Daniel Vlček

Effects of supercypermethrin, a synthetic developmental pyrethroid, on four biological test systems

The genotoxic potential of the insecticide supercypermethrin, a second-generation pyrethroid, was studied on four different test systems. It was non-mutagenic to Salmonella typhimurium strains TA1535, TA100, TA1538, TA98 and TA97 in the presence and absence of S9 mixture. It induced gene conversion at the tryptophan locus and induced point mutations at the isoleucine locus in Saccharomyces cerevisiae cells. A slight increase in the frequency of aberrant anaphases and telophases in root tips of Hordeum vulgare and Vicia faba was observed, but no genotoxic effects were detected in Drosophila melanogaster.

General characteristics, molecular and genetic analysis of two new UV-sensitive mutants of Chlamydomonas reinhardtii

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.

Chlamydomonas reinhardtii: a convenient model system for the study of DNA repair in photoautotrophic eukaryotes

The green alga Chlamydomonas reinhardtii is a convenient model organism for the study of basic biological processes, including DNA repair investigations. This review is focused on the studies of DNA repair pathways in C. reinhardtii. Emphasis is given to the connection of DNA repair with other cellular functions, namely the regulation of the cell cycle. Comparison with the results of repair investigations that are already available revealed the presence of all basic repair pathways in C. reinhardtii as well as special features characteristic of this alga. Among others, the involvement of UVSE1 gene in recombinational repair and uniparental inheritance of chloroplast genome, the specific role of TRXH1 gene in strand break repair, the requirement of PHR1 gene for full activity of PHR2 gene, or encoding of two excision repair proteins by the single REX1 gene. Contrary to yeast, mammals and higher plants, C. reinhardtii does not appear to contain the ortholog of RAD6 gene, which plays an important role in DNA translesion synthesis and mutagenesis. Completed genome sequences will be a basis for molecular analyses allowing to explain the differences that have been observed in DNA repair of this alga in comparison with other model organisms.

Genotoxicity and antigenotoxicity evaluation of non-photoactivated hypericin.

The potential genotoxicity and antigenotoxicity of non‐photoactivated hypericin was investigated in five experimental models. Hypericin was non‐mutagenic in the Ames assay, with and without metabolic activation. It did not exert a protective effect against mutagenicity induced by 9‐aminoacridine. In a yeast (Saccharomyces cerevisiae) assay, hypericin did not increase the frequency of mitotic crossovers or total aberrants at the ade2 locus, the number of convertants at the trp5 locus, or the number of revertants at the ilv1 locus. In combined application with 4‐nitroquinoline‐1‐oxide, it significantly enhanced the number of revertants at the ilv1 locus at the highest concentration used. Hypericin was not mutagenic in the alga Chlamydomonas reinhardtii. However, in combined application with methyl methane sulfonate, toxicity and mutagenicity were slightly reduced. In a chromosome aberration assay using three mammalian cell lines, hypericin did not alter the frequency of structural chromosome aberrations, and in the DPPH radical scavenging assay, it did not exert any antioxidant effects. Copyright

New DNA repair-deficient mutants of Chlamydomonas reinhardtii

Two new UV-sensitive mutants of Chlamydomonas reinhardtii, uvs12 and uvs13, were characterized. Genetic analysis proved that they were non-allelic. They complemented the other repair-deficient mutants uvs8, uvs9, uvs10 and uvs11. While uvs12 may have an impaired excision-repair pathway, uvs13 is, by its UV sensitivity under non-photoreactivating conditions, very similar to uvsE1 and uvs10, but differs in the effect of caffeine on its survival. After UV, survival of some repair-deficient mutants was, under photoreactivating conditions, much lower than that of phr1, while survival of other repair-deficient mutants did not differ from that of a wild-type strain. A lower UV survival of some dark-repair-defective mutants of Chlamydomonas reinhardtii, under photoreactivating conditions, can perhaps be used as an additional criterion for mutants defective in the excision-repair pathway.

The alga Chlamydomonas reinhardtii UVS11 gene is responsible for cell division delay and temporal decrease in histone H1 kinase activity caused by UV irradiation

The aim of the present work was to study the possible role of the UVS11 gene of the alga Chlamydomonas reinhardtii, in regulation of the cell cycle. To characterize the defect of a uvs11 mutant in respect to DNA damage-dependent cell cycle arrest, we examined first the influence of the tubulin-destabilizing drug methyl benzimidazole-2-yl-carbamate (MBC) on inhibition of mitosis in response to UV 254nm. Then the growth and reproductive processes and activity of cyclin-dependent kinases (CDK)-like kinases during the cell cycle of C. reinhardtii were investigated. In both, the wild type and the uvs11 mutant strain were compared under standard conditions and after DNA damage caused by UV 254nm. We assume the green alga C. reinhardtii possesses control mechanisms allowing to stop the cell cycle progression before mitosis in response to DNA damage. The results indicate that the uvs11 mutant is not able to stop the cell cycle after UV irradiation. We suggest that a product of the UVS11 gene affects cell response to DNA damage and influences a decrease in histone H1 kinase activity.

Interactions between photolyase and dark repair processes in Chlamydomonas reinhardtii

The participation of DNA photolyase in dark repair processes has been reported in some heterotrophic organisms. To assess the role of photolyase in dark repair in photoautotrophs, double mutants of Chlamydomonas reinhardtii deficient in dark repair and photoreactivation were constructed and assayed for UV sensitivity in different posttreatment light conditions (with or without subsequent photoreactivation). We found that a functional PHR1 gene enhanced dark survival in the excision deficient (uvs9, uvs12) and in the recombination deficient (uvs10) genetic backgrounds but failed to do so in the strain deficient in a repair pathway other than excision and recombination (uvs13). Therefore we can conclude that photolyase may stimulate dark repair processes in C. reinhardtii also via pathway(s) other than nucleotide excision repair. The fact that some of the double mutants deficient in dark repair and photoreactivation survived better in the light than in the dark supports the idea that additional photorepair might be active and may enhance survival in a specific genetic background.

Mutagenic activity of phosmet, the active component of the organophosphorus insecticide Decemtione EK 20 in Salmonella and Saccharomyces assays

Phosmet, the active component of the organophosphorus insecticide Decemtione EK 20, was shown to be mutagenic in the standard Ames Salmonella/mammalian microsome assay in the absence and presence of metabolic activation. It appears to be a direct mutagen inducing base substitution mutations (TA100) as well as a weak frameshift mutagen (TA97). This compound was genotoxic in the Saccharomyces cerevisiae D7 strain. It significantly increased reverse mutation, mitotic crossing-over and slightly, but not significantly, increased gene conversion at the highest concentration used.

A Chlamydomonas reinhardtii UV-sensitive mutant uvs15 is impaired in a gene involved in several repair pathways

In this report, three DNA repair-deficient mutants of Chlamydomonas reinhardtii (uvs13, uvs14, uvs15) were characterized by using genetic, mutational and biochemical analyses. The mutant strain uvs15 belongs to the most sensitive repair-deficient mutants following exposure to all agents used. It is deficient in the nuclear excision-repair pathway, whereas uvs13 and uvs14 are not blocked in removal of pyrimidine dimers. Mutation study also revealed differences among strains. The mutant uvs15 does not mutate after UV and X-ray irradiation, and there is very low mutation rate after MNNG. These findings might indicate the involvement of UVS15 gene product in regulation of several repair pathways. Contrary to this, uvs14 showed higher mutation frequency, both spontaneous and induced after UV and MNNG treatments. Tetrad dissection proved that the uvs13 and uvs14 genes are located on the right arm of the linkage group I in the vicinity of the previously mapped uvs10 gene. Both mutants belong to the same repair pathway, which is different from that of uvs10 and uvs15.

induction of new uv-sensitive mutants of chlamydomonas reinhardtii

Summary In this work the induction of new UV-sensitive mutants of green single-celled algae Chlamydomonas reinhardtii after MNNG-treatment is described. Seven new isolated mutants were characterized by their inactivation curves, and reaction on a caffeine after UV-irradiation. Genetic analysis showed the monogenic determination of five mutants and two mutants were probably double ones.