Genetic effects of 5-azacytidine in Saccharomyces cerevisiae

The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in different organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.     

Tests for recombinagens in fungi.

Three types of mitotic recombination can be studied in Aspergillus nidulans and Saccharomyces cerevisiae: (1) The classical type of reciprocal mitotic crossing-over which can be detected when it occurs between non-sister chromatids at the four-strand stage followed by co-segregation of a crossing-over and a non-crossing-over chromatid in the subsequent mitotic division. Consequently, mitotic crossing-over reflects cellular responses to primary genetic damage in the G2 phase of the cell cycle. (2) Mitotic gene conversion is a unidirectional event of a localized transfer of genetic information between non-sister chromatids which in yeast can extend to segments of up to 18 cM and even beyond 22 cM in Aspergillus nidulans. Mitotic gene conversion can also occur between unreplicated chromatids and lead to the expression of the newly created genotype without any need for a subsequent mitotic cell division. It reflects a cellular response in G1. (3) Mitotic sister-strand gene conversion can be studied in a recently constructed strain with the same technical ease as classical non-sister chromatid gene conversion. It can be induced by chemicals which do not induce mutation in the Salmonella system and non-sister chromatid gene conversion. Mitotic segregation in Saccharomyces cerevisiae results almost exclusively from crossing-over and gene conversion whereas mitotic chromosomal malsegregation contributes only very little. In contrast to this, in Aspergillus nidulans, both processes contribute considerably so that mitotic segregants always have to be tested for their mechanistic origin.     

Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae

The treatment of diploid cultures of yeast with ultraviolet light (UV), γ-rays, nitrous acid (NA) and ethyl methane sulphonate (EMS) results in increases in cell death, mitotic gene conversion and crossing-over. Acridine orange (AO) treatment, in contrast, was effective only in increasing the frequency of gene conversion. The individual mutagens were effective in the order UV > NA > γ-rays > AO > EMS. Prior treatment of yeast cultures in starvation medium produced a significant reduction in the yield of induced gene conversion.The results have been interpreted on the basis of a general model of mitotic gene conversion which involves the post-replication repair of induced lesions involving de novo DNA synthesis without genetic exchange. In contrast mitotic crossing-over appears to involve the action of a repair system independent from excision or post-replication repair which involves genetic exchange between homologous chromosomes.     

A novel yeast-based tool to detect mutagenic and recombinogenic effects simultaneously

In this work, we describe a new yeast-based assay to allow efficient detection of a comprehensive spectrum of genotoxicity events. The constructed diploid Saccharomyces cerevisiae strain allows the simultaneous monitoring of forward mutations, mitotic recombination events and chromosome loss or non-disjunction by direct selection in an easy and highly reproducible approach. The strain contains a DNA module consisting of a single functional copy of the URA3 gene and the kanMX4 gene inserted at the ADE2 locus on the right arm of chromosome XV. The changes of the genotype within the marker region were primarily selected on 5-fluoroorotic acid (5-FOA) agar plates. Further simple phenotypic tests of the 5-FOA-resistant ura3 clones make it possible to analyze the genetic configuration in detail (e.g. point mutations in URA3, gene conversion, crossing-over and chromosome loss). We demonstrate the successful application of our test system by studying the effects of well-known genotoxic agents (UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, aniline and benomyl). We found that the various agents induced mutations and recombination events with different relative frequencies. The integration of the module has generated a hot spot region of mutation and recombination at the borders of the artificially integrated URA3 kanMX4 cassette, which makes the system more sensitive towards DNA-damaging agents. Unlike other test systems, our S. cerevisiae strain is capable to detect a mutagenic effect caused by aniline.     

Genetic effects of N-nitroso-N-methylurea on Saccharomyces cerevisiae yeasts. I. The influence of radiosensitivity mutations on lethal and recombinogenic effects]

Effect of mutations rad2 and rad54 in homozygous state on survival, mitotic segregation and crossing-over induced by NMU in yeast was studied. Mutation rad2 did not influence on these effects of NMU. The mutation rad54 increased sensitivity to the lethal effect, the frequencies of NMU-induced segregation and crossing-over were decreased in the strain rad54 rad54. The recombinogenic effect of NMU on yeast was lower than under the action of UV and gamma rays.     

Genotoxicity of selenite in diploid yeast

Selenite, a chemical of industrial importance and also an antimutagenic/anticarcinogenic agent, was tested for mutagenic and recombinogenic effects in 2 diploid yeast strains, Saccharomyces cerevisiae BZ 34 and D7. Selenite induced gene conversion and toxicity in BZ 34 and a variety of genetic events, viz. back-mutation, gene conversion, mitotic crossing-over, aberrant colony formation and also toxicity in the D7 strain. In both strains, the genetic effects of selenite showed a peak and a decline during 5 h of treatment while its toxicity increased marginally during 1-5 h. In the BZ 34 strain, the presence of glutathione (GSH) during selenite treatment greatly enhanced the convertogenic and toxic effects of selenite.     

Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH

The genetic effects of variation in pH in culture media and in suspension tests were examined in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. Deviation from the normal pH of 6.24 in the liquid culture medium, has a significant effect on cellular growth and on mitotic gene conversion at the trp5 locus. Frequencies of reversion at the ilv I-92 locus and of mitotic crossing-over at the ade2 locus are not significantly influenced. Suspension tests, performed using phosphate buffer (pH 5.8), strongly confirm the original results. Our data suggest that the increase in mitotic gene conversion under various conditions of pH is due to a specific effect of pH itself on the cells of S. cerevisiae. In fact, increases were obtained using the same pH in both cellular growth and non-growth conditions. The maximum effect detected with both procedures was obtained at pH 5.8; in the growth test, at this pH, gene conversion frequency appeared to be most pronounced, being about 10 times higher than that of the control. These results suggest that pH exerts its specific action both on growing and non-growing yeast cells, and the difference in induction of genetic effect between these two conditions is probably due to a time factor.     

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.     

Mitotic recombination and inactivation in Saccharomyces cerevisiae induced by UV-radiation (254 nm) and hyperthermia depend on UV fluence rate

In experiments with wild-type diploid yeast cells of Saccharomyces cerevisiae, the synergistic interaction of ultraviolet (UV) light (wavelength, 254 nm) and heat (45--60 degrees C) was studied both for mutagenic and inactivation effects. Simultaneous hyperthermia and UV light treatments increase the frequency of UV-induced mitotic intergenic recombination (crossing-over) and cell inactivation. The enhancing effect was a function of UV light fluence rate. It is concluded that the effect of hyperthermia on low fluence UV or high fluence UV irradiation results in comparable effects on survival and mitotic recombination suggesting similar modulation by hyperthermia of the effects induced by UV at different fluence rates. The interpretation of the data obtained was carried out within the widely accepted point of view considering the synergistic effects as a result of repair ability damage.     

Long-Tract Mitotic Gene Conversion in Yeast: Evidence for a Triparental Contribution during Spontaneous Recombination

In Saccharomyces cerevisiae, spontaneous mitotic gene conversion at one site is statistically correlated with recombination at other loci. In general, coincident conversion frequencies are highest for tightly linked markers and decline as a function of intermarker distance. Paradoxically, a significant fraction of mitotic gene convertants exhibits concomitant nonreciprocal segregation for multiple and widely spaced markers. We have undertaken a detailed genetic analysis of this class of mitotic recombinants. Our results indicate that mitotic gene conversion in yeast is frequently associated with nonreciprocal segregation of markers centromere-distal to the selected site of conversion. In addition, distal markers are often found to be mosaic within the product colonies. These observations, and others described here, suggest that a percentage of gene conversion in vegetative yeast cells is coupled to a chromosome break and repair mechanism. This hypothesis was further tested using a strain trisomic for chromosome VII which was specially marked to detect homolog-dependent repair events. An association between mitotic gene conversion events and the production of broken chromosomes which are repaired by a homologous-pairing-copy mechanism was supported.     

Aneuploidy and other genetic effects induced by hydroxyurea in Saccharomyces cerevisiae

Hydroxyurea induces mitotic gene conversion, mitotic crossing-over, reverse mutation, respiration-deficient petite mutants and aneuploidy in growing cultures of Saccharomyces cerevisiae. Evidence is presented indicating that induction rather than selection is responsible for the increase in frequency of the genetic end points measured. Complications concerning the detection of aneuploidy in the presence of other genetic effects are described, and the need for following the complete protocol for confirmation of the aneuploids in any chemical screening program is emphasized.     

Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program

The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.     

Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae

Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, beta-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv(+) revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state.     

Cell-cycle variation in the induction of lethality and mitotic recombination after treatment with UV and nitrous acid in the yeast, Saccharomyces cerevisiae.

Exponentially growing yeast cultures separated into discrete periods of the cell cycle by zonal rotor centrifugation show cyclic variation in both UV and nitrous acid induced cell lethality, mitotic gene conversion and mitotic crossing-over. Maximum cell survival after UV treatment was observed in the S and G2 phases of the cell cycle at a time when UV induction of both types of mitotic recombination was at a minimum. In contrast, cell inactivation by the chemical mutagen nitrous acid showed a single discrete period of sensitivity which occurred in S phase cells which are undergoing DNA synthesis. Mitotic gene conversion and mitotic crossing-over were induced by nitrous acid in cells at all stages of the cell cycle with a peak of induction of both events occurring at the time of maximum cell lethality. The lack of correlation observed between maximum cell and the maximum induction of mitotic intragenic recombination suggest that other DNA-repair mechanisms besides DNA-recombination repair are involved in the recovery of inactivated yeast cells during the cell cycle.     

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.     

Induction and repair of gene conversion in UV-sensitive mutants of yeast

Summary Photoreactivation effect on UV-induced allelic recombination has been examined using various combinations of leu 1 alleles in UV-sensitive and wild type diploid yeast, Saccharomyces cerevisiae. The frequencies of UV-induced heteroallelic reversion in UV-sensitive strains, presumably lacking dark-repair, are strikingly enhanced compared to those in wild type at the same doses under dark condition. However, these enhanced frequencies of reversion are diminished by photoreactivation almost to the level of those in wild type. The induced frequencies of homoallelic reversion (mutation) of relevant alleles are apparently lower than those of heteroallelic reversion. Phenotypic analysis for linked gene leu 1 on UV-induced heteroallelic revertants has shown that most of the revertants are of the nonreciprocal type recombination (mitotic gene conversion). These results would indicate that most of the dark-repairable damage leading to mitotic gene conversion after UV-light is due to pyrimidine dimers.On leave of absence from Radiation Center of Osaka Prefecture, Shinke-cho Sakai, Osaka, Japan.     

Exposure to low dose of gamma radiation enhances the excision repair in Saccharomyces cerevisiae

The effect of low doses of ionizing and nonionizing radiation on the radiation response of yeast Saccharomyces cerevisiae toward ionizing and nonionizing radiation was studied. The wild-type strain D273-10B on exposure to 54 Gy gamma radiation (resulting in about 10% cell killing) showed enhanced resistance to subsequent exposure to UV radiation. This induced UV resistance increased with the incubation time between the initial gamma radiation stress and the UV irradiation. Exposure to low doses of UV light on the other hand showed no change in gamma or UV radiation response of this strain. The strains carrying a mutation at rad52 behaved in a way similar to the wild type, but with slightly reduced induced response. In contrast to this, the rad3 mutants, defective in excision repair, showed no induced UV resistance. Removal of UV-induced pyrimidine dimers in wild-type yeast DNA after UV irradiation was examined by analyzing the sites recognized by UV endonuclease from Micrococcus luteus. The samples that were exposed to low doses of gamma radiation before UV irradiation were able to repair the pyrimidine dimers more efficiently than the samples in which low gamma irradiation was omitted. The nature of enhanced repair was studied by scoring the frequency of induced gene conversion and reverse mutation at trp and ilv loci respectively in strain D7, which showed similar enhanced UV resistance induced by low-dose gamma irradiation. The induced repair was found to be essentially error-free. These results suggest that irradiation of strain D273-10B with low doses of gamma radiation enhances its capability for excision repair of UV-induced pyrimidine dimers.     

Conservation of mitotic controls in fission and budding yeasts

In fission yeast, the initiation of mitosis is regulated by a control network that integrates the opposing activities of mitotic inducers and inhibitors. To evaluate whether this control system is likely to be conserved among eukaryotes, we have investigated whether a similar mitotic control operates in the distantly related budding yeast S. cerevisiae. We have found that the protein kinase encoded by the mitotic inhibitor gene wee1+ of fission yeast, which acts to delay mitosis, is able also to delay the initiation of mitosis when expressed in S. cerevisiae. The wee1+ activity is counteracted in S. cerevisiae by the gene product of MIH1, a newly identified gene capable of encoding a protein of MW 54,000, which is a structural and functional homolog of the cdc25+ mitotic inducer of fission yeast. Expression of wee1+ in a mih1- strain prevents the initiation of mitosis. These data indicate that important features of the cdc25+-wee1+ mitotic control network identified in S. pombe are conserved in S. cerevisiae, and therefore are also likely to be generally conserved among eukaryotic organisms.     

Genetic effects of chlorinated ethylenes in the yeast Saccharomyces cerevisiae

The chlorinated ethylenes 1,1-dichloroethylene (vinylidene chloride), trans-1,2-dichloroethylene, trichloroethylene, and tetrachloroethylene (perchloroethylene) were assayed for their ability to induce mitotic gene conversion and point mutation as well as mitotic aneuploidy in diploid strains of the yeast Saccharomyces cerevisiae. From strain D7 late logarithmic-phase cells grown in 20% glucose liquid medium, containing a high level of cytochrome P-450, as well as stationary-phase cells combined with an exogenous metabolic activating system (S9) were used, in order to activate the chlorinated compounds and to produce electrophilic mutagenic intermediates. Only 1,1-dichloroethylene exhibited a dose-dependent genetic activity, while the other ethylenes did not. The 2 ways of metabolic activation were compared and were found to cause approximately the same effect. In contrast to the findings with strain D7, vinylidene chloride, trans-1,2-dichloroethylene, and trichloroethylene induced, without metabolic activation, mitotic chromosomal malsegregation in strain D61.M. The presence of liver homogenate as an activating system did not enhance the respective frequencies of chromosome loss. In the case of tetrachloroethylene, sufficient data have not become available, since this compound showed a highly toxic effect towards yeast cells, decreasing the rate of surviving cells to less than 30% at a concentration of 9.8 mM.     

Analysis of non-linearities in frequency curves for UV-induced mitotic recombination in wild-type and excision-repair-deficient strains of yeast

Frequency curves for UV-induced mitotic recombination often are linear at low doses. As dose increases, these curves either increase at higher powers of dose and/or reach a maximum induced frequency and then decline. Similar dose-response patterns have been observed previously for mutation. The non-linearities can arise from higher order effects inherent in the molecular mechanisms of mutagenesis and/or from 'delta-effects' (Eckardt and Haynes, 1977a), i.e., differential probabilities of clone formation for mutant and non-mutant cells. Previously, we have shown that one can distinguish between these two possibilities by plotting the ratio of the induced mutant yield to the linear component of frequency as a function of dose (Haynes et al., 1985). In this study, we have used this ratio, a quantity we call 'apparent survival', to analyse the non-linear regions of the dose-response curves for UV-induced mitotic crossing-over and gene conversion in wild-type (RAD) and excision-repair-deficient (rad3) strains of yeast. Plots of apparent survival versus dose reveal the existence of a positive, non-linear component associated with UV-induced gene conversion in RAD, but not rad3, cells. A high dose decline in frequency, which is observed for UV-induced recombination in both strains, can be attributed to delta-effects.