Wieslawa Prazmo

Induction by manganese of mitochondrial antibiotic resistance mutations in yeast.

SummaryManganese added to the growth medium of Saccharomyces cerevisiae to a final concentration of 4–8 mM induces not only mitochondrial respiratory-deficient mutations (Fig. 1), but also mitochondrial mutations to chloramphenicol- and erythromycin-resistance (Fig. 1, Tables 1–3). This is the first mutagen shown to be capable of inducing mitochondrial antibiotic resistance mutations in yeast. It is assumed that manganese induces mutations through its interaction with mitochondrial DNA polymerase.

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.

Mutations Affecting the Sulphur Assimilation Pathway in Aspergillus nidulans: Their Effect on Sulphur Amino Acid Metabolism

Several sul-reg mutants of Aspergillus nidulans isolated as constitutive for arylsulphatase were studied with respect to the regulation of enzymes involved in cysteine and homocysteine synthesis and to the pool of sulphur amino acids. All mutants examined showed a decreased concentration of glutathione as compared with the wild type, and all mutants, with one exception, had a decreased total pool of sulphur amino acids. The results suggest that the mutants are leaky in the sulphate assimilation pathway. They show derepression of cysteine synthase, homocysteine synthase, cystathionine beta-synthase and gamma-cystathionase. In spite of having derepressed homocysteine synthase, the enzyme which constitutes an alternative pathway for homocysteine synthesis, the sul-reg mutations do not suppress lesions in genes required for the main homocysteine-synthesizing pathway. This indicates that the derepression of homocysteine synthase is not in itself sufficient for physiological functioning of this enzyme, but seems to depend also on the effectiveness of cysteine synthesis and sulphide formation.

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.

On the specificity of caffeine effects

SummaryCaffeine in 0.1% or higher concentration reversibly inhibits E. coli and yeast growth. It inhibits RNA and protein synthesis (Tables 1+3) within a few minutes after being added to the incubation medium (Fig. 2). The suggestion is made that these effects of caffeine, as well as its synergism with some mutagens, are due to its ability to costack with free purines and to form complexes with single-stranded nucleic acids.

Regulation of homocysteine metabolizing enzymes in Aspergillus nidulans

SummaryMutants of Aspergillus nidulans blocked in the main pathway of cysteine synthesis show an elevated level of the enzymes involved in the synthesis of cysteine from homocysteine i.e. cystathionine β-synthase and γ-cystathionase and a depressed level of homocysteine methyltransferase. This results in a considerable change in the sulfur amino acids pool as compared to the wild type. Upon addition of cysteine to the growth medium the first two enzymes are repressed while the level of the third one increases. These data indicate that the two diverging pathways of homocysteine metabolism are anti-coordinately regulated.

Mutations affecting cysteine synthesis in Aspergillus nidulans : characterization and chromosome mapping

Selection and mapping of mutations affecting cysteine synthesis in Aspergillus nidulans was carried out. A new locus, cysE , is described, the mutants of which are deficient in in vivo conversion of O-acetylserine to cysteine, a step mediated by cysteine synthase. Three loci ( cysB, C and E ) were thus found to control this step in vivo , apparently without affecting the enzyme activity in vitro . By scoring for propargylglycine sensitivity of cys mutants, chromosomal map positions were obtained for all five cysteine loci ( A, B, C, D and E ).

Chapter 2 Manganese Mutagenesis in Yeast

Publisher Summary The chapter discusses the manganese mutagenesis in yeast. Tests for the mutagenicity of manganese toward yeast seem to be useful for two reasons. First, the results answer the question whether or not the cation is mutagenic toward eukaryotes. Second, in Saccharomyces cerevisiae , not only nuclear but also mitochondrially inherited mutations conferring either a respiratory deficiency or resistance to antibiotics can be easily scored. The search for the latter type of mitochondrial mutations was of particular interest, since a few years ago there was no evidence that any factor could induce such mutations. Most experiments were performed using 6-8 mM manganese and small inocula. Under these conditions, in all strains tested, the percentage of rho – mutants remained unchanged during the first 6–8 hours of incubation in the mutagenic medium. The increase in rho – mutants started after 12–24 hours, depending on the strain tested, and after 48–72 hours up to 99% of the cells developed a respiratory deficiency. The chapter discusses induction of nuclear mutations, the uptake of manganese and its general effects on yeast cells, and other mutagens inducing mutations nonlethal for rho factor.