Use of lycorine and DAPI staining inSaccharomyces cerevisiaeto differentiate betweenrho0andrho-cells in acce1/Δcce1 nuclear background

Abstract

In the yeast Saccharomyces cerevisiae, mutants are viable with large deletions (rho-), or even complete loss of the mitochondrial genome (rho0). One class of rho-mutants, which is called hypersuppressive, is characterised by a high transmission of the mutated mitochondrial genome to the diploid progeny when mated to a wild-type (rho+) haploid. The nuclear gene CCE1 encodes a cruciform cutting endonuclease, which is located in the mitochondrion and is responsible for the highly biased transmission of the hypersuppressive rho-genome. CCE1 is a Holliday junction specific endonuclease that resolves recombination intermediates in mitochondrial DNA. The cleavage 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 are rho0. In this paper we demonstrate the usefulness of lycorine, together with staining by 4',6-diamidino-2-phenylindole (DAPI), to assay for the mitotic stability of a variety of mitochondrial genomes. We have found that rho+and rho-strains that contain CT sequences produce a large fraction of rho0progeny in the absence of CCE1 activity. Only those rho-mitochondrial genomes lacking the CT recognition sequence are unaffected by the cce1 allele.Key words: yeast, mitochondria, hypersuppressive, Saccharomyces cerevisiae, lycorine.