Analysis of replication of DEB-alkylated DNA in yeast: bypass replication in a rad3 mutant of Saccharomyces cerevisiae.

We presented indirect evidence that in an excision-deficient rad3 mutant of yeast exposed to diepoxybutane (DEB), DNA synthesis continued past the damaged sites. This bypass replication was confined to the first post-treatment round of replication and was followed by inhibition of DNA synthesis. Analyses by alkaline sucrose gradient sedimentation and by alkaline elution from filters revealed that in mutant cells the first post-treatment round of replication proceeded at a similar rate to that in untreated cells and was not accompanied by strand scission of template DNA. The post-treatment synthesis was presumably of an error-prone type, as the frequency of reversion to ade2-1 prototrophy was increased. In contrast, in the isogenic wild-type strain, the post-treatment incorporation of radioactivity into DNA was slightly reduced and newly replicated DNA fragments were of lower molecular weight than in control cells. There was also some strain scission in template DNA, presumably resulting from excision-repair.

Analysis of yeast DNA by alkaline filter elution.

The method of analysis of DNA in mammalian cells by alkaline elution from filters (Kohn et al. 1974) was adapted for studies on yeast DNA. By this technique spheroplasts obtained from yeast cells are lysed on filters and single-stranded DNA fragments selectively eluted by alkaline solutions. The procedure was applied to monitor the occurrence of replication intermediates and production of DNA single-strand breakage by MMS, and its repair in growth medium.

DNA replication in a diploid strain of Saccharomyces cerevisiae homozygous for the rad6-1 mutation.

The generation time of a diploid strain homozygous for the rad6-1 mutation was 160 min, and the duration of the S phase was 80 min; in the parental heterozygote, these values were 90 and 40 min, respectively. Analysis of DNA sedimentation in an alkaline sucrose gradient revealed that heterozygote high-molecular-weight DNA appeared after 60 min, and homozygote high-molecular weight DNA only after a 100-min pulse.

Abnormalities in cell division induced by diepoxybutane in rad1-1 and rad3 mutants of Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants rad1-1 and rad3 differ from the wild-type and from other UV-sensitive rad mutants in their behaviour after transfer from medium containing 1,2:3,4-diepoxybutane (DEB) to DEB-free medium. In both mutants several post-treatment cell cycles proceed in the absence of cell wall separation, resulting in the formation of multicellular chains or aggregates. In this study, electron and light microscopy revealed that at least one post-treatment budding cycle is accompanied by nuclear division while subsequent cell cycles can proceed in the absence of regular nuclear cycles. At low percentage survival levels, the first post-treatment budding cycle was not delayed and was accompanied by significant incorporation of radioactivity into DNA and protein. In contrast, subsequent cell cycles were found to be accompanied by only protein synthesis and not DNA synthesis. The wild-type strain, unlike the mutants, responded to DEB treatment by a dose-dependent lag in the onset of macromolecular synthesis and cell proliferation, and after prolonged incubation in mutagen-free medium the culture consisted of single budded and unbudded cells.

cdc and prt Mutants of Saccharomyces cerevisiae with increased sensitivity to diepoxybutane and ultraviolet.

The sensitivity to UV and DEB of nine temperature-sensitive mutants was investigated. All prt and four cdc mutants (cdc4, cdc7, cdc8, cdc28) showed a higher level of sensitivity to UV than the wild type strain. Three mutants cdc7, cdc9 and prt1 are more sensitive than the wild type to DEB, but only two cdc7 and prt1 are sensitive to UV and DEB. Treatment with restrictive temperature increases the sensitivity after UV treatment of cdc9, cdc21, prt1, prt3 and after DEB treatment of cdc28 and all prt mutants.

Energy requirement for liquid holding recovery from UV- and DEB-induced damage in rad mutants of Saccharomyces cerevisiae.

Respiratory deficient (rho o) strains respond to liquid holding in buffer alone with a sharp decrease of cell survival. In buffer with 0.02% glucose rho o strains behave similarly as rho+ strains. After UV or DEB inactivation rho o strains derived from rho+ rad mutants capable of LHR in buffer alone, manifest this capacity in glucose buffer. In some respiratory proficient rad mutants LHR is enhanced or induced by holding in glucose buffer, e.g. DEB inactivated rad3, rad6 and rad14. In such mutants the number of mitochondrial profiles was found to be 3 to 4 times lower than in the wild-type RAD or in rad mutants with LHR insensitive to glucose. This suggests that mutants with glucose-enhanced LHR are defective in energy metabolism and require exogenous energy supply for initiation of liquid holding repair capacity. In UV-irradiated JG-1 (rad1-1), rad3 and rad11 as well as in DEB-inactivated rad7, rad11, rad19 and rad20 the inability of LHR is a constitutive phenomenon and cannot be overcome by exogenous energy supply.

Relation between liquid-holding recovery, DNA repair, and mitotic recombination in the rad3 mutant of Saccharomyces cerevisiae after treatment with diepoxybutane (DEB).

The rad3 mutant is characterized by a high level of liquid-holding recovery after DEB treatment. The recovery is abolished when the treated cells are postincubated in growth medium, but the effect can be cancelled by suppression of DNA and protein synthesis by specific inhibitors. Alkaline sucrose gradient sedimentation revealed that DEB induces single strand breaks in DNA which are not repaired during post-treatment incubation in growth medium or during LH. Effective repair takes place only when LH is followed by incubation in growth medium. Split-dose treatment applied to test the possible inducibility of repair by LH did not confirm this presumption. In a diploid homozygous for rad3 mutation, DEB induces mitotic inter- and intragenic recombination with very high frequency. Liquid-holding recovery (LHR) was found to be accompanied by an increase in molecular weight of DNA and by a sharp decrease in the frequency of mitotic recombination. The data suggest that recombination events are not involved in LHR pathway.

Comparison of sensitivity and liquid holding recovery in rad mutants of Saccharomyces cerevisiae inactivated by UV and DEB.

Twenty one UV-sensitive rad mutants were tested for their sensitivity towards DEB. All mutants were more sensitive to this treatment than the wild type. Seven mutants were classified as supersensitive to DEB (rad 1-1, 2,3, 6, 15 and 18-2), while only rad2 and rad3 can be classified as supersensitive to UV. For all mutants ability for liquid holding recovery (LHR) after UV and DEB was compared. Mutants rad 1-1, 3, 5, 6, 9 and 11 differ in their response to LH after the two treatments. Survival of rad1-1 and rad3 increases signficantly during LH after DEB but not after UV exposure. In contrast rad5, 6, 11, and 22 show marked LHR after UV but no increase of survival after DEB treatment.

Cytotoxic effects of maleic hydrazide.

Since 1950, maleic hydrazide (MH) has been introduced into agriculture as a major commercial herbicide and a depressant of plant growth in numerous circumstances such as suppression of sprouting of vegetables and stored food crops, control of sucker growth on tobacco plants, ratardation of flowering and prolongation of dormancy period. Since 1951 MH has been known as an effective chromosome-breaking agent in higher plants, in sharp contrast with its low effect on the chromosomes and general health of tested mammals. The selectivity of action of MH in plants and animals was obviously the main reason of low interest devoted to the chemical by people working the field of environmental mutagenesis. In early works the inhibitory effects of MH on plant growth were mainly considered to result from the suppression of plant metabolism (inhibition of enzymic activity) and interference of the compound with plant hormones and growth regulators. More recently, numerous experiments performed with various plant species have shown that MH acts as an inhibitor of the synthesis of nucleic acids and proteins. Similar results have been obtained with animal tumour cells. The chromosome-breaking effect of MH on plant chromosomes resembles very closely the chromosome-breaking properties of alkylating agents and other mutagenic compounds such as mitomycin C. MH-induced chromosomal aberrations have also been recorded in grasshoppers, fish and mice, although tests with some mammalian cell lines gave negative results. Among higher plants, selective sensitivity to the toxic effects of MH is well proved. This phenomenon seems to be due to the differential ability of various plant species to detoxicate the chemical. Plants can break down MH into several products, one of which, hydrazine, is a well-known mutagen and carcinogen. MH does not seem to be toxic to bacteria and fungi. The compound is degraded by soil microflora and hence can be utilized as a source of nitrogen nutrition. MH proved to be of low toxicity to mammals, but in some instances it decreased the fertility of rats. The reported carcinogenic effects of MH in mice and rats raise the question of its risks to man.

Alkaline sucrose sedimentation studies of MMS-induced DNA single-strand breakage and rejoining in the wild type and in UV-sensitive mutants of Saccharomyces cerevisiae.

MMS-induced DNA single-strand breakage and rejoining was studied in the RAD strain and in rad6 and rad21 mutants, both very sensitive to this treatment as compared with the wild type. Alkaline sucrose gradient centrifugation showed that MMS treatment reduced the molecular weight of DNA in the RAD strain and in rad6 and rad21 mutants to the same extent. Four hours of post-incubation in synthetic growth medium after treatment with a dose of 0.4% MMS which reduces cell survival of RAD, rad21 and rad6 to 50, 20 and less than 0.01%, respectively, resulted in a significant increase in the molecular weight of DNA in the wild type, but in only slight increase in mutant strains. When the strains were exposed to a lower dose of MMS (0.04%) which led to 100% survival of RAD and 50 and 20% survival of rad21 and rad6, respectively, wild-type DNA sedimented to the position of control DNA, while in both mutants the increase in molecular weight of DNA was less pronounced.

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.

Effect of caffeine on recovery from DEB-induced cell inactivation in UV-sensitive mutants of Saccharomyces cerevisiae.

The UV-sensitive yeast mutants rad3 and rad6 are highly sensitive to diepoxybutane (DEB) as compared with the RAD strain. The two mutants show differential response to liquid holding (LH) after exposure to DEB and UV. The survival of rad3 increases markedly after DEB and decreases after UV. Caffeine significantly affects LH recovery of DEB-treated RAD strain, slightly decreases recovery of rad3 and has almost no effect on survival of rad6. When DEB-treated cultures are plated immediately on caffeine-containing medium, survival of rad3 decreases more significantly than that of the RAD strain, whereas survival of rad6 is only slightly decreased as compared with the untreated cultures. Possible mechanisms of recovery from DEB-induced cell damage are discussed.

Ultrastructural changes in zygote formation and autoradiographic study of DNA replication during conjugation in Saccharomyces cerevisiae.

The following stages of mating reaction were analysed in electron micrographs: initial contact of the conjugating cells, formation of a tube-like structure between the mating partners, nuclear fusion and formation of first diploid bud. Nuclear fusion was observed to take place within the conjugation tube, the fusion nucleus, however, was often localised in one of the initial conjugants. The stage of fusion nucleus, preceding the first diploid bud formation, is of longest duration in the mating process. The second longest stage is the formation of a tube-like structure. The whole conjugation process lasts at 25 degrees approximately 60 min. According to the autoradiographic data, DNA synthesis begins after nucelar fusion and proceeds during formation of the first diploid bud.

Cytogenetical relationships between Rumex tuberosus, R. thyrsiflorus and R. acetosa and occurrence of polyploidy among their hybrids.

Reciprocal crosses were performed between Rumex tuberosus and two other taxa belonging to the Acetosa group: R. acetosa and R. thyrsiflorus. All three taxa are dioecious with the same chromosome numbers, ♀ XX + 12A, ♂ XYY+12A, but they differ from one another in the morphology of some autosomes. R. tuberosus and R. thyrsiflorus are similar in external morphology, while R. acetosa differs greatly from the other two taxa in external morphology as well as ecological characteristics.The F 1 and F 2 hybrids were fully vigorous and viable but their fertility was greatly reduced. Meiotic irregularities were observed in hybrids derived from all crosses. The most remarkable meiotic irregularity was diad formation after first meiotic division. The unreduced gametes derived from diads were responsible for the high frequency of polyploidy observed among the F 2 hybrids. In the cross R. tuberosus × R. acetosa the whole F 2 generation was tetraploid.It is concluded from the analysis performed that R. tuberosus, R. acetosa and R. thyrsiflorus are separate species, closely related to one another but already isolated by genetic and ecological barriers.