Genome instability phenotypes of zeocin-sensitive diploid strains lacking vesicular trafficking genes.

The analysis was performed with de novo-constructed strains in a 2n background. (A) Results of the DNA content analysis via FACS. Yeast cells were stained with propidium iodide and and Methods. WT 1n and 2n strains served as DNA content controls. (B) PFGE analysis of yeast chromosome integrity after zeocin treatment. Exponentially growing cells were synchronized for 3 hours with 15 μg per ml nocodazole (N) or treated with 150 μg per ml zeocin for one hour (Z), and after the removal of zeocin, they were allowed to recover for 3 additional hours of growth in medium supplemented with 15 μg per ml nocodazole (R). The 1n (WT) strain served as a positive control strain, and the rad52/rad52 and xrs2/xrs2 strains served as negative controls that were unable to recover from zeocin stress. yku70/yku70 was included in the analysis as a zeocin-insensitive strain. (C-E) Induction of nuclear Rad52-YFP foci by zeocin in the haploid strains with deleted vesicular trafficking genes in the BY4741 background. (C) Microscopic images of cells expressing the Rad52-YFP fusion protein from the pWJ1344 plasmid visualized after one hour of treatment with 150 μg/ml zeocin. The formation of Rad52-YFP foci in WT (left column) and two selected strains: vps51, showing an elevated level of spontaneous formation of Rad52-YFP foci (middle column), and vps63, displaying an increase in the number of cells with Rad52-YFP foci. Bright-field images displaying the distribution of cells (upper row) and fluorescent pseudocolored monochrome images with Rad52-YFP in green (lower row) are shown. (D-E) Quantification of Rad52 foci. All focal planes were inspected for Rad52 foci in at least 600 cells per analyzed strain. The number of foci observed before and after zeocin treatment was counted, and the percentage of cells with at least one Rad52-YFP focus (D) and average number of foci per cell (E) were calculated.