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KMID : 0578319970070060800
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Molecules and Cells
1997 Volume.7 No. 6 p.800 ~ p.806
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Isolation of Synthetic lethal Mutants of rsa1 in Schizosacchromyces pombe
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Chung, Kyung-Sook
Kim, Kyu-Won/Yoo, Hyang-Sook
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Abstract
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Ras proteins are membrane-associated guanine nucleotide-binding proteins that werve as molecular switches for signal transduction pathways in a diverse arrary of organisms. Various cellular factors are known to interact with Ras proteins. In order to find the novel cellular factors that are associated with Ras function, we have constructed synthetic lethal mutants of the rasl^(+) gene in Schizosaccharomyces plmbe and used them to identify the gene that are functionally dependent on the Ras1. We first constructed S. prove strains in which chromosomal rasl^(+) gene is placed under the nnt1 promoter that is regulated by thiamine. This strain shows rasl^(+) phenotype in the absence of thiamine, whereas it shows rasl^(-) phenotype in the presence of thiamine. Second, we mutated the constructed strains with ultraviolet lihgt (UV) and selected two synthetic lethal mutants that could not grow when Rasl funtion was repressed(rasl^(-). One of the mutants, KSC3, showed a swollen cell shape, aberrant deposition of septum materials, and aberrant nuclei. The other mutant, KSC4, showed sensitivity to hyper-osmolarity when Rasl fuction is absent. These mutants, however, grow normally when Rasl is expressed (rasl^(+)). These two novel synthetic lethal mutant of rasl provide the means to isolate the corresponding gene that fuction in association ith Rasl in S. pombe. Screening of a genomic library of S. pombe complementing the mutant phenotype allowed us to identify several novel genes associated with Rasl of S. pombe.
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