Search for genes that alter mtDNA instability using Barcode Sequencing (BarSeq) of Yeast KnockOut Collection
Saccharomyces cerevisiae is a facultative anaerobe capable of sustaining viability under both respiratory and fermentative growth regimes. Respiratory competence is contingent upon the presence of functional mitochondrial DNA (mtDNA); however, certain selfish mtDNA variants can outcompete and functionally supplant wild-type mtDNA, thereby rendering host cells obligately dependent on glycolytic metabolism. Crossing a yeast knockout collection (YKOC) strain with a strain harboring such a selfish mtDNA variant yields heterozygous diploid progeny bearing a deletion of one allele of a given nuclear gene, coupled with mtDNA heteroplasmy - that is, the coexistence of multiple mtDNA variants within a single cell. These heteroplasmic strains can subsequently be propagated under fermentable (glucose-based) and non-fermentable (glycerol-based) carbon source conditions, followed by genomic DNA extraction and sequencing of the unique molecular barcode inserted at the locus of the deleted gene. Comparative analysis of barcode read counts across the two growth conditions permits identification of nuclear genes required for the maintenance and stability of functional mtDNA.