Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae

Ethidium bromide is known to be a powerful mutagen for the induction of cytoplasmically inherited petite mutations in yeast. The effect of ethidium bromide on the degree of suppressiveness of the induced mutants as a function of exposure time is described. The mitochondrial DNA of 20 ethidium bromide-induced petite mutants has been studied to determine its absence or presence and its buoyant density. Ten mutants, in which we were not able to detect any mitochondrial DNA, were neutral petites. The 10 remaining mutants with mitochondrial DNA simultaneously showed a measurable degree of suppressiveness. It was not possible to correlate the buoyant density of the mutant mitochondrial DNA with the degree of suppressiveness.This study was supported in part by USPHS grant GM 10017. G.M. received a Fulbright Travel Grant.     

Lethality of the petite mutation in petite negative yeasts

Yeast species from which respiratory deficient, or petite, mutants cannot easily be obtained (petite negative species) give rise to micro-colony-producing mutants when subjected to an acriflavine treatment that induces the production of petite mutants in several other yeasts (petite positive species). As a rule, the micro-colonies die before becoming visible to the naked eye. Sometimes they can be subcultured and the respiratory deficiency of the mutants can then be demonstrated. The results of growth experiments under anaerobic conditions suggest that the functioning of a respiratory system is more important to the petite negative yeasts than it is to the petite positive yeasts. An incidental lethal side-effect of acriflavine, specifically on petite negative yeasts, is improbable since mutagenic treatment with supraoptimum temperatures induced viable petite mutants in petite positive yeasts only, and again a lethal mutation in petite negative yeasts.     

Kinetics of petite mutation and thermal death in Saccharomyces cerevisiae growing at superoptimal temperatures.

Mass formation of petite mutants took place in a strain of Saccharomyces cerevisiae when grown at superoptimal temperatures. After an initial period of exponential growth, a second period followed during which exponential death and net exponential petite mutation concurred with exponential growth. The specific rates of the three exponential processes were of the same order of magnitude and varied with the temperature. Net exponential petite mutation did not occur during the deathless first period of growth at superoptimal temperatures nor at any time during growth at suboptimal temperatures. Mitochondria are discussed as possible targets of thermal death in mesophilic yeasts.     

Induction of the cytoplasmic petite mutation in Saccharomyces cerevisiae by the antibacterial antibiotics erythromycin and chloramphenicol

Summary Low concentrations of erythromycin and chloramphenicol (0.3 mg/ml) specifically affect intra-mitochondrial protein synthesis in most strains of Saccharomyces cerevisiae, thereby preventing growth on non-fermentable substrates. This effect is reversible, the genetic capacity for respiration in the absence of the drug being unaffected. However, we now show that exposure of growing cells to high concentrations (1.3–3.0 mg/ml) of either antibiotic generates a high frequency of cytoplasmic petite (respiratory-deficient) mutants with a concomitant loss of the cytoplasmic genetic determinant for respiration known as the rho factor. In one strain in which the effect of erythromycin was examined, the entire population abruptly underwent mutation but only after exposure to the drug for several generations. Mitochondrial DNA was synthesised normally during the silent pre-mutational period, but was rapidly lost, by a process partly dependent on degradation, at the time of the mutational event. Intra-mitochondrial protein synthesis was inhibited only about 67% by the lower levels of erythromycin but was completely (99%) inhibited by the higher petite-inducing levels. These results are interpreted as evidence that the normal maintenance of mitochondrial DNA in this organism requires a protein(s) whose assembly in the mitochondria is completely blocked only by high erythromycin concentrations. This protein is normally present in excess and on exposure to high drug levels replication of mitochondrial DNA is unaffected until the supply runs out. When this happens, replication ceases, existing molecules are degraded, and rho factors are destroyed.     

Repression of In Vivo Synthesis of the Mitochondrial Elongation Factors T and G in Saccharomyces fragilis

In vivo synthesis of the mitochondrial elongation factors T and G in the yeast Saccharomyces fragilis can be repressed. Enzymatic activity assays and immunochemical titration methods reveal that cells grown in the presence of 8% glucose or in the absence of oxygen contain relatively lower amounts of mitochondrial elongation factors than cells grown in the presence of lactate. In contrast, in vivo production of the cytoplasmic elongation factors 1 and 2 does not respond to such a change of extracellular conditions. The rate of growth does not affect the level of the mitochondrial elongation factors. Production of both enzymes is almost constant during logarithmic growth, but decreases when the stationary phase is reached. Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not block but, rather, seems to enhance the in vivo synthesis of mitochondrial T or G.