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Proc Natl Acad Sci U S A. Jun 1988; 85(12): 4426–4430.
PMCID: PMC280442

Dominant negative protein kinase mutations that confer a G1 arrest phenotype.

Abstract

The CDC28 gene of Saccharomyces cerevisiae encodes a protein kinase that is required for passage through the G1 phase of the cell cycle. We have used an inducible promoter fused to the CDC28 coding sequence to isolate conditionally dominant mutant alleles of CDC28. Overexpression of these dominant alleles causes arrest in the G1 phase of the cell cycle but permits the distinctive asymmetric growth that is characteristic of recessive temperature-sensitive cdc28 mutants. The dominant alleles encode products with no detectable protein kinase activity, and their phenotypic effects can be suppressed by simultaneous overproduction of the wild-type protein. DNA sequence analysis showed that the mutant site in at least one of the dominant alleles is in a residue that is highly conserved among protein kinases. These properties are best understood if the dominant mutation results in the catalytic inactivation of the protein kinase but still allows the binding of another component needed for CDC28 function. By this model, high levels of the mutant protein arrest cell division by denying the wild-type protein access to this other component. Suppressors that may encode this other component have been isolated on high-copy-number plasmids.

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  • DeFeo-Jones D, Scolnick EM, Koller R, Dhar R. ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature. 1983 Dec 15;306(5944):707–709. [PubMed]
  • Lörincz AT, Reed SI. Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes. Nature. 1984 Jan 12;307(5947):183–185. [PubMed]
  • Peterson TA, Yochem J, Byers B, Nunn MF, Duesberg PH, Doolittle RF, Reed SI. A relationship between the yeast cell cycle genes CDC4 and CDC36 and the ets sequence of oncogenic virus E26. Nature. 1984 Jun 7;309(5968):556–558. [PubMed]
  • Vogt PK, Bos TJ, Doolittle RF. Homology between the DNA-binding domain of the GCN4 regulatory protein of yeast and the carboxyl-terminal region of a protein coded for by the oncogene jun. Proc Natl Acad Sci U S A. 1987 May;84(10):3316–3319. [PMC free article] [PubMed]
  • Hartwell LH, Mortimer RK, Culotti J, Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. [PMC free article] [PubMed]
  • Bücking-Throm E, Duntze W, Hartwell LH, Manney TR. Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor. Exp Cell Res. 1973 Jan;76(1):99–110. [PubMed]
  • Reid BJ, Hartwell LH. Regulation of mating in the cell cycle of Saccharomyces cerevisiae. J Cell Biol. 1977 Nov;75(2 Pt 1):355–365. [PMC free article] [PubMed]
  • Hartwell LH. Saccharomyces cerevisiae cell cycle. Bacteriol Rev. 1974 Jun;38(2):164–198. [PMC free article] [PubMed]
  • Reed SI, Hadwiger JA, Lörincz AT. Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4055–4059. [PMC free article] [PubMed]
  • Mendenhall MD, Jones CA, Reed SI. Dual regulation of the yeast CDC28-p40 protein kinase complex: cell cycle, pheromone, and nutrient limitation effects. Cell. 1987 Sep 11;50(6):927–935. [PubMed]
  • Orr-Weaver TL, Szostak JW, Rothstein RJ. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 1983;101:228–245. [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • Hartwell LH. Macromolecule synthesis in temperature-sensitive mutants of yeast. J Bacteriol. 1967 May;93(5):1662–1670. [PMC free article] [PubMed]
  • Broach JR, Strathern JN, Hicks JB. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. [PubMed]
  • Johnston M, Davis RW. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. [PMC free article] [PubMed]
  • Wittenberg C, Richardson SL, Reed SI. Subcellular localization of a protein kinase required for cell cycle initiation in Saccharomyces cerevisiae: evidence for an association between the CDC28 gene product and the insoluble cytoplasmic matrix. J Cell Biol. 1987 Oct;105(4):1527–1538. [PMC free article] [PubMed]
  • Peden KW, Nathans D. Local mutagenesis within deletion loops of DNA heteroduplexes. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7214–7217. [PMC free article] [PubMed]
  • Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Hutter KJ, Eipel HE. Microbial determinations by flow cytometry. J Gen Microbiol. 1979 Aug;113(2):369–375. [PubMed]
  • Hindley J, Phear GA. Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases. Gene. 1984 Nov;31(1-3):129–134. [PubMed]
  • Lee MG, Nurse P. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature. 1987 May 7;327(6117):31–35. [PubMed]
  • Hunter T. A thousand and one protein kinases. Cell. 1987 Sep 11;50(6):823–829. [PubMed]
  • Beach D, Durkacz B, Nurse P. Functionally homologous cell cycle control genes in budding and fission yeast. Nature. 1982 Dec 23;300(5894):706–709. [PubMed]
  • Simon M, Seraphin B, Faye G. KIN28, a yeast split gene coding for a putative protein kinase homologous to CDC28. EMBO J. 1986 Oct;5(10):2697–2701. [PMC free article] [PubMed]
  • Mellor J, Dobson MJ, Roberts NA, Tuite MF, Emtage JS, White S, Lowe PA, Patel T, Kingsman AJ, Kingsman SM. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene. 1983 Sep;24(1):1–14. [PubMed]
  • Lörincz AT, Reed SI. Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28. Mol Cell Biol. 1986 Nov;6(11):4099–4103. [PMC free article] [PubMed]
  • Nasmyth KA, Tatchell K. The structure of transposable yeast mating type loci. Cell. 1980 Mar;19(3):753–764. [PubMed]
  • Murray AW, Szostak JW. Pedigree analysis of plasmid segregation in yeast. Cell. 1983 Oct;34(3):961–970. [PubMed]
  • Gibbs JB, Ellis RW, Scolnick EM. Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12. Proc Natl Acad Sci U S A. 1984 May;81(9):2674–2678. [PMC free article] [PubMed]

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