Date of Award
Doctor of Philosophy (PhD)
Cross Fred Laboratory
Progression through the eukaryotic cell cycle is controlled by the CDC28 protein kinase and its homologs. In the budding yeast Saccharomyces cerevisiae, this kinase is required for cell cycle S T A R T (commitment to cell cycle progression late in Gl), and for entry into mitosis. Although constitutively present, CDC28 is only periodically activated. The activation of CDC28 involves its physical association with proteins called cyclins. Budding yeast have three CLN genes (CLNs 1 - 3), which have limited cyclin homology. At least one of the three is required for cell cycle START. Four B cyclins are known in yeast (CLBs 1-4); two have been shown to function in mitosis. This thesis reports the discovery of three genes which, when either mutated or overexpressed, relieve the requirement for CLN genes for the execution of START. The first gene was discovered as a mutation which bypasses the requirement for CLN genes, and which we have named the CLN bypass mutation (CBM). 15 distinct isolates of C B M were obtained; all were dominant, and 12 were shown to be linked or allelic with one another. Two of the isolates caused single division meiosis as an unselected, dominant phenotype. Two CEN plasmid clones were isolated which bypassed the CLN requirement. The first of these contained a novel B cyclin, which we have named CLBS. CLBS transcript abundance peaks in Gl, coincident with CLN2 transcript, but earlier than CLB2 transcript. CLBS deletion does not cause lethality, either alone or in combination with other CLN or CLB deletions. However, strains deleted for CLBS require more time to complete S phase, suggesting that CLBS promotes some step in D N A synthesis. CLBS is the only yeast cyclin whose deletion lengthens S phase. CLBS may also have some role promoting the Gl/S transition, since clnl cln2 strains require both CLN3 and CLBS for viability on glycerol media, and clnl, 2, 3' strains require CLBS for rescue by the D. melanogaster cdc2 gene. The other CEN plasmid clone rescuing the clnl, 2, 3' genotype contained MPKl, already known as a gene which could function in this context. We have generated a null allele of MPKl, and shown that while mpkl strains are only mildly impaired at START execution, mpkl cln3 strains are very sick, and mpkl cln3 spores are inviable. MPKl clnl cln2 and mpkl clnl cln2 strains are both robust, suggesting that MPKl and CLN1, 2 supply substantially the same function in the cln3 background. MPKl appears to be required for the CDC28 independent phase of pre-START CLNl and CLN2 transcription, which is nearly essential to viability in the cln3 background.
Epstein, Charles B., "A Genetic and Molecular Analysis of the Control of the Start of the Cell Cycle in Saccharomyces cerevisiae" (1992). Student Theses and Dissertations. 357.