Norio Gunge

Fusion of mitochondria with protoplasts in Saccharomyces cerevisiae

SummaryProtoplasts prepared from a neutral petite haploid BO60AF-1 (a ade2 arg4 leu2 trp COEOOO ύO ρO) were mixed with mitochondria isolated from an oligomycin resistant respiring haploid ANROR 12D (a his4 leu2 thr4 CSESOIIRύ+ ρ+) and treated with 30% polythylene glycol and CaCl2. When the treated protoplasts were spread and incubated on selective agar plates, oligomycin resistant respiration-sufficient colonies appeared with low frequency. All of these colonies carried the mitochondrial genotype of CSESOIIRύ+ ρ+ and showed the same mating type and nutritional requirements as did BO60AF-1, thus evidencing the mitochondrial transfer into protoplasts. Recombination and transmission of the mitochondrial drug resistance markers were studied in crosses involving the strains issued from mitochondria accepted protoplasts.

Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomou sreplication of hybrid plasmids in Saccharomyces cerevisiae

SummaryBy use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker. The replication properties of hybrid plasmids in yeasts were investigated. We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids. Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.

Genetic analysis of unequal transmission of the mitochondrial markers in Saccharomyces cerevisiae

SummaryThe presence of mitochondrial sex factor, ω, was demonstrated in haploid strains of yeast Saccharomyces cerevisiae which came from our laboratory. Transmission and recombination of the mitochondrial genes (CR/CS, ER/ES and OR/OS), conferring the resistance/sensitivity to chloramphenicol, erythromycin and oligomycin, respectively, were non-polar in homosexual crosses and highly polar in heterosexual crosses.Different results were obtained in crosses involving an erythromycin resistant mutant G706E11 (CSEROS) which was found to contain cellular DNA of diploid level. This strain was ω− and showed no recombination polarity when crossed to ω− haploid strains having the genotype CRESOR, but there was a highly polar transmission, that is, the alleles from G706E11 (CS, ER and OS) were transmitted to the zygote progeny in preference to the CR, ES and OR alleles. When crossed to ω+ haploid strains, there was a highly polar recombination, but no transmission was seen for the E and O alleles. Polar transmission of markers from ω+ haploid parental strain, characteristic of heterosexual crosses, was noticed only for the C allele. The crosses of G706E11 toω+ haploids featured an increase in the recombination frequency.The values of % suppressiveness of ϱ− petite mutants were relatively low when determined by crossing to G706E11 or to ϱ+ diploid strain M2-8C rather than by crossing to ϱ+ haploid strains, indicating that there is a positive correlation between the polar transmission of drug resistance markers and the suppressiveness degrees.Genetic mechanism of the anomalous behaviors if mitochondrial genes in crosses involving G706E11 was discussed and interpreted as due to an unbalanced supply of mitochondrial genomes from parental strains.

Effects of elevation of strain-ploidy on transmission and recombination of mitochondrial drug resistance genes in Saccharomyces cerevisiae

SummaryIn order to study the effects of strainploidy on the transmission and recombination of the mitochondrial genes C, E and O conferring the resistance to chloramphenicol, erythromycin and oligomycin, respectively, haploids were crossed to diploids and the results of genetic analysis were compared with those from haploidxhaploid crosses. All haploidx diploid crosses showed an increased transmission of diploid derived alleles, relative to haploid derived ones, but the pattern of increase differed between homosexual and heterosexual crosses. In ω− haploid x ω− diploid homosexual crosses, the increase was of roughly equal magnitude at the C, E and O loci: there was a polar co-transmission of the diploid derived alleles. In ω+ haploid x ω− diploid heterosexual crosses, on the contrary, a differential increase was observed at the different loci, the magnitude being the smallest at the C locus and the largest at the O locus. As a result, there was a preferential transmission in favor of the haploid derived C alleles and of the diploid derived O alleles. A near equal transmission from both parents was observed for the E alleles. A decrease and an increase in the recombination frequency were noticed in the above haploidxdiploid homosexual and heterosexual crosses, respectively.The above phenomena were ascribed to different dosages of mitochondrial genomes from parents. Experimental data were well accorded with the theoretical expectations which were obtained on the assumptions that diploids contain twice as many mitochondrial genomes as haploids, and that random pairing and recombination would occur among mitochondrial genomes from parents. The elevation of strain-ploidy did not affect the recombination polarity which is under the control of the ω gene.It was theoretically predicted that a preferential transmission in favor of diploid derived alleles at all the C, E and O loci would be seen in ω− haploid x ω+ diploid heterosexual crosses as well as in ω+; haploid x ω+; diploid homosexual crosses, but that the magnitude of the polar transmission would vary depending upon the loci in the former crosses, while it would be the same at all the loci in the latter ones. The recombination frequency was predicted to decrease in both of these crosses.