A new sensitive test based on yeast cells for studying environmental pollution.

Different tests based on yeast cells were developed for determination of mutagenic/carcinogenic action; however, they all showed lower sensitivity compared to bacterial tests, the main reason for this being the limited permeability of yeast cells. We found that general permeability of Saccharomyces cerevisiae cells can be increased by mutation and on this basis we developed a more sensitive test. The aim of this study was to prove the applicability of our test, called D7ts1, in environmental studies. Soil, water and air samples were taken during 1998 from regions in Bulgaria with declared low, average or high pollution levels and investigated for presence of mutagenic/carcinogenic activities in the bacterial test of Ames, the yeast D7 test of Zimmermann and our new D7ts1 test. Results obtained evidenced the following conclusions: (1) the usage of D7ts1 test instead of D7 test permits a clearer measurement of positive samples and detects mutagenic/carcinogenic activities undetectable by D7 test; (2) all samples with positive Ames test were positive in the D7ts1 test; however, some samples, clearly positive in the D7ts1, were negative in the Ames test; therefore, the simultaneous usage of D7ts1 and Ames tests in environmental studies is advantageous because it detects dangers for the human health activities to which bacterial cells do not respond; and (3) regions in Bulgaria declared clean were found to be polluted; particularly troubled are the whole-year positive data in the three tests for air samples from a 'clean' region.

Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160.

The VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time-, temperature- and energy-dependent and consistent with a fluid-phase mechanism of endocytosis. The rate of steady-state accumulation of the dye at 37 degrees C is 145 ng/micrograms DNA per h for VY1160 mutant and 23 ng/micrograms DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild-type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24 degrees C and shifted at 38 degrees C shows that ts1 strains, but not srb1 and wild-type strains, increase ten-fold the internalization of lucifer yellow 2 h after the shift at 38 degrees C. The analysis of ts1 x wild-type crosses provides evidence that the temperature-sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might explain the generally increased permeability of VY1160 mutant cells.

Increased extracellular secretion in fragile mutants of Saccharomyces cerevisiae.

Evidences are presented for a generally increased extracellular secretion capability of the fragile mutants of S. cerevisiae. Proteins secreted in wild type yeasts to the periplasmic space can not be retained by the defective cell wall of the fragile cells and are released into the medium. Changes in the structure of non-mannan components of the cell wall might be the reason for the extracellular release of the periplasmic proteins.

Relaxed mutant of Saccharomyces cerevisiae: proper maturation of ribosomal RNA in absence of protein synthesis.

We have characterized a relaxed yeast mutant, S. cerevisiae SY15, isolated by mutagenesis with ethylmethanesulfonate of strain A364A. Starvation for a required amino acid or treatment with cycloheximide blocks protein synthesis in both parental and mutant strains, while the synthesis of total RNA is inhibited by 72% in A364A and 23% in SY15 cells. In the absence of protein synthesis, the transcription of 37S primary precursor to rRNA is not inhibited in the SY15 mutant, and the rRNA transcripts are correctly processed, although at a reduced rate, and are almost free of ribosomal proteins. The relaxed phenotype in yeast is accompanied by alteration in the regulation of rRNA biosynthesis at transcriptional and posttranscriptional levels.