Genomic stability of Saccharomyces cerevisiae baker's yeasts.

The objective of this study has been to gather data on genomic stability of baker's yeast strains during long-term mitotic growth under restrictive conditions so that comparisons could be made to other studies indicating genomic instability during meiosis. The work describes the analysis of mitotic stability of the nuclear and mitochondrial genomes in the baker's yeast strain V1 during incubation in continuous culture for 190 generations (300 days). The cells were cultured in complete medium containing 2% glucose and 8 to 12% ethanol, as a mutagenic agent specific for mtDNA. The high concentration of ethanol severely limited the growth rate of the cells. DNA samples were monitored for chromosomal pattern, polymorphisms in selected nuclear genes (SUC2, MALIT, ADH1) and mobile genetic elements (Ty1 and Y'), and for RFLPs in mtDNA. The results show that both the nuclear and mitochondrial genomes of grande cells were very stable. However, the frequency of petite mutants in the population varied dramatically during the course of the experiment, reaching as high as 87% petite during the first 27 days of the experiment and declining to 5.8% petite at the end. This decline can be attributed to selection against petite mutants in media containing high concentrations of ethanol. Moreover, when samples and the parental strain were compared at the end of the experiment, no change could be observed in parameters such as their growth rate in different media, capacity to leave doughs, viability in ethanol or frequency of petite mutants. Results therefore indicated that the majority of the cells in the population were very similar to the parental throughout the experiments, with no apparent molecular or phenotypical changes.

Lysine-overproducing mutants of Saccharomyces cerevisiae baker's yeast isolated in continuous culture.

Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected. The baker's yeast strain was growth in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC). The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to alpha-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids. Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene. The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway. This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to alpha-aminoadipate, AEC, and lysine can be used as a dominant marker.

Characterization of genetically transformed Saccharomyces cerevisiae baker's yeasts able to metabolize melibiose.

Three transformant (Mel+) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized. The strains, which originally lacked alpha-galactosidase activity (Mel-), had been transformed with a DNA fragment which possessed an ILV1-SMR1 allele of the ILV2 gene and a MEL1 gene. The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media. The alpha-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel. The yield, YX/S (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel. When molasses was used, YX/S (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls. Whereas no viable spores were recovered from either DADI-Mel or VS-Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci. Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS-Mel was 2n and DADI-Mel was 1.5n. Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors Which Affect the Frequency of Sporulation and Tetrad Formation in Saccharomyces cerevisiae Baker's Yeasts.

Vol. 61, no. 2, p. 635, legend to Fig. 3, line 1: "VS ( )" should read "VS ((symbl))." Legend to Fig. 4, line 3: "DS81-D ( )" should read "DS81-D ((symbl))." [This corrects the article on p. 630 in vol. 61.].

Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts.

To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)