Aleksandra Putrament

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. II. Conditions of induction and characteristics of mitochondrial respiratory deficient Saccharomyces cerevisiae mutants induced with manganese and cobalt.

Manganese and cobalt are capable of inducing ρ − mutations* in non-growing cells of Saccharomyces cerevisiae , but their mutagenic action is much stronger in growing cells. At a given concentration cobalt and manganese can be either strongly mutagenic or non-mutagenic, depending on the cell density. Most of the ρ − mutants induced with manganese and a considerable proportion of those induced with cobalt are suppressive and/or transmit drug resistance markers, so they must still carry mitochondrial DNA. Cobalt can decrease suppressiveness with low efficiency and eliminate drug resistance markers from established ρ − clones.

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

SummaryFour types of mit− mutations induced with manganese are found in the following relative proportions: oxi3− > cob-box− > oxi2− ⩾ 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 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.

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 V. Ethyl methanesulfonate

Abstract Mitochondrial genomes are more sensitive to the lethal action of EMS than are nuclear genomes of S. cerevisiae . EMS induces efficiently only some types of mutation in nuclear genomes of yeast, and probably the same is true for induction of mutations non-lethal to the mitochondrial genomes.

Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae

SummaryTen nuclear suppressors (nam mutations) of the mitochondrial oxi1-V25 ochre mutation are characterized. They restore to some extent the functional form of cytochrome oxidase, as judged by the results of growth tests, cytochrome spectra, cytochrome oxidase activities, and electrophoresis of the products of mitochondrial translation. The nam mutants can suppress some mit− mutations mapping in four mitochondrial genes. They act on a number of chain-terminating mit− mutations. When grown on glycerol medium some double mutants namx-V25 show an increased sensitivity to paromomycin, while the growth of others is stimulated by the drug. The nam mutants are probably omnipotent suppressors resulting from mutations in nuclear gene(s) specifying mitoribosomal protein(s).

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 oxil− 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 nam x/NAM3+, NAM+ x/nam3−1 the oxil − (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.