Use of lycorine and DAPI staining in Saccharomyces cerevisiae to differentiate between rho0 and rho- cells in a cce1/delta cce1 nuclear background.
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 rho0 progeny in the absence of CCE1 activity. Only those rho- mitochondrial genomes lacking the CT recognition sequence are unaffected by the cce1 allele.
Massardo
D R
DR
Istituto Internazionale di Genetica e Biofisica, CNR, Naples, Italy.
Zweifel
S G
SG
Gunge
N
N
Miyakawa
I
I
Sando
N
N
Del Giudice
A
A
Wolf
K
K
Del Giudice
L
L
D047151:Amaryllidaceae Alkaloids; D001482:Base Composition; D002467:Cell Nucleus / Q000235:genetics*; D003433:Crosses, Genetic; D004272:DNA, Mitochondrial / Q000302:isolation & purification; D004171:Diploidy; D004352:Drug Resistance, Microbial; D004706:Endodeoxyribonucleases / Q000235:genetics*; D017353:Gene Deletion; D045565:Holliday Junction Resolvases; D007211:Indoles; D008928:Mitochondria / Q000235:genetics* / Q000648:ultrastructure; D009693:Nucleic Acid Hybridization; D010617:Phenanthridines / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013194:Staining and Labeling / Q000379:methods*
D047151:Amaryllidaceae Alkaloids; D004272:DNA, Mitochondrial; D007211:Indoles; D010617:Phenanthridines; D029701:Saccharomyces cerevisiae Proteins; C007293:DAPI; D004706:Endodeoxyribonucleases; C473486:CCE1 protein, S cerevisiae; D045565:Holliday Junction Resolvases; C015330:lycorine