CHARACTERIZATION OF THERMOSENSITIVE AUTOLYTIC MUTANTS FROM DIPLOID SACCHAROMYCES-CEREVISIAE

In order to carry out a systematic search for mutants affected in cell integrity, the diploid strain Saccharomyces cerevisiae D1 was subject ed to mutagenesis with ethyl methane sulphonate (EMS), and mutant clon es were screened for thermosensitive autolytic phenotypes. The screeni ng was based on examination of cell populations, from individual mutan t clones, stained with propidium iodide to establish the proportion of cells lysing under non-permissive conditions by means of flow cytomet ry. Osmotic remediation of the autolytic phenotype in the presence of 1 M sorbitol was also checked. Out of 13300 clones surviving mutagenes is, 34 were confirmed to be thermosensitive autolytic and 7 of them sh owed some osmotic complementation with regard to growth and cell lysis . The osmotic remediation in the other strains was negligible or affec ted only one of the two parameters. The expression of the mutant pheno type in the strains isolated led to a sporulation defect (40% of the s trains) and significant alterations in morphology, such as cells in ch ains (35%), altered buds (25%) that eventually might elongate, round u nbudded and highly vacuolated cells (12%) and large-sized cells (12%). These observations show that alterations in functions related to cell integrity can be correlated with an altered morphology. Genetic analy sis of the mutant strains that could sporulate showed that in many ins tances the mutant phenotype was the result of more than one mutation, the mutations being individually recessive. However, at least one muta nt strain, 933, carried a single mendelian mutation that was dominant in the diploid but haploid segregants were non-viable. Dominance of th is mutation was also confirmed in tetraploids obtained by means of pro toplast fusion.