Induction of Petite Mutation in Yeast by Starvation in Glycerol

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Abstract
SUMMARY Starvation of baker's yeast, Saccharomyces cerevisiae, in 2 % glycerol induces high levels of petite mutants. The rate of mutation is highest when exponential phase cells are used to inoculate the starvation medium. The starved cells are exceedingly sensitive to ethidium bromide mutagenesis. Cycloheximide and sodium nalidixate enhance starvation-induced mutation (although nalidixate reduces the ethidium bromide induction). Starvation-induced mutation is inhibited by rifampicin or erythromycin. The appearance of petite mutants is preceded by a fall in both Qo, and mitochondrial DNA. Considerable interest is shown in the cytoplasmically-inherited petite mutation in the yeast Saccharomyces cerevisiae. Petite mutants exhibit multiple deficiencies in mitochon- drial function and it is established that the primary lesion is in mitochondrial DNA. This may be either grossly altered in base composition (Mounolou, Jakob & Slonimski, 1966) or totally lost (Moustacchi & Williamson, I 966). Generally, yeast cultures maintain levels of 0.1 to 1.0 % petite mutants which have arisen spontaneously. It is possible to raise this level to roo % petite mutants by growing the yeast in the presence of ethidium bromide, acriflavine, 5-fluorouracil or erythromycin. Wallis, Ottolenghi & Whittaker (1972) have recently reported that high levels of petite mutants can be induced in the absence of a muta- genic drug when yeast cells are incubated in 2 % glycerol. This lability of the mitochondrial genome was not associated with any apparent damage to the nuclear genome. The characteristics of the mutation process are reported. METHODS Yeast strain and growth conditions. The diploid, prototrophic strain of Saccharomyces cerevisiae, NCYC~~~, was used in all experiments ; NCYC283 (diploid) and NCYC~~~ (haploid) were also used. Liquid and agar growth media used were those described by Luha, Sarcoe & Whittaker (1971). A working stock was prepared as follows: a loopfull of yeast from an agar slope was inoculated into 10 ml glucose yeast liquid medium (Luha et al. 1971) and incu- bated at 30 "C without shaking for 16 h. An appropriate volume of this culture was used to inoculate a further 10 ml of the glucose yeast liquid medium to a concentration of 4-5 x 104 cells/ml and again incubated at 30 "C without shaking for 16 h before storage at 4 "C for up to 7 days. Inocula prepared in this way gave reproducible growth on glycerol yeast liquid medium (Luha et al. 1971). For the mutation experiments 50 or 250 ml yeast liquid medium in 250 ml or 2 1 conical flasks respectively were inoculated from the working

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