC CCA GAC ATC ATG TCA GAA TTA CAC TTT CAG AAC AAT GAG ATC ATC 2400 Asn Pro Asp He Met Ser Glu Leu His Phe Gin Asn Asn Glu He He 785 790 795 800 TTT AAA AAT GGG GAT GAT TTA CGG CAA GAT ATG CTA ACC CTT CAG ATT 2448 Phe Lys Asn Gly Asp Asp Leu Arg Gin Asp Met Leu Thr Leu Gin He 805 810 815 ATT CGC ATT ATG GAA AAT ATC TGG CAA AAT CAA GGT CTT GAT CTT CGA 2496 He Arg He Met Glu Asn He Trp Gin Asn Gin Gly Leu Asp Leu Arg 820 825 830 ATG TTA CCT TAT GGA TGT CTG TCA ATC GGT GAC TGT Gl'G GGA CTT ATC 2544 Met Leu Pro Tyr Gly Cys Leu Ser He Gly Asp Cys Val Gly Leu He 835 840 845 GAG GTG GTG AGA AAT TCT CAC ACT ATA ATG CAG ATT CAG TGT AAA GGA 2592 Glu Val Val Arg Asn Ser His Thr He Met Gin He Gin Cys Lys Gly 850 855 860 GGC CTG AAA GGT GCA CTG CAG TTT AAC AGC CAC ACA CTC CAT CAG TGG 2640 Gly Leu Lys Gly Ala Leu Gin Phe Asn Ser His Thr Leu His Gin Trp 865 870 875 880 CTC AAA GAC AAG AAC AAG GGG GAA ATA TAT GAT GCG GCC ATC GAT TTG 2688 Leu Lys Asp Lys Asn Lys Gly Glu He Tyr Asp Ala Ala He Asp Leu 885 890 895 TTT ACA CGA TCA TGT GCT GGA TAT TGT GTT GCC ACC TTC ATT TTG GGA 2736 Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe He Leu Gly 900 905 910 ATT GGA GAT CGT CAC AAT AGT AAT ATC ATG GTT AAA GAT GAT GGA CAA 2784 He Gly Asp Arg His Asn Ser Asn He Met Val Lys Asp Asp Gly Gin 915 920 925 CTG TTT CAT ATA GAT TTT GGA CAC TTT TTG GAT CAC AAG AAG AAA AAA 2832 Leu Phe His He Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940 TTT GGT TAT AAA CGA GAG CGC GTG CCG TTT GTT TTG ACA CAA GAT TTC 2880 Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gin Asp Phe 945 950 955 960 TTA ATA GTG ATT AGT AAA GGA GCC CAA GAA TGC ACA AAG ACA AGA GAA 2928 Leu He Val He Ser Lys Gly Ala Gin Glu Cys Thr Lys Thr Arg Glu 965 970 975 TTT GAG AGG TTT CAG GAG ATG TGT TAC AAG GCT TAT CTA GCT ATT CGG 2976 Phe Glu Arg Phe Gin Glu Met Cys Tyr Lys Ala Tyr Leu Ala He Arg 980 985 990 CAG CAT GCC AAT CTC TTC ATA AAT CTT TTC TCA ATG ATG CTT GGC TCT 3024 Gin His Ala Asn Leu Phe He Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005 GGA ATG CCA GAA CTG CAA TCT TTT GAT GAT ATT GCA TAC ATT CGA AAG 3072 Gly Met Pro Glu Leu Gin Ser Phe Asp Asp He Ala Tyr He Arg Lys 1010 1015 1020 ACC CTA GCT TTA GAT AAA ACT GAG CAA GAG GCT TTG GAG TAT TTC ATG 3120 Thr Leu Ala Leu Asp Lys Thr Glu Gin Glu Ala Leu Glu Tyr Phe Met 1025 1030 1035 1040 AAA CAA ATG AAT GAT GCA CAC CAT GGT GGC TGG ACA ACA AAA ATG GAT 3168 Lys Gin Met Asn Asp Ala His His Gly Gly Trp Thr Thr Lys Met Asp 1045 1050 1055 TGG ATC TTC CAC ACA ATT AAG CAG CAT GCT TTG AAC TGAAATGATA 3214 Trp He Phe His Thr He Lys Gin His Ala Leu Asn 1060 1065 ACTAAAAGCT CAGTATCTGG ATTCTACACT GCACTGTTAA TAACTGTCAA CAGGCAAAGA 3274 CTGATTGCAT AGGAATTGCA CAATCCATGA ACAGCATTAG AATTTACAGC AAGAACAGAA 3334 ATAAAATACT ATATAATTTA AATAATGTAA ACGCAAACAG GGTTTGATAG CACTAAACTA 3394 GTTCATTTCA AAATTAAGCT TTAGAATAAT GCGCAATTTC ATGTTATGCC TTAAGTCCAA 3454 AAAGGTAAAC TTTAAAGATT GTTTGTATCT TTCCTTTAAA AAAA 3498 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1068 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Pro Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly He His Leu Met 1 5 10 15 Pro Pro Arg He Leu Val Glu Cys Leu Leu Pro Asn Gly Met He Val 20 25 30 Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu He Thr He Lys His Glu 35 40 45 Leu Phe Lys Glu Ala Arg Lys Tyr Pro Leu His Gin Leu Leu Gin Asp 50 55 60 Glu Ser Ser Tyr He Phe Val Ser Val Thr Gin Glu Ala Glu Arg Glu 65 70 75 80 Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gin 85 90 95 Pro Phe Leu Lys Val He Glu Pro Val Gly Asn Arg Glu Glu Lys He 100 105 110 Leu Asn Arg Glu He Gly Phe Ala He Gly Met Pro Val Cys Glu Phe 115 120 125 Asp Met Val Lys Asp Pro Glu Val Gin Asp Phe Arg Arg Asn He Leu 130 135 140 Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His 145 150 155 160 Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175 Leu Pro Lys His He Tyr Asn Lys Leu Asp Lys Gly Gin He He Val 180 185 190 Val He Trp Val He Val Ser Pro Asn Asn Asp Lys Gin Lys Tyr Thr 195 200 205 Leu Lys He Asn His Asp Cys Val Pro Glu Gin Val He Ala Glu Ala 210 215 220 He Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gin Leu Lys 225 230 235 240 Leu Cys Val Leu Glu Tyr Gin Gly Lys Tyr He Leu Lys Val Cys Gly 245 250 255 Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gin Tyr Lys Tyr 260 265 270 He Arg Ser Cys He Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285 Ala Lys Glu Ser Leu Tyr Ser Gin Leu Pro Met Asp Cys Phe Thr Met 290 295 300 Pro Ser Tyr Ser Arg Arg He Ser Thr Ala Thr Pro Tyr Met Asn Gly 305 310 315 320 Glu Thr Ser Thr Lys Ser Leu Trp Val He Asn Ser Ala Leu Arg He 325 330 335 Lys He Leu Cys Ala Thr Tyr Val Asn Val Asn He Arg Asp He Asp 340 345 350 Lys He Tyr Val Arg Thr Gly He Tyr His Gly Gly Glu Pro Leu Cys 355 360 365 Asp Asn Val Asn Thr Gin Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380 Glu Trp Leu Asn Tyr Asp He Tyr He Pro Asp Leu Pro Arg Ala Ala 385 390 395 400 Arg Leu Cys Leu Ser He Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415 Glu Glu His Cys Pro Leu Ala Trp Gly Asn He Asn Leu Phe Asp Tyr 420 425 430 Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445 Pro His Gly Leu Glu Asp Leu Leu Asn Pro He Gly Val Thr Gly Ser 450 455 460 Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe 465 470 475 480 Ser Ser Val Val Lys Phe Pro Asp Met Ser Val He Glu Glu His Ala 485 490 495 Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His Ala Gly 500 505 510 Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525 Glu Gin Leu Arg Ala He Cys Thr Arg Asp Pro Leu Ser Glu He Thr 530 535 540 Glu Gin Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr 545 550 555 560 He Pro Glu He Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575 Arg Asp Glu Val Ala Gin Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590 He Lys Pro Glu Gin Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605 Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620 Asp Asp Lys Leu Ser Gin Tyr Leu He Gin Leu Val Gin Val Leu Lys 625 630 635 640 Tyr Glu Gin Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655 Ala Leu Thr Asn Gin Arg He Gly His Phe Phe Phe Trp His Leu Lys 660 665 670 Ser Glu Met His Asn Lys Thr Val Ser Gin Arg Phe Gly Leu Leu Leu 675 680 685 Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700 Gin Val Glu Ala Met Glu Lys Leu He Asn Leu Thr Asp He Leu Lys 705 710 715 720 Gin Glu Lys Lys Asp Glu Thr Gin Lys Val Gin Met Lys Phe Leu Val 725 730 735 Glu Gin Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gin Gly Phe Leu 740 745 750 Ser Pro Leu Asn Pro Ala His Gin Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765 Cys Arg He Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780 Asn Pro Asp He Met Ser Glu Leu His Phe Gin Asn Asn Glu He He 785 790 795 800 Phe Lys Asn Gly Asp Asp Leu Arg Gin Asp Met Leu Thr Leu Gin He 805 810 815 He Arg He Met Glu Asn He Trp Gin Asn Gin Gly Leu Asp Leu Arg 820 825 830 Met Leu Pro Tyr Gly Cys Leu Ser He Gly Asp Cys Val Gly Leu He 835 840 845 Glu Val Val Arg Asn Ser His Thr He Met Gin He Gin Cys Lys Gly 850 855 860 Gly Leu Lys Gly Ala Leu Gin Phe Asn Ser His Thr Leu His Gin Trp 865 870 875 880 Leu Lys Asp Lys Asn Lys Gly Glu He Tyr Asp Ala Ala He Asp Leu 885 890 895 Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe He Leu Gly 900 905 910 He Gly Asp Arg His Asn Ser Asn He Met Val Lys Asp Asp Gly Gin 915 920 925 Leu Phe His He Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940 Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gin Asp Phe 945 950 955 960 Leu He Val He Ser Lys Gly Ala Gin Glu Cys Thr Lys Thr Arg Glu 965 970 975 Phe Glu Arg Phe Gin Glu Met Cys Tyr Lys Ala Tyr Leu Ala He Arg 980 985 990 Gin His Ala Asn Leu Phe He Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005 Gly Met Pro Glu Leu Gin Ser Phe Asp Asp He Ala Tyr He Arg Lys 1010 1015 1020 Thr Leu Ala Leu Asp Lys Thr Glu Gin Glu Ala Leu Glu Tyr Phe Met 1025 1030 1035 1040 Lys Gin Met Asn Asp Ala His His Gly Gly Trp Thr Thr Lys Met Asp 1045 1050 1055 Trp He Phe His Thr He Lys Gin His Ala Leu Asn 1060 1065 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2199 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: SCHIZOSACCHAROMYCES POMBE (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAAAATCTCA ACACATGTGA ATGATCAGAA AATTATCGCC ATAAAAGACA GAATAAGTCA 60 TCAGCGGTTG TTTCATTTCC TATATTTTTT TTTTATTTTT TTATTTTTTA ATAAGGGAAA 120 ATTTAACGTC TAAGGATACA GAAGATTGTT AGCACATTAA AGTAATAAAG GCTTAAGTAG 180 TAAGTGCCTT AGCATGTTAT TGTATTTCAA AGGACATAAT CTAAAATAAT AACAATATCA 240 TTTCTCACAA GTTATTCAAT TTTCTTTTTT TTTTCTAATA ATATCAAGAA TGTATTATTT 300 GTTTGACATA AGTCAACTAA TTTATTTAAT ATGCTGGATT AATCTTGCAG ACATGTAAAT 360 TAACAAGTTT TAGTCAAATA ACGTTGAAGT TTCAATGAAC TCAAATAATT TCTCTTTTTT 420 TTTATATAAC CATATGTCTA ATCTGATTTA TATTTTCCGC AGGATCAACT GAAGTTATGA 480 CATTTGGATT GGATCACTTA TAACCTTGGT CGCCAAATAA TACAAAAATC AGCGTTATAA 540 AACAAAGAAG GTTTTTGTTA AGAAATTAAT CCTCTTTCTT GATAAGAAAG TTGAACCGAA 600 ATTGCAGATA CTGATATATG AAAATAATAC CCACAATTTT GGGAATAGCG CAAGCCTCAA 660 TTTAAACAAT AGGTGAGGAC ACATGATAAT GACCTCAATG ATTGTTAGAA GAAAAGAGCC 720 TCATTACAAA ATCGAAAAAT GAATGGTTGG GTACAAGTTT CCAAAACATG GTAAAGTGGA 780 CTTTGCGTAT GAGACGTAAA TAGAAAAAAA CACTTGTTAT ATGTTTTCTA GAATTATTGT 840 TGTCTCTTTA TGGTTGGATG ATGCAAAATA GTAATTTCGG TTAGTTGCTG TAAAACACCA 900 CGAGACAAAT AGATATGGAT ATTTATTAAA TCAGGAAAAA CGTAACTCTC GGCTACTGGA 960 TGGTTCAGTC ACCCAACGAT TACTGGGGAG AGAAAACAGG GCAAAAGCAA AGCTTAAAGG 1020 AATCCGATTG TCATTCGGCA ATGTGCAGCG AAACTAAAAA CCGGATAATG GACCTGTTAA 1080 TCGAAACATT GAAGATATAT AAAGGAAGAG GAATCCTGGC TGAATTAATT ATTTCAATCT CATTCTCACT TTCTGACTTA AGGAATGTCT CCCTTGCCAG TACTGCTAGG GTTTTTCTTT AAGCTGCATA TTACGATTTT GTTTTTCGCT TTTAGAAAGT AAAATTCTTG ATCTCCGACA ACGAGTACTT TTATTTTTTT ATTAGCTCGA AATCGTAGAA ACGTAGACGG GTGCGGGATA TTTTTATAAA CCACGTGGCC CAAAAATATG AACCCAAAAC TAAGAAGGCT ATACCCCTTC GTGTTAGATG TAGTTTTAGC GCTTGACTTC AGATGTAGAA GGCATTAAAT CGTTTTGAAT AATTAAATAT TTAAAAGCAA TCATACGCTG AAAATTTAGT ATGGAAATGC CATAAAAGTG ACTTTGACAA AAAAAAAAGT TCTACTTCAT TGACTTTGTT TACAGCATGT GGAAGGAGGA GTTTAACATT CAATAAGTAA TACTATTGAA ATTCGACAAG AGAGGCATTT TGCTTTGGGA GAATTAGTTC AAATTAGAAC GGCAAAAATG TCGGATTGAG ATCGTAAAAG TTCGCTCGTC TTTCATGGAT ACATCTTGCT GGATATTTAA ATTTTAGTAC AATGTTTTAT CACCCAAACC TGTTAGCGCC TTCTTAATTC CTGAGACTAC TTCTTGGACT TTCACTACTT GTTAGTTATA GTGAAATAAT CCTTTCTATT AGTAATGCAA ACAAAAATC (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2707 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CTCGAGCTGA AGAACCAGCG AGGCGGCGAG GCAGCCCCCG CGGCTTGCAG CGGAGGCGAC 60 AGCTCGTCTC CTGCCGTGGA GGTGTCGCCG GTGGTGGGGG GGAGAGACTT GCTCCAAAAA 120 AACGGACGTC TCCAGCTCTC CCCCCTCCCT GTTTTCCGTT AGGAATCCGG CGAGGAAATA 180 CATGCACTCG CTGAGAATCG GCGGCGCCAG GAGGCAGCGC CACAAGGTGT AGCGAGTGAG 240 TGGGGTGGGG CAAGAGGGGA CCCAGGAGTC CCCCAGGCTC CCGGCGCGCC TGCTCCTGCT 300 CCCGCACAAG CGCCTGGGCT GTGTGGCAGC GCAGAACGGG TCCATTCTTC AAGGAGATTG ACTGGGTACT GCTGGAGCAG CAAGCCGAGA ATTAAAACCA AAAGAGATGT CAATAACTTT AGAGCCAATA CTTACACTTG TGGATGAAGC AATCATTAAG CAAAGGCTTC TCCTACTTTG GTGAAGACCT GATGCCCTGA GAGCTCATGC TGCAAGGACG GTGTTGAGAT ACTCCCAAGC CAACTCCTCC TCCTCCTCCC CCTCCCCAGA GCCCCAGTCC TTGCATT (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2167 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGCCCCTGTT CTGCAGAAAG GGGGCTCTGA GGCAGAAGGT ACAAGTTCAC CGCTCGCTTC TTCAAGCAGC CGACCTTCTG TATGGGGGAT TGGAAAACAG GGTCTGCAAT GTCAAGTCTG GATGCCACGA GTTTGTGACC TTCGAGTGTC CAGGCGCTGG ATCCCCGGAA CAAGCACAAG TTCCGTCTGC ACAGCTACAG ACTGTGGCTC CCTGCTCTAC GGGCTGGTGC ACCAGGGCAT TGAACGTGCA CCGGCGCTGT GTGCGCAGCG TGCCCTCTCT AGCGCCGGGG CCGCCTGCAG CTGGAGATCC GGGCGCCCAC CGGTTGGCGA GGCCCGGAAC CTCATCCCAA TGGACCCCAA TGAAGCTGAA GCTCATCCCA GACCCTCGGA ATTTGACCAA AAGCTACGCT AAACCCTGTG TGGAACGAGA CCTTTGTGTT TGGAGCGCCG GCTCAGCGTG GAGGTGTGGG ACTGGGACCG TGGGCGCCAT GTCCTTCGGC GTCTCGGAGC TGCTCAAGGC AGTTACTGAA CCAGGAGGAG GGCGAGTATT ACAATGTGCC GCAACCTCCT CCAGAAGTTC GAGGCCTGTA ACTACCCCCT GGACGGGTCC CTCTTCATCT CCCATCCCCT CCCCATCCCC GCTGTTTCTT CGGGGCCAGC CCTGGACGAC TGCACATCTC TTCTAGGAAA AGGCAGTTTT GGGAAGGTGA TGCTGGCCGA TCTACGCCAT CAAGATCCTG AAGAAAGACG TGATCGTCCA CCCTGGTGGA GAAACGCGTG CTGGCTCTGG GGGGCCGAGG TCCTCACCCA GCTTCACTCC ACCTTCCAGA CCCCGGATCG ATGTCACCGG GGGCGACTTG ATGTACCACA TTCAACAGCT ACGCAGCGTT CTACGCTGCA GAAATCGCCA TCGGCCTCTT TTATCTATCG GGACCTGAAA CTGGACAACG TGATGCTGGA TCACCGACTT CGGCATGTGT AAGGAGAACG TCTTTCCCGG GCGGGACCCC GGACTACATA GCCCCCGAGA TCATTGCCTA TGGATTGGTG GTCCTTTGGG GTTCTGCTCT ACGAGATGTT ATGGAGAAGA TGAGGAGGAG CTGTTTCAAG CCATCATGGA AGTCGCTTTC CCGGGAAGCT GTGGCCATCT GCAAGGGGTT AGCGCCTGGG CTCAGGCCCC GATGGAGAGC CCACCATCCG GGATCGACTG GGACAGGCTG GAACGATTAG AGATCGCGCC GTGGCCGCAG CGGCGAGAAC TTCGACAAGT TCTTCACTCG CCCCTGACCG CCTGGTTCTG GCCAGCATCG ACCAGGCTGA TCAACCCGGA TTTCGTGCAC CCGGATGCCC GCAGCCCCAT TCATGTAATC CCACCTGCCG CCACCAGGCG TCCCCACGGC TGGCCCTCGC CTCACCATGC CACCCGCCTT TCCAATTCTA TGGCCTC (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2891 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGCGGCGGCC GCGGGGATCC CGCGAGCGGC CCCTGAACAT CTACCCTTCT TGCCGGGACC 60 CGGGAGGTCC CCACTGGCCT CCGGGCCCGT CCTGATCAGA CTCGTGTCGA CCTCCCCGTC 120 CACGCGCATC CGGGAGAGCC GCGCCACGAG ACGGACCCGG GCCCGCCGGG ACCCCTGGTG 180 CTATCAACTG GAACCTGCTG GAGAAGCGGA AGGTGGAGCC AATCCCCTTC AGACTACAGC AACTTTGACC CAGAGTTCCT CCTTCAGTGA CAAGAACCTC ATCGACTCTA TGGACCAGAC TTGTGAACCC CAAATATGAG CAATTCCTGG AATAGTGAGC CCCCGGCAGA GTAGGCCCAT CTGCCCTGGT TTGCATCCTC GGTGACTGGT GATTCCTGCT GCTGCCCCCT CTTCCTCGGA GGGCTCACAG TACTTCCTCT GTGAACTGTT TGTGAATTTG GGAAACTGTA AATCCTGTGT GTCATTACTT GAATGTAGTT TATGCACATA TATATAATAG GCTGTATATA TTGCTCAGTA GGTGATGTGT TGACCTTTTT TTAAAAAAAA CCATATGTAT ACACACGTAT ACATATATGT ATGTATGTAT GTATGTATGT AAAGAGAGCA CAAGCTACCT GAACCACAGG ATTGTTTATG ATGGTAAAAA A (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2176 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TCCGGGTTCC CCAGTGCCAG CCAGCGCGGC CCCCTCGGGG TGTCGTCCGG CACGATGAAG TTCAATGGCT ATCTGAGGGT GGCTGCAGCC CACCCGCTGG TCCCTGCGGC ACTCGCTCTT TGGACCCCTA CCTGACGGTG AGCGTAGACC AGGTACGCGT AGAAGACCAA CAAACCCACC TACAACGAGG AGTTCTGCGC ACCTGGAGCT AGCCGTCTTC CACGAGACGC CCCTGGGTTA GCACGCTGCA GTTCCAGGAG CTGTTGCGCA CGGCTGGTAC GGGTGGATCT GGAGCCTGAG GGGAAAGTGT TTGTGGTAAT CTGAAGCCAC TCTCCAGAGA GACCGCATCT TCAAGCATTT CTATGCGAAG ACGAGTCCAT CAAGTGAACG GACATAAGTT AGCCCACCTA CTGCTCTCAT TGCCGAGAGT TCATCTGGGG ATCAATGCCA AGTGTGCACC TGCGTCGTCC ATAAACGCTG CCTGCACTTG CCAAAACAAT ATTAACAAAG TGGATGCCAA GCATCAACAT CCCACACAAG TTCAACGTTC ACAATTACAA ACTGTGGCTC CCTGCTCTGG GGGATAATGC GACAAGGACT TGAATGTACA TATTCGGTGT CAGGCGAACG TGGCCCCAAA AGCTTGCCAA GACCCTGGCA GGGATGGGTC TCCAACCCGG AACTCATTTC CAGGTCGACA CTAAGACGGC AGGGAAAGGA GGATCGGTGT TAACTCTTCC AGCAGATTCG GCATCGACAA TGGGGAAGGG GAGCTTCGGG AAGGTGATGC TTGCCAGGAT ACGCCGTGAA GGTGCTGAAG AAGGACGTGA TTCTGCAGGA TGACTGAGAA GAGGATCCTG TCCTTGGCTC GCAACCACCC GCTGCTTTCA GACTCCAGAC CGTCTGTTCT TTGTCATGGA TGATGTTCCA CATCCAAAAG TCCCGTCGTT TCGATGAAGC CGGAGATCAT TTCTGCACTC ATGTTCCTAC ATGAGAAAGG AACTGGACAA TGTGCTATTG GACCACGAAG GTCACTGTAA GCAAGGAGGG GATTTGTAAT GGGGTCACCA CAGCCACCTT TTGCCCCAGA GATCCTTCAG GAGATGCTGT ATGGACCTGC GCGTGTTGCT TTATGAGATG CTGTGCGGAC ATGCGCCCTT ACCTTTTTGA GGCCATACTG AATGATGAAG TCGTCTACCC CCAGAGGGAT CCTCAAGTCT TTCATGACCA AGAACCCCAC CTCAGGGAGG AGAGCATGAG ATCCTGAGAC ACCCTTTCTT AGTTGAACCA TCGCCAGTTA GAGCCGCCTT TCCGACCTAG TCAGCAATTT TGACCCAGAC TTTATAAAAG AAGAGCCCGT GACATCTTCC TATGATTAAC CAGGATGAGT TTAGAAACTT TGCAACTGTA GCCTTATGGG GAGTCAGAAC CAAAGGGGAA TCTTATGTGG GAATTC (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Asp Trp He Phe His Thr (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AARATGGAYT GGATHTTYCA YAC 23 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Asp Asp Gly Gin Leu Phe His He Asp Phe Gly His Phe 1 5 10 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GATGATGGCC ARCTGTTYCA YATWGAYTTT GGCCAYTT 38 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AATTCACACA CTGGCATGCC GAT 23 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GACTCGAGTC GACATCGATT TTTTTTTTTT TTTTT 35 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TTTAAGCTTA GGCATTCTAA AGTCACTATC ATCCC 35 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GACTCGAGTC GACATCGA 18

Claims: CLAIMS 1. A eukaryotic cell transformed with a DNA construct comprising a coding sequence encoding a polypeptide having the activity of a mammalian phospholipid kinase or a mammalian protein kinase activated by a phospholipid or its metabolite and regulatory elements to allow transcription of the coding sequence in the said cell wherein the regulatory elements include a repressible or inducible promoter and the expression of the said coding sequence is lethal or growth inhibitory to the cell. 2. A cell according to Claim 1 wherein the cell is a yeast cell. 3. A cell according to Claim 2 wherein the yeast is Schizosaccharomyces. 4. A cell according to Claim 3 wherein the promoter is the nmt promoter. 5. A Schizosaccharomyces cell transformed with a DNA construct comprising a coding sequence encoding a polypeptide having the activity of a mammalian phospholipid kinase or a mammalian protein kinase activated by a phospholipid or its metabolite and regulatory elements to allow transcription of the coding sequence in the said cell wherein the regulatory elements include a constitutive promoter and the expression of the said coding sequence is lethal or growth inhibitory to the cell. 6. A Schizosaccharomyces cell according to Claim 5 wherein the promoter is the adh promoter. 7. A cell according to any one of the preceding claims wherein the phospholipid kinase is an inositol phospholipid kinase. 8. A cell according to any one of Claims 1 to 6 wherein the protein kinase activated by a phospholipid or its metabolite is a protein kinase C. 9. A cell according to Claim 7 wherein the phospholipid kinase is selected from the group consisting of phosphatidyl inositol 3-kinase, phosphatidyl inositol 4-kinase and phosphatidyl inositol-5-kinase. 10. A cell according to Claim 9 wherein the phospholipid kinase is phosphatidyl inositol 3-kinase. 11. A cell according to Claim 8 wherein the protein kinase C is selected from any one of PKC-γ, PKC-δ, PKC-η or PKC-e. 12. An assay for detecting whether a compound is involved in cell growth regulation, the assay comprising (1) a cell according to any one of the preceding claims, (2) a container for the said cell, (3) a growth medium for the said cell and (4) means to detect the viability of the cell. 13. A kit comprising a eukaryotic cell as defined in Claim 1 and culture medium such that the cell will divide and grow. 14. A method for assaying for a compound that is involved in cell growth regulation the method comprising (1) culturing a cell as defined in Claim 1, (2) adding a compound and (3) determining the cell growth rate in the presence of the compound. 15. A compound identified by the assay of Claim 12 or the method of Claim 14 as being involved in cell growth regulation.

Inventors: GOODE NIGEL THOMAS, NURSE PAUL MAXIME, PARKER PETER JOSEPH JACQUES, WATERFIELD MICHAEL DEREK, GOODE, NIGEL, THOMAS, NURSE, PAUL, MAXIME, PARKER, PETER, JOSEPH, JACQUES, WATERFIELD, MICHAEL, DEREK
Applicants: GOODE NIGEL THOMAS, IMP CANCER RES TECH, NURSE PAUL MAXIME, PARKER PETER JOSEPH JACQUES, WATERFIELD MICHAEL DEREK, IMPERIAL CANCER RESEARCH TECHNOLOGY LIMITED
Application year: 1993
Main classification: C12N15
IPC codes: A61K38, C12M1, C12N1, C12N9, C12N15, C12Q1, C12R1