Z. Swietlińska

Induction of chromosome aberrations by diepoxybutane and caffeine in root meristems and germinating seeds of Vicia faba

Abstract In seedling root meristems and germinating seeds of V. faba , caffeine posttreatment was most effective in increasing aberration yields induced by diepoxybutane (DEB) when, after alkylation, cells underwent their first passage through the S phase. In seedling root meristems caffeine given in pretreatment was not effective, whereas embryonic radicle meristems of germinating seeds responded to caffeine both in pre- and posttreatment. The effect in pretreatment was observed up to 48h after germination. The results obtained so far suggest that sensitivity to caffeine pretreatment is confined to the first postdormancy cell cycle.

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.

High frequency of chromosome aberrations induced by DEB with caffeine posttreatment in Vicia faba var. minor

SummaryIn root tips treated with DEB and caffeine as a posttreatment aberration yield was almost four times higher than after DEB alone. Caffeine applied in simultaneous treatment with DEB was less effective.

Analysis of yeast DNA by alkaline filter elution.

SummaryThe 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.

Induction of mitotic recombination by UV and diepoxybutane and its enhancement by hydroxyurea in Saccharomyces cerevisiae

Abstract Mitotic inter- and intra-genic recombination was induced by UV-irradiation and treatment with diepoxybutane (DEB) in 2 heteroallelic diploid strains of Saccharomyces cerevisiae SBTD and D7. Induction of the events tested was strongly potentiated by plating of mutagen-treated cells on growth media containing 0.03 M hydroxyurea (HU).

The effect of diepoxybutane on chromosomes of Vicia faba

SummaryThe frequency of aberrations induced by DEB depends on the temperature and concentration of the agent. The effect of DEB is delayed. Pretreatment with EDTA increased the frequency of aberrations. The frequency of aberrations was also increased after simultaneous treatment with DEB and chloramphenicol or streptomycin. Pre- and posttreatment with protein synthesis inhibitors had a reverse effect, the frequency of aberrations was decreased.

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

SummaryTwenty 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 (radl-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 radl-1, 3, 5, 6, 9 and 11 differ in their response to LH afterr the two treatments. Survival of radl-1 and rad3 increases significantly 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.

Relation Between Liquid-Holding Recovery, DNA Repair, and Mitotic Recombination in the rad3 Mutant of Saccharomyces cerevisiae after Treatment with Diepoxybutane (DEB)

SummaryThe 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. Splitdose 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.

Study on liquid-holding recovery in DEB-inactivated rad3 mutant of Saccharomyces cerevisiae.

SummaryMaximal liquid-holding recovery (LHR) of the DEB-treated rad3 mutant occurs at 30° C in buffer supplemented with glucose. Addition of cycloheximide (CHX) to the buffer, the increase in cell density above 2 × 107/ml as well as lowering of temperature during liquid holding (LH) below 27° C decrease considerably the cell capacity for recovery. LHR does not take place at 5° C. No measurable DNA synthesis or degradation occurs in cells held in buffer alone, while addition of 0.02% glucose results in incorporation of radioactivity into DNA both of DEB-treated and control cells. Similarly, protein synthesis was observed only in cultures held in buffer supplemented with glucose. Cells transfered to growth medium directly after treatment complete one round of DNA replication and at least one division cycle, but further DNA replication and cell division are inhibited. Cells placed in growth medium after 5 days LH show an increased rate of DNA replication and cell division. Completion of the first posttreatment round of DNA replication in growth medium abolishes ishes the cell capacity for LHR. DEB treatment results in abnormal cell division of the rad3 mutant, giving ‘colonies’ consisting of several cells, usually abnormal in shape, held together by common cell walls.

Effect of caffeine on recovery from DEB-induced cell inactiation 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.