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.     

Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro.

We previously described a 5'-3' exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5'-3' exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair.     

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.     

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).     

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 effect of liquid holding on chemical induced lethality and mitotic gene conversion in Saccharomyces cerevisiae

Summary Mitotic gene conversion was induced with a variety of chemical mutagens in a double heteroallelic strain of Saccharomyces cerevisiae. Cells were treated with various mutagens and plated immediately onto selective and nonselective growth medium or else they were subject before plating to liquid holding in buffer for various lengths of time. In respiratory competent cells liquid holding caused a decrease in lethality and in conversion frequencies. Respiratory deficient cells, unable to use a non-fermentable substrate as an energy source, behaved different. Untreated cells started to die in buffer after two days of storage, and moreover, there was a considerable increase in potential convertants i.e. cells giving rise to gene convertants when plated on selective growth media. Respiratory deficient cells treated with various chemical mutagens were still more sensitive to liquid holding. After low, sublethal doses cells started to die after one day of liquid holding already and when plated on media selective for convertants, showed an increasing frequency of gene convertants. Addition of very low concentrations of glucose to the liquid holding buffer post-poned the lethal and convertogenic effects. Higher concentrations of glucose completely abolished sensitivity to liquid holding-induced lethality and genetic alterations. The results are interpreted to mean that in respiratory deficient cells no repair activities are possible to an accumulation of spontaneous lethal damage and genetic alterations which are expressed as gene conversion when an energy source becomes available. Such a repairless condition causes an increased sensitivity to genetically active agents, and provides a useful system to detect genetic effects of slowly reacting agents.     

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.     

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.     

Recombinogenicity and mutagenicity of saccharin in Saccharomyces cerevisiae.

Diploid yeast grown in the presence of a commercial lot of saccharin exhibited reproducible, dose-dependent increases in intergenic and intragenic recombination, and mutation. Cells grew to nearly the same titer in media without saccharin and containing 2 or 20 mg saccharin/ml, although cell viability was somewhat reduced in saccharin-containing media. At the high test dose of 100 mg/ml, titers and cell viability were more markedly lowered. Differences between this study and previous (negative) tests of saccharin in yeast are described.     

The gene for a major exopolyphosphatase of Saccharomyces cerevisiae.

The gene encoding a major exopolyphosphatase (scPPX1) in Saccharomyces cerevisiae (H. Wurst and A. Kornberg, J. Biol. Chem. 269:10996-11001, 1994) has been isolated from a genomic library. The gene, located at 57 kbp from the end of the right arm of chromosome VIII, encodes a protein of 396 amino acids. Overexpression in Escherichia coli allowed the ready purification of a recombinant form of the enzyme. Disruption of the gene did not affect the growth rate of S. cerevisiae. Lysates from the mutants displayed considerably lower exopolyphosphatase activity than the wild type. The enzyme is located in the cytosol, whereas the vast accumulation of polyphosphate (polyP) of the yeast is in the vacuole. Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase activity in the vacuole. This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteolytic activation. A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patterns of expression during growth and activation by arginine. With regard to the levels of polyP in various mutants, those deficient in vacuolar ATPase retain less than 1% of the cellular polyP, a loss that is not offset by additional mutations that eliminate the cytosolic exopolyphosphatase and the vacuolar polyphosphatases dependent on vacuolar protease processing.