Haploid RSC deletion mutants spontaneously form stable diploids. (a) Heterozygous rscΔ strains were sporulated (i) and dissected (ii). A resulting colony was used to inoculate a liquid culture (iii), which was grown to saturation (iv). Saturated cultures were diluted and grown for an additional 4 h and then fixed for flow cytometry (v), and the OD600 of the saturated culture was measured (vi). The saturated culture was then diluted 1 in 1,000 (vii) to repeat steps iv to vii for 10 cycles, which represents ∼100 generations of growth. (b) Flow cytometric analysis of samples taken from panel a for the indicated strains. Histograms represent the distributions of ∼10,000 cells after growth for the indicated number of generations after germination. Positions of cells with 1C, 2C, and 4C DNA content are indicated on the x axis, which reflects fluorescence intensity on a linear scale. The y axis of each graph, which represents cell frequency, has been scaled to represent the percentage of the maximum bin contained in that graph. (c) Diploidized rscΔ strains were subjected to a mating-type test with a MATa and a MATα strain. Growth on minimal medium signifies a mating event. Wild-type MATa (BY4741), MATα (BY4742), and MATa/α (BY4743) strains were included as controls. (d) The maximum doubling time was calculated from growth curves depicted in Fig. S1 d for three technical replicates. The P values from a one-sided Student’s t test are shown. Data distributions were assumed to be normal, but were not formally tested. Black bars represent the means. (e) Viability was assessed using methylene blue staining of logarithmically grown cells for three technical replicates. Haploids and diploids represent cells grown for 10 and 100 generations after germination. Tetraploids were obtained by mating diploidized cells. At least 200 cells were scored as alive (white) or dead (blue), and the percentage of viable cells is indicated. Black bars indicate the means. The htl1Δ strain cannot be maintained as a haploid. (f) Ploidy of the indicated tetraploid strains was monitored by flow cytometry as in panel b, except the x-axis is on a logarithmic scale.
