Search for Cell Cycle Control Genes and the Characterization of CLN3^+ in Saccharomyces Cerevisiae

Abstract

Cell cycle control genes in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been studied in detail in the past few years. The cdc25+ and wee1+ genes of S. pombe play key roles in the commitment to division. Genes homologous to the mitotic inducer cdc25+ and the mitotic inhibitor wee1+ of Schizosaccharomyces pombe were searched for in Saccharomyces cerevisiae using DNA cross-hybridization as the method of detection. Such homologs were not found in Saccharomyces cerevisiae by this method. Attention was therefore turned towards sequencing and partially characterizing a previously cloned gene, WHI1+, and its mutant form WHI1-1 (now call CLN3+ and CLN3-1 respectively). Sequence analysis showed that CLN3+ is a cyclin homolog. Cyclins are probably present in all eukaryotes and play an important role in controlling the onset of mitosis. However, unlike these mitotic cyclins, CLN3+ functions in G1. CLN3-1 cells enter a new round of the cell cycle at an aberrantly small cell size and are α-factor resistant. Sequence analysis showed that the CLN3-1 protein was a truncated form of CLN3+ caused by a nonsense mutation in the CLN3+ gene. Cells overexpressing CLN3+ had the same phenotype as CLN3-1 cells, suggesting that the truncated CLN3-l protein was a hyperactive form ofthe wild-type protein. CLN3+ and CLN3-1 were placed downstream of the yeast GAL1 promoter in a shuttle plasmid. Cells transformed with these plasmids and grown in the presence of galactose and the absence of glucose produced CLN3+ or CLN3-1 in large amounts. Cell size was reduced in such cells. These cells were also α-factor resistant.