Use of lycorine and DAPI staining in Saccharomyces cerevisiae to differentiate between rho(0) and rho(-) cells in a cce1/Delta cce1 nuclear background

In the yeast Saccharomyces cerevisiae, mutants are viable with large deleti ons (rho(-)), or even complete loss of the mitochondrial genome (rho(0)). O ne class of rho(-) mutants, which is called hypersuppressive, is characteri sed by a high transmission of the mutated mitochondrial genome to the diplo id progeny when mated to a wild-type (rho(+)) haploid. The nuclear gene CCE 1 encodes a cruciform cutting endonuclease, which is located in the mitocho ndrion and is responsible for the highly biased transmission of the hypersu ppressive rho(-) genome. CCE1 is a Holliday junction specific endonuclease that resolves recombination intermediates in mitochondrial DNA. The cleavag e activity shows a strong preference for cutting after a 5'-CT dinucleotide . In the absence of the CCE1 gene product, the mitochondrial genomes remain interconnected and have difficulty segregating to the daughter cells. As a consequence, there is an increase in the fraction of daughter cells that a re rho(0). In this paper we demonstrate the usefulness of lycorine, togethe r with staining by 4',6-diamidino-2-phenylindole (DAPI), to assay for the m itotic stability of a variety of mitochondrial genomes. We have found that rho(+) and rho(-) strains that contain CT sequences produce a large fractio n of rho(0) progeny in the absence of CCE1 activity. Only those rho(-) mito chondrial genomes lacking the CT recognition sequence are unaffected by the cce1 allele.