Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae. Genetic analysis of the suppressors: absence of complementation between non-allelic mutants.

Ten informational nuclear suppressors of the oxi1- mitochondrial mutation of Saccharomyces cerevisiae are recessive. They are linked to each other, but their allelism is uncertain. Some of them unfavourably affect functions of standard (mit+) mitochondrial genomes. One suppressor severely impairs or entirely prevents mitochondrial functions of the spore clones carrying it. The spectrum of mit- mutations on which these suppressors act is similar to that exhibited by nam3-1. In double heterozygotes namx/NAM3+, NAM+x/nam3-1 the oxi1- (and box3-) mutation is suppressed, yet one of our suppressors (R705) and nam3-1 show independent segregation in tetrads. This indicates that there may be absence of complementation between non-allelic suppressors.

Mitochondrial mutagenesis in Saccharomyces cerevisiae : V. Frequencies of different mit (-) mutants and loss of their mit (+) alleles in rho (-) clones.

Four types of mit (-) mutations induced with manganese are found in the following relative proportions: oxi3 (-) > cob-box (-) > oxi2 (-) [Symbol: see text] oxi1 (-1). The frequences of loss of their respective mit (+) alleles in manganese-induced rho (-)] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.

Mitochondrial mutagenesis in Saccharomyces cerevisiae. III. Nitrous acid.

Nitrous acid (NA) induced mutations efficiently in mitDNA, conferring resistance to erythromycin and weakly induces mit- mutations. In some strains of yeast it also enhanced rho- mutations. The frequencies of nuclear and mitochondrial mutations induced with NA are compared.

Mitochondrial mutagenesis in Saccharomyces cerevisiae. II. Methyl methanesulphonate and diepoxybutane.

In Saccharomyces cerevisiae, methyl methanesulphonate and diepoxybutane produced efficiently lethal, as well as mutagenic, damage in nuclear DNA. However, in the same conditions, these agents did not induce cytoplasmic petite mutations and poorly induced point mutations (resistance to erythromycin and chloramphenicol) in mitochondrial DNA. Possible reasons for these differences are discussed.

Mitochondrial mutagenesis in Saccharomyces cerevisiae. I. Ultraviolet radiation.

UV efficiently induces mutations in mitDNA , conferring resistance to erythromycin. Mitochondrial chloramphenicol-resistant mutants are probably also induced by UV, but almost 90% of mutants with such phenotype are non-mitochondrial; therefore it is possible to estimate accurately the frequences of the induced presumptive mitochondrial capr mutations.

Spontaneous and induced non-specific drug resistance in Saccharomyces cerevisiae.

Nitrous acid, diepoxybutane and methyl methane sulfonate induce effectively non-mitochondrial chloramphenicol-resistant mutants cross-resistant to other drugs. HNO2 induces also unstable erythromycin resistant mutants. The ability of the mutants to grow on antibiotic media can be modified by detergents, guanidine hydrochloride or increased osmotic pressure of the medium. This suggests that the resistance is due to changes in cell membrane permeability similar to those described by Rank, Robertson and Philips (1975b). Multiple drug-resistant mutants selected for chloramphenicol resistance show an increased sensitivity to ethidium bromide in glucose medium. Therefore the mutations involved increase probably nuclear envelope permeability to the latter drug. Results of genetic analyses of non-mitochondrial capr and eryr mutants suggest strongly that in most, if not all, cases the resistance is determined by interaction between nuclear and extranuclear factors.

Manganese mutagenesis in yeast. VI. Mn2+ uptake, mitDNA replication and ER induction: comparison with other divalent cations.

A medium was found in which manganese efficiently induces erythromycin-resistant mitochondrial mutations, and which is suitable for measuring Mn2+ uptake and the labelling of DNA (fig. 1). Mn2+ uptake is stimulated by glucose and slowed down by cycloheximide (Fig 2). Mg2+ competes with Mn2+ uptake much stronger than does Zn2+ (Fig. 3). All of the conditions which favour Mn2+ uptake also favour induction of erythromycin-resistant mutations (Tables 3, 4). Mn2+ strongly inhibits protein synthesis (Table 1). Nuclear DNA replication is also strongly inhibited by this cation, while mitochondrial DNA replication is only weakly inhibited during the first 3 h of labelling, but there is small if any increase of the label incorporation between the 3rd 6th h of labelling (Table 2). The relation between label incorporation into mitDNA and mutation induction by manganese is not straightforward (Table 5). From among 11 divalent cations tested, only Mn2+ was capable of inducing mitochondrial erythromycin-resistant mutations (Table 6).

Manganese mutagenesis in yeast. A practical application of manganese for the induction of mitochondrial antibiotic-resistant mutations.

When yeast cells were incubated for 4 to 8 h in yeast extract-peptone-glucose medium, pH 6, containing 8 mM-manganese, and then plated on selective media, there was a strong induction of antibiotic-resistant mutations. Indirect evidence suggests that practically all resistant mutants selected were of independent origin. The analysis of manganese-induced resistant mutants showed that most were extranuclear, while those tested showed recombination with known mitochondrial markers. Our results suggest that manganese can be considered as a mutagen which specifically induces mitochondrial mutations in Saccharomyces cerevisiae.

Manganese mutagenesis in yeast. IV. The effects of magnesium, protein synthesis inhibitors and hydroxyurea on AntR induction in mitochondrial DNA.

The induction of antibiotic-resistant mutations in yeast mitochondrial DNA by manganese is decreased when the manganese-containing medium is additionally supplemented with magnesium. At equimolar concentrations of manganese and magnesium the former is no longer mutagenic. Amino acid starvation, cycloheximide, chloramphenicol and erythromycin have very little, if any, effect on the mutagenicity of manganese. Hydroxyurea itself seems to be slightly mutagenicity of manganese. Our results show that manganese acts as an error-producing factor in DNA replication probably through a direct interaction with mitDNA polymerase(s).

Studies on cytochrome b2 synthesis in Saccharomyces cerevisiae.

Synthesis of cytochrome b2 together with that of other hemoproteins is induced by oxygen. It is further stimulated by L-lactate. This is true for the enyzme in mitochondrial as well as cytoplasmic cell fractions. Chloramphenicol and erythromycin do not effect cytochrome b2 biosynthesis, whereas cycloheximide prevents it in aerobicallly adapting cells. Mutants lacking cytochrome b2 activity still exhibit the activities of D-lactate dehydrogenase and D-2hydroxyacid dehydrogenase.

A search for Saccharomyces cerevisiae mutants with an increased sensitivity to nitrous acid.

Six strains with an increased nitrous acid sensitivity were isolated (Fig. 1). The putative HNO2-sensitive mutants, as well as the parental strain 55R5 behaved abnormally in crosses, so that studies on the segregation of the sensitivity were difficult and unreliable. During 1.5 years the oversensitivity of the mutants gradually decreased to disappear completely (Tab. V). The differences in HNO2 sensitivity between respiratory-sufficient and cytoplasmic respiratory-deficient strains (Tab. I), as well as between different respiratory-sufficient strains (Tab. II-IV) are analysed.