Searching for mutations allowing survival of petite-negative cells lacking mitochondrial fusion. Saccharomyces cerevisiae W303

We carried out a novel selection for mutations that allow Saccharomyces cerevisiae strains with defective mitochondrial fusion to maintain mitochondrial DNA (mtDNA). For this selection, strains deleted of chromosomal FZO1 and AAC2, but carrying a counter-selectable plasmid harboring FZO1, were generated. Clones with either a spontaneously generated or ultraviolet light-generated mutation allowing loss of FZO1 in the background of the petite-negative aac2∆ mutation were identified. Seven strains carrying independently isolated suppressor mutations were crossed to a wild-type strain lacking mtDNA and carrying plasmid-borne FZO1. The resulting seven diploids were then sporulated to generate meiotic products. Of fzo1∆ aac2∆ pFZO1 progeny, those either exhibiting or lacking the ability to lose the counter-selectable FZO1 plasmid were identified. For the identification of each individual suppressor mutation, equal volumes of saturated cultures of suppressed or of brethren non-suppressed segregants were mixed, and genomic DNA was isolated and sequenced in a single lane on the Illumina HiSeq2000 platform. Further analysis allowed identification of mutations private to the suppressor-containing pools. All suppressor containing strains contained a mutation of either PDR1 or PDR3, two transcription factors key for activation of pleiotropic drug resistance.