Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.     

Induction of mitotic gene conversion in Saccharomyces cerevisiae by N-nitrosated pesticides

The herbicide N-(2-benzothiazolyl)-N′-methylurea (Benzthiazuron, Gatnon®) the insecticides 2-isopropoxyphenyl-N-methylcarbamate (Propoxur, Unden®) and 1-naphthyl-N-methylcarbamate (Carbaryl, Sevin®), together with their N-nitroso derivatives, were examined for genetic activity. A diploid strain of Saccharomyces cerevisiae heteroallelic at the gene loci ade2 and trp5, was used to test for the induction of mitotic gene conversion in these two unlinked gene loci. The non-nitrosated compounds had no influence on the frequency of mitotic gene conversions. The nitrosated substances, however, displayed marked convertogenic activities. N-nitrosopropoxur (N-nitrosopropoxur, N-nitrosocarbaryl) were much more active than the substituted N nitrosomethylurea (N-nitrosobenzthiazuron). N-Nitrosocarbaryl induced the greatest number of mitotic gene conversions.     

Genetic effects of herbicides: induction of mitotic gene conversion in Saccharomyces cerevisiae

32 herbicides have been tested for their induction of mitotic gene conversion in a diploid strain of the ascomycete Saccharomyces cerevisiae heteroallelic at two loci. Two of these herbicides showed weak genetic activity: Reglone (1,1′-ethylene-2,2′-dipyridylium dibromide, Diquat) and U 46 D-Fluid (2,4-dichlorophenoxyacetic acid, 2,4-D).     

Effect of post-irradiation inhibition of protein synthesis on UV-induced mitotic gene conversion in Saccharomyces cerevisiae

The effect of post-irradiation inhibition of protein synthesis with cycloheximide was studied on UV-induced mitotic gene conversion in yeast. The frequency of UV-induced mitotic gene convertants as well as survival were reduced when post-irradiation protein synthesis was inhibited beyond 8 h. It is concluded that proteins required for mitotic recombination are not induced by UV irradiation and are already present in mitotic cells.     

Fine-resolution mapping of spontaneous and double-strand break-induced gene conversion tracts in Saccharomyces cerevisiae reveals reversible mitotic conversion polarity.

Spontaneous and double-strand break (DSB)-induced gene conversion was examined in alleles of the Saccharomyces cerevisiae ura3 gene containing nine phenotypically silent markers and an HO nuclease recognition site. Conversions of these alleles, carried on ARS1/CEN4 plasmids, involved interactions with heteroalleles on chromosome V and were stimulated by DSBs created at HO sites. Crossovers that integrate plasmids into chromosomes were not detected since the resultant dicentric chromosomes would be lethal. Converted alleles in shuttle plasmids were easily transferred to Escherichia coli and analyzed for marker conversion, facilitating the characterization of more than 400 independent products from five crosses. This analysis revealed several new features of gene conversions. The average length of DSB-induced conversion tracts was 200 to 300 bp, although about 20% were very short (less than 53 bp). About 20% of spontaneous tracts also were also less than 53 bp, but spontaneous tracts were on average about 40% longer than DSB-induced tracts. Most tracts were continuous, but 3% had discontinuous conversion patterns, indicating that extensive heteroduplex DNA is formed during at least this fraction of events. Mismatches in heteroduplex DNA were repaired in both directions, and repair tracts as short as 44 bp were observed. Surprisingly, most DSB-induced gene conversion tracts were unidirectional and exhibited a reversible polarity that depended on the locations of DSBs and frameshift mutations in recipient and donor alleles.     

The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae.

Summary Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.     

Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae

We have stimulated mitotic and meiotic gene conversion between non-tandem direct repeats of ADE4 by a defined double-strand break imparted in vivo to one of two copies of the gene. The experimental design permitted us to distinguish unambiguously between reciprocal intra-chromosomal crossing over and non-reciprocal break-join events that could accompany the induced conversions. We observed that (1) less than 10% of the induced conversion events are accompanied by intra-chromosomal crossing over in both mitosis and meiosis; (2) non-reciprocal break-join is not stimulated by the double-strand breaks; (3) a double-strand break in meiosis is repaired off intra-chromosomal homology (if available) with approximately sevenfold preference over repair off the homologous chromosome. Our observations, analyzed in the light of previous investigations of spontaneous inter and intra-chromosomal crossing over and gene conversion, lead to the view that chromosomal configuration constrains intra-chromosomal crossing over accompanying conversion between closely spaced repeated genes during resolution of the conversion intermediate.     

A novel Arabidopsis gene causes Bax-like lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.     

The effect of him mutations characterized by enhanced induced mutagenesis on the spontaneous mitotic recombination in the ADE2 gene in Saccharomyces cerevisiae

We have studied the influence of him1, him2, him3 and himX mutations on the frequency of spontaneous mitotic gene conversion in the yeast Saccharomyces cerevisiae using the set of heteroallelic combinations in the ADE2 gene. Data obtained on the HIM/HIM, him/him homozygotes and HIM/him heterozygotes indicate that the him1 mutation is recessive with respect to conversion, whereas the him2, him3 and himX mutations are semidominant. Gene conversion was increased in the majority of heteroalleles of mutant diploids him1/him1. On the contrary, the him2, him3 and himX mutants have hypo-rec phenotypes on mitotic conversion. The him mutations do not affect some heteroalleles, moreover, for some heteroalleles, the effects of the him mutations was opposite. On the basis of the sum of genetical data and, particularly, of conversion event pattern in the him mutants, we suggest that him mutations analysed affect the repair pathway for mismatch correction.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.