Cloning and characterization of a sulphite-resistance gene of Saccharomyces cerevisiae.

In this paper we describe the cloning and sequencing of the gene (SUL1) responsible for sulphite resistance in a Saccharomyces cerevisiae mutant (Casalone et al., 1992). The deduced amino acid sequence predicted that the gene codes for a zinc-finger protein with five fingers. Comparison of wild-type and mutant gene sequences demonstrated that the mutation event was a transversion from C to G; as a consequence of the mutation a histidine substituted an aspartic acid, affecting directly the fourth finger structure. The SUL1 gene sequence corresponds to that of FZF1 gene (Breitwieser et al., 1993) to which no function was attributed.

Mechanism of resistance to sulphite in Saccharomyces cerevisiae.

Growth inhibition and cell killing caused by sulphite were reduced in seven Saccharomyces cerevisiae sulphite-resistant independent mutants, compared to their parental strains. Genetic analysis showed that in the seven mutants resistance was inherited as a single-gene dominant mutation and that all the analyzed mutations were allelic, thus identifying a major gene responsible for sulphite resistance in S. cerevisiae. Two of the mutants, MBS20-9 and MBS30, were further characterized. 35S-sulphite uptake experiments showed that the ability to accumulate sulphite was markedly reduced in the two resistant strains. No difference between resistant and sensitive strains with respect to glyceraldehyde-3-phosphate dehydrogenase sensitivity to sulphite, or to intracellular glutathione content, were revealed. In contrast, the extracellular acetaldehyde concentration was higher in the resistant mutants, both in the presence and in the absence of sulphite.

1,8-Naphthalic anhydride antidote enhances the toxic effects of captan and thiram fungicides on Azospirillum brasilense cells.

The effects of ten fungicides, six herbicides and four insecticides on the nitrogen-fixing bacterium Azospirillum brasilense were examined. The fungicides captan and thiram were the most toxic among the compounds tested. Cell growth and nitrogenase activity of the bacterium were markedly inhibited by low concentrations of the two fungicides. Antidote 1,8-naphthalic anhydride increased by a factor of 2 the cellular level of glutathione. The addition of the antidote in the presence of captan or thiram caused a similar increase in the glutathione content, but at the same time enhanced the toxicity of the two fungicides.

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to sulphite.

Several spontaneous and UV-induced sulphite resistant mutants of Saccharomyces cerevisiae have been isolated and characterized. Some of the UV-induced mutants appeared to be much more resistant than the spontaneous ones, as judged by their plating efficiency and cellular growth in the presence of increasing concentrations of sulphite. All the resistant mutants seemed to have an intracellular glutathione content and glutathione reductase activity higher than and an extracellular glutathione concentration lower than the parental strain.

Gene dosage mutants at adenine phosphoribosyltransferase locus induced by colcemid in Chinese hamster V79-AP4 cells.

Pseudodiploid Chinese hamster V79-AP4 cells, functionally diploid at the adenine phosphoribosyltransferase (aprt) locus, were treated with colcemid, a well-known aneuploidizing agent, under various experimental conditions. Aneuploid and tetraploid cells and variants resistant to 10 micrograms/ml of 2,6-diaminopurine (DAP), which selects for presumptive aprt+/- heterozygotes in the untreated cells, were induced. Many of the induced variants were hypotetraploid with three (rather than four) chromosomes carrying the aprt gene. Dot-blot and Southern analysis of the DNA of these clones confirmed that they had three copies of the aprt gene. Their APRT specific enzymatic activity was 60-80% of that of wild-type V79-AP4. The results of these and other experiments suggest that in these variants resistance to DAP is due to an altered aprt gene dosage and point to a possible genetic effect of colcemid and other aneuploidizing agents in somatic mammalian cells.

A genetic analysis of the adenine phosphoribosyl transferase locus in Chinese hamster V79-AP4 cells: relevance to mutagenesis studies.

The chromosomal location of the autosomal locus aprt has been investigated in the permanent Chinese hamster cell line V79-AP4 by standard somatic cell genetics methodologies. Aprt is functionally dizygous in V79-AP4 and the 2 alleles map on 2 chromosome 3 homologs, in agreement with the chromosome assignment of the gene in Chinese hamster primary cells. Chromosome G-banding and a Southern blot analysis of V79-AP4 DNA, using as a probe the cloned Chinese hamster aprt gene, have not revealed any structural alteration at either of the 2 aprt alleles. One of the chromosomes 3 has, however, a terminal deletion in its long arm and is therefore morphologically marked. These findings could make V79-AP4 an interesting cell system for the study of mutational mechanisms at the aprt locus in Chinese hamster.

Qualitative analysis of chromosomal evolution in a colcemid-treated Chinese hamster population.

Colcemid is known to inhibit the spindle formation and to induce polyploidy and chromosomal nondisjunction. Using a V79 Chinese hamster cell line, we have shown that colcemid is able to induce the formation of cells that are numerically diploid but whose karyotype, when analyzed with the G-banding technique, differs from that of the untreated ones. Even though these cells have a normal chromosomal constitution, they carry alterations in the chromosomal balance and, consequently, in gene dosage. This could result in an abnormal expression of cellular genes or in the expression of new or preexisting recessive mutations, even in a diploid chromosomal constitution.

Analysis by BrUdR-labelling technique of induced aneuploidy in mammalian cells in culture.

To study the origin of induced aneuploid cells, the BrUdR-labelling technique was applied to V79/AP4 Chinese hamster cells treated with colcemid or benomyl. In this way we were able to recognize the cells which had undergone one cellular division after the treatment since their chromosomes exhibited sister-chromatid differentiation. The results showed that the induced aneuploid cells can have either a few or numerous additional chromosomes depending on the concentrations of the drug. Moreover, it could be established that aneuploid cells with numerous additional chromosomes were obtained mainly when polyploid cells were also present in the treated population. This strongly suggests that the excess of additional chromosomes found in the aneuploid cells induced by the highest concentrations may be derived by disturbances of the whole mitotic apparatus rather than by a multiplicity of errors affecting individual chromosomes.

Increased inhibition of HGPRT by IMP and GMP and higher levels of PRPP in an 8-azaguanine - hat resistant mutant of Chinese hamster cells.

In this paper an 8-azaguanine resistant mutant of Chinese hamster cells able to grow in HAT medium is analyzed. Kinetic analysis of hypoxanthine-guanine-phosphoribosyl-transferase show that the enzyme of the mutant has an altered affinity for both phosphoribosyl-pyrophosphate and the inhibitors IMP and GMP. Furthermore we show that mutant cells exposed to inhibitors of de novo purine synthesis have increased levels of phosphoribosyl-pyrophosphate probably due to decreased utilization by the de novo pathway. These results explain the ability of the mutant to grow both in 8-azaguanine and HAT medium.

8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells.

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.

Thioguanine resistance, ouabain resistance and sister chromatid exchanges in V79/AP4 Chinese hamster cells treated with potassium dichromate.

The biological activity of potassium dichromate (K2Cr2O7) was assayed in V79/AP4 Chinese hamster cells by measuring two mutational end points, thioguanine (TG) resistance and ouabain (OUA) resistance, and two non-mutational end points, cytotoxicity and sister chromatid exchanges (SCE). By exposing the cells for 1 h to the chemical, all biological end points examined were affected by the treatment in a dose-dependent manner. Moreover the combined use of the two selective systems indicated that chromium induces base-pair substitutions in mammalian cells in culture.

A comparison of the 8-azaguanine and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells.

The forward mutation selection system based on resistance to 8-azaguanine has been widely used with cells cultured from a diversity of species and with a variety of mutagens. Ouabain resistance is an alternative selective system. Both systems show a substantial influence of expression time on the number of resistant variants observed after addition of the selective agent such that the frequency reaches a maximum which is dose dependent, and then declines rapidly. Metabolic cooperation has been propsed as the mechanism responsible for this decline with the 8-azaguanine system, but it is less likely to account for the loss of ouabain-resistant variants where it is necessary to invoke generalised effects on the viability of variants due to overcrowding on the plates. A comparison of the two selective systems showed that, with the exception of gamma-irradiation, which was apparently non-mutagenic in the ouabain system, there was broad agreement between the two systems for each mutagen tested. Ethyl methanesulphonate was the most efficient mutagen by a substantial factor. Ouabain resistance permitted greater discrimination particularly between weak mutagens because of the low frequency of spontaneous variants (4 x 10(-7) and also produced data with less intrinsic variability. The absence of gamma ray induced mutation in the ouabain system shows that it may fail to detect certain types of mutagens. Thus the two systems should be used to complement each other. Mutation by the fungicide captan was evaluated using both systems and the positive results indicate that it may pose a hazard to man.