Miller P J, Johnson D I
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405, USA.
Yeast. 1997 May;13(6):561-72. doi: 10.1002/(SICI)1097-0061(199705)13:6<561::AID-YEA114>3.0.CO;2-X.
Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1ts allele and identified multiple mutations in the coding region and 5' promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23 degrees C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42W97R mutant displayed a cold-sensitive, los-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42T58A and cdc42S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe: these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts.
Cdc42p是一种高度保守的GTP酶，参与控制细胞极性和使肌动蛋白细胞骨架极化。CDC42基因最初是通过酿酒酵母中温度敏感的细胞分裂周期突变体cdc42-1ts鉴定出来的。我们已经确定了cdc42-1ts等位基因的DNA和预测的氨基酸序列，并在编码区和5'启动子区鉴定出多个突变，从而限制了其在遗传筛选中的用途。因此，我们在酿酒酵母和粟酒裂殖酵母Cdc42p的高度保守氨基酸残基中产生了额外的温度条件致死等位基因。酿酒酵母中的cdc42W97R温度敏感等位基因表现出与cdc42-1ts突变体相同的细胞分裂周期停滞表型（大的、圆形的未出芽细胞）。然而，在23摄氏度的允许温度下，它表现出芽位选择缺陷和异常的芽形态。这些表型表明，Cdc42p在形态发生过程早期的芽位选择中起作用，并且在该过程后期导致正确芽形态发生的极化生长模式中也起作用。在粟酒裂殖酵母中，当在抑制条件下从硫胺素可抑制的nmt1启动子表达时，cdc42W97R突变体表现出冷敏感的功能丧失表型。此外，cdc42T58A和cdc42S71P突变体在粟酒裂殖酵母中表达时表现出温度敏感的功能丧失表型：这些突变体在酿酒酵母中表达时不表现出条件表型。这些新的条件致死cdc42等位基因将是进一步剖析两种酵母细胞极性途径的重要试剂。
