Induction of petite mutants in Saccharomyces cerevisiae by ICR-170 and their genetic characterization.

The strain employed was S288C, a genetically well-studied respiration-sufficient haploid that forms predominantly single cells and cells with one bud during log-phase growth and single cells in stationary phase. The respiratory genetic makeup of spontaneous and induced petites was determined by tetrad analysis and by complementation tests (PITTMAN et al. 1960; SHERMAN and EPHRUSSI 1962) using segregational (pet p f), cytoplasmic (PET p -), and double mutant ( p e t p -) tester stocks. We thank Professors MAURICE OGUR, ROBERT MORTIMER and FRED SHERMAN for supplying many of the tester cultures. Formulas are presented elsewhere for the glucose nutrient agar and broth (PITTMAN et al. 1960), lactate nutnent agar (OGUR and ST. JOHN 1956), triphenyl tetrazdium chloride (TTC) overlay agar (OGUR, ST. JOHN and NAGAI 1957), and sodium phosphate starvation buffer (BRUSICK 1970). All conditions for growth of cultures, cell starvation, incubation, treatment, and platings were at 30°C. ICR-I70 was kindly supplied by Dr. H. J. CREECH (Institute for Cancer Research, Philadelphia) and designated Sample E in our department. The mutagen was dissolved in glass-distilled water just prior to use, although we find that solutions stored at 5°C remain active over several weeks All steps involving mutagen were carried out under dim yellow light Mutagen-containing fluids and materials were treated rvith 15-30% KOH f3r 2-3 weeks before discarding or cleaning. Mutation induction was studied in both lag-phase and stationary-phase cells since BRUSICK (1970) observed that the former are highly sensitive to the killing and mutagenic action of ICR-170. Our protocol for growth and treatment of the two cell types is shown in Figure 1. Cells were grown aerobically from a starting inoculum of 1-2 x 1W cells/ml to mid-log phase (11-12 hr; lo7 cclls/ml) and to stationary phase (24 hr; 3 x 108 cells/ml), harvested, washed, starved for 12 hr in buffer, and treated as shown. Control and treated suspensions were shaken, sampled at prescribed time intervals (Figure 2), diluted as required in water, and plated on glucose nutrient agar. From 6 to 20 plates were spread per dilution at each time interval. Colony counts were made after plates had incubated 3-4 days. Plates were then separated into two sets for each dilution and treatment period. One set was overlayed with TTC agar and &eudther set was replica plated onto glucose nutrient agar and lactate nutrient agar. The frequencies of whole-colony mutants and distinctly sectored calonies obtained by replica plating were comparabfe to those obtained by TTC overlay. Mutation-dose response curves are based on the percent mutants and “total mutants” (whole-colony mutants plus distinctly sectored colonies). Colonies that failed to grow when replica plated onto lactate and which vciere classified as presumptive whole-colony mutants were collected from master plates, retested, and catalogued as to treatment time and mutagen concentration. About 90% of the whole-colony mutants that