Date of Award
Doctor of Philosophy (PhD)
Progression through the yeast cell cycle is controlled in the G1 phase at a point called Start. One prerequisite for the completion of start is growth to a critical cell size. Once critical cell size is obtained and other conditions have been satisfied, cells complete Start and become committed to a full cell cycle. One approach to identifying genes which regulate the cell cycle is to find mutants which confer a small cell size because they proceed through Start prematurely. For example, the CLN3-1 mutant has precisely these properties and encodes a stable hyperactive G1 cyclin variant that alters the timing of Start through premature activation of the Cdc28 protein kina 'e.
A tagged Ty-1 transposon mutagenesis procedure coupled with size enrichment was used to isolate a novel cell size mutant called whi3. The whi3 mutation is partially dominant and causes cells to commit to cell division at a reduced cell size. whi3 mutants are still capable of modulating cell size depending on the nutritional conditions. The size effects caused by the whi3 mutation are multiplicative with those of CLN3-1. In addition, the whi3 mutant is more a-factor resistant than the isogenic wild-type strain and has a slightly reduced mating efficiency. The whi3 Cln3-1 double mutant is nearly sterile and is largely defective for mating factor induced transcription.
The WHI3 gene was cloned and was able to complement the size defect of the mutant as well as increase the cell size of a wild-type strain. Extra copies of the gene carried on single copy vectors or integrated into the yeast genome increase cell size in a dose-dependent manner. Thus, WHI3 may encode a cellular metric used to measure cell size or mass. Sequence analysis indicates that this 71 kDa protein contains a domain common to a family of RNA binding proteins, the RNP motif. The deduced sequence of the protein is rich in serine residues and contains a small region rich in glutamine residues. Deletion of the WHI3 gene results in a small cell size phenotype, increased a-factor resistance and reduced mating efficiency, phenotypes shared with the Ty induced mutation. When WHI3 is placed under the control of the GALI promoter and induced, cells arrest in the G1 phase of the cell cycle. These results suggest that WHI3 encodes a dose-dependent inhibitor of Start by affecting the critical cell size requirement. It is hoped that these studies will increase our understanding of Start through the identification of molecules which regulate this transition.
Nash, Robert Stuart, "Analysis of Cell Size Control in the Teast Saccharomyces Cerevisie: Isolation and Characterization of WHI3, A Dose-Dependant Inhibitor of Start" (1993). Open Access Dissertations and Theses. Paper 2822.