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 subjected to mutagenesis with ethyl methane sulphonate (EMS), and mutant clones were screened for thermosensitive autolytic phenotypes. The screening was based on examination of cell populations, from individual mutant clones, stained with propidium iodide to establish the proportion of cells lysing under non-permissive conditions by means of flow cytometry. Osmotic remediation of the autolytic phenotype in the presence of 1 M sorbitol was also checked. Out of 13,300 clones surviving mutagenesis, 34 were confirmed to be thermosensitive autolytic and 7 of them showed some osmotic complementation with regard to growth and cell lysis. The osmotic remediation in the other strains was negligible or affected only one of the two parameters. The expression of the mutant phenotype in the strains isolated led to a sporulation defect (40% of the strains) and significant alterations in morphology, such as cells in chains (35%), altered buds (25%) that eventually might elongate, round unbudded 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 analysis of the mutant strains that could sporulate showed that in many instances the mutant phenotype was the result of more than one mutation, the mutations being individually recessive. However, at least one mutant strain, 933, carried a single mendelian mutation that was dominant in the diploid but haploid segregants were non-viable. Dominance of this mutation was also confirmed in tetraploids obtained by means of protoplast fusion.