Summary
The cdc2 + gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis. Previously the cdc2 + gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity. Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time. We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughtout the cdc2 protein. Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2 + activity and regulation. These include regions which may be involved in the interaction of the cdc2 + gene product with the proteins encoded by the wee1 +, cdc13 + and suc1 + genes.
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Aves S, Durkacz B, Carr A, Nurse P (1985) Cloning, sequencing and transcriptional control of the Schizosaccharomyces pombe cec10 + start gene. EMBO J 4:457–463
Bach ML (1987) Cloning and expression of the OMP decarboxylase gene ura4 from Schizosaccharomyces pombe. Curr Genet 12:527–534
Beach D, Nurse P (1981) High frequency transformation of the fission yeast Schizosaccharomyces pombe. Nature 290:140–142
Beach D, Durkacz B, Nurse P (1982) Functionally homologous cell cycle control genes in budding and fission yeast. Nature 300:706–709
Booher R, Beach D (1986) Site-specific mutagenesis of cdc2 +, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe. Mol Cell Biol 6:3523–3530
Booher R, Beach D (1987) Interaction between cdc2 + and cdc13 + in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2 + protein kinase. EMBO J 6:3441–3447
Booher R, Beach D (1988) Involvement of cdc13 + in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules. EMBO J 8:2321–2327
Borst P, Greaves DR (1987) Programmed gene rearrangements altering gene expression. Science 235:658–667
Brizuela L, Draetta G, Beach D (1987) p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J 6:3507–3514
Bryant BL, Parsons JT (1984) Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity. Mol Cell Biol 4:862–866
Draetta G, Beach D (1988) Activation of the cdc2 protein kinase during mitosis in human cells: cells cycle dependent phosphorylation and subunit rearrangement. Cell 54:17–26
Draetta G, Brizuela L, Potashkin J, Beach D (1987) Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2 + and suc1 +. Cell 50:319–325
Dunphy WG, Brizuela L, Beach D, Newport J (1988) The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54:423–431
Durkacz B, Beach D, Hayles J, Nurse P (1985) The fission yeast cell cycle control gene cdc2: structure of the cdc2 gene region. Mol Gen Genet 201:543–545
Durkacz B, Carr A, Nurse P (1986) Transcription of the cdc2 cell cycle control gene of the fission yeast Schizosaccharomyces pombe. EMBO J 5:369–373
Fantes P (1979) Epistatic gene interactions in the control of division in fission yeast. Nature 279:428–430
Fantes P (1981) Isolation of cell size mutants of a fission yeast by a new selective method: characterisation of mutants and implications for division control mechanisms. J Bacteriol 146:746–754
Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Fisher CR (1969) Enzymology of pigmented adenine-requiring mutants of Saccharomyces and Schizosaccharomyces. Biochem Biophys Res Commun 34:306–310
Garnier J, Osguthorpe DJ, Robson B (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120:97–120
Gautier J, Norbury C, Lohka M, Nurse P, Maller J (1988) Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2 +. Cell 54:433–439
Goebl M, Byers B (1988) Cyclin in fission yeast. Cell 54:739–740
Grimm C, Kohli J, Murray J, Maundrell K (1988) Genetic engineering in Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet 215:81–86
Gutz H, Heslot H, Leupold U, Loprieno N (1974) Schizosaccharomyces pombe. In: King RC (ed) Handbook of Genetics, vol 1. Plenum, New York, pp 395–446
Hagan IM, Hayles J, Nurse P (1988) Cloning and sequencing of the cyclin-related cdc13 + gene and a cytological study of its role in fission yeast mitosis. J Cell Sci 91:587–595
Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42–45
Hartwell LH (1974) Saccharomyces cerevisiae cell cycle. Bacteriol Rev 38:164–198
Hawthorne DC, Leupold U (1974) Suppressor mutations in yeast. Curr Top Microbiol Immunol 64:1–47
Hayles J, Beach D, Durkacz B, Nurse P (1986a) The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function. Mol Gen Genet 202:291–293
hayles J, Aves S, Nurse P (1986b) suc1 is an essential gene involved in both the cell cycle and growth in fission yeast. EMBO J 5:3373–3379
Hindley J, Phear G (1984) Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases. Gene 31:129–134
Hiraoka Y, Toda T, Yanigida M (1984) The nda3 + gene of fission yeast encodes β-tubulin: a cold sensitive nda3 mutation reversibly encodes β-tubulin: a cold sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell 39:349–358
Hofer F, Hollerstein H, Janner F, Minet M, Thuriaux P, Leupold U (1979) The genetic fine structure of nonsense suppressors in Schizosaccharomyces pombe. Curr Genet 1:45–61
Hottinger H, Pearson D, Yamao F, Gamulin V, Cooley L, Cooper T, Soll D (1982) Nonsense suppression in Schizosaccharomyces pombe: the S. pombe sup3-e tRNAScr gene is active in S. cerevisiae. Mol Gen Genet 188:219–224
Kohli J (1987) Genetic nomenclature and gene list of the fission yeast Schizosaccharomyces pombe. Curr Genet 11:5750589
Labbe JC, Lee MG, Nurse P, Picard A, Doree M (1988) Activation at M-phase of a protein kinase encoded by a starfish homologue of the cell cycle control gene cdc2 +. Nature 335:251–254
Lee M, Nurse P (1987) Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327:31–35
Lee M, Norbury CJ, Spurr NK, Nurse P (1987) Regulated expression and phosphorylation of a possible mammalian cell cycle control protein. Nature 333:676–679
Lorincz AT, Reed SI (1984) Primary structure homology between the product of the yeast cell division control gene CDC28 and vertebrate oncogenes. Nature 307:183–185
Lorincz AT, Reed SI (1984) Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28. Mol Cell Biol 6:4099–4103
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Nurse P (1985) Cell cycle control genes in yeast. Trends Genet 1:51–55
Nurse P, Bissett Y (1981) Gene required in G1 for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature 292:558–560
Nurse P, Thuriaux P (1980) Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe. Genetics 96:627–637
Nurse P, Thuriaux P, Nasmyth KA (1976) Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet 146:167–178
Orr-Weaver TK, Szostak JW, Rothstein RJ (1983) Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol 101:228–245
Oshima Y (1982) Regulatory elements for gene expression: the metabolism of galactose and phosphate. In: Strathern JN, Jones EW, Broach JR (eds) The Molecular Biology Of The Yeast Saccharomyces: Metabolism and gene expression. Cold Spring Harbor Laboratory Press, New York, pp 159–180
Piggott JR, Rai R, Carter BLA (1982) A bifunctional yeast gene involved in two stages of the cell cycle. Nature 298:391–393
Rothstein RJ (1983) One step gene disruption in yeast. Methods Enzymol 101:202–211
Russell P, Nurse P (1986) cdc25 + functions as an inducer in the mitotic control of fission yeast. Cell 45:145–153
Russell P, Nurse P (1987) Negative regulation of mitosis by weel +, a gene encoding a protein kinase homolog. Cell 49:559–567
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5469
Simanis V, Nurse P (1986) The cell cycle control gene cdc2 + of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell 45:261–268
Solomon M, Booher R, Kirschner M, Beach D (1988) Cyclin in fission yeast. Cell 54:738–739
Thuriaux P, Nurse P, Carter B (1978) Mutants altered in the control coordinating cell division with cell growth in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet 161:215–220
Toh-e A, Tanaka K, Uesono Y, Wickner RB (1988) PHO85, a negative regulator of the PHO system, is a homolog of the protein kinase gene, CDC28, of Saccharomyces cerevisiae. Mol Gen Genet 214:162–164
Uemura T, Ohkura H, Adachi Y, Morino K, Shiozaki K, Yanigida M (1987) DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. Cell 50:917–925
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Communicated by C.P. Hollenberg
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Carr, A.M., MacNeill, S.A., Hayles, J. et al. Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: Implications for cdc2 + protein structure and function. Mol Gen Genet 218, 41–49 (1989). https://doi.org/10.1007/BF00330563
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DOI: https://doi.org/10.1007/BF00330563