ETHANOL HYPERSENSITIVE AND ETHANOL-DEPENDENT CDC(-) MUTANTS IN SCHIZOSACCHAROMYCES-POMBE

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schi zosaccharomyces pombe wild-type cells. Genetic analysis of these ets s trains demonstrated that the ets phenotype is associated with mutation s in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature co nditional method; accordingly, we isolated some ets non-ts cdc(-) muta nts, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cd c(-) mutants were tested for their ethanol sensitivity; among them, cd c1-7 and cdc13-117 exhibited a tight ets phenotype. Ethanol sensitivit y was also tested in strains bearing different alleles of the cdc2 gen e, and we found that some of them were ets, but others were non-ets; t hus, ethanol hypersensitivity is an allele-specific phenotype. Based o n the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34(cdc 2) gene product can produce this ets phenotype, and that ethanol hyper sensitivity is probably due to the influence of this alcohol on the se condary and/or tertiary structure of the target protein. Ethanol-depen dent (etd) mutants were also identified as mutants that can only be pr opagated on ethanol-containing media. This novel type of conditional p henotype also covers many unrelated genes. One of these etd mutants, e td1-1, was further characterized because of the lethal cdc(-) phenotyp e of the mutant cells under restrictive conditions (absence of ethanol ). The isolation of extragenic suppressors of etd1-1, and the compleme ntation cloning of a DNA fragment encompassing the etd1(+) wild-type g ene (or an extragenic multicopy suppressor) demonstrate that current g enetic techniques may be applied to mutants isolated by using ethanol as a selective agent.