Current methods for Saccharomyces cerevisiae. II. Sporulation.
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Carbon↗
Biomedical subjects
Z Olempska-Beer
Publications and source records attributed to Z Olempska-Beer.
Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.
Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.
Cyclic AMP↗
Optimal extraction conditions for high-performance liquid chromatographic determination of nucleotides in yeast.
Different extraction methods of nucleotides from the yeast Saccharomyces cerevisiae were compared. A new extraction solution--formic acid saturated with 1-butanol--was found to be more effective than the commonly used solutions of trichloroacetic acid, perchloric acid, or formic acid alone. Using this solution the optimal extraction conditions were established. Nucleotide recovery was evaluated by adding standard nucleotides to the extraction medium and carrying them together with the cells through the whole extraction procedure. Nucleotides were separated and quantitated by high-performance liquid chromatography on an anion-exchange column.
1-Butanol↗
Initiation of meiosis and sporulation of Saccharomyces cerevisiae by sulfur or guanine deprivation.
Homothallic Saccharomyces cerevisiae, growing exponentially in a synthetic acetate medium, could be initiated to undergo meiosis and subsequent sporulation by removal of sulfur from the medium or by partial purine deprivation of purine auxotrophs or, most efficiently, by guanine deprivation of a guanine auxotroph. In contrast, partial uracil deprivation of uracil auxotrophs did not cause sporulation. Under any of the above and other sporulation conditions, the intracellular concentrations of GTP and, usually at some time later, S-adenosylmethionine (SAM) decreased; the concentrations of the other nucleoside triphosphates decreased under some but increased under other sporulation conditions. The addition of 1 mM methionine or, more effectively, of SAM or the combination of adenine plus methionine greatly increased the intracellular concentration of SAM and reduced or prevented sporulation, even when GTP decreased. However, differentiation can be inhibited by an excess of many metabolites which do not specifically control the initiation process; in particular, SAM is known to inhibit yeast metabolism (e.g., transamination). Therefore, we cannot yet decide whether the deficiency of GTP or SAM (or related compounds) serves as a signal for the initiation of meiosis/sporulation.
Culture Media↗
UV-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities.
Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D0 = 0.7 and 5.5 J/m2; n = 6.0 and 2.0 for LY-R and LY-S cells, respectively). In this study, the frequency of hypoxanthine-guanine-phosphoribosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 +/- 0.6) X 10(-5) TGr mutants per viable cell, and the UV induction rate was (2.2 +/- 0.8) X 10(-4) TGr mutants per viable cell, per J/m2. Both spontaneous and induced mutation frequencies were much lower for LY-S cells. The spontaneous mutation frequency for these cells were too low to make its measurement practicable (less than 0.0013 X 10(-5) TGr mutants per viable cell). Mutation induction rate was (4.2 +/- 2.2) X 10(-7) TGr mutants per viable cell, per J/m2. These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.
Animals↗