[Genetic toxicology: findings and challenges].

The review highlights the history of genetic toxicology as a distinct research area, as well as the issues of genetic toxicology and development of its methodology. The strategies and testing patterns of genotoxic compounds are discussed with the purpose of identifying potential human carcinogens, as well as compounds capable of inducing heritable mutations in humans. The main achievements of genetic toxicology in the 20th century are summarized and the challenges of the 21st century are discussed.

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus.

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.

The influence of mutation rad57-1 on the fidelity of DNA double-strand gap repair in Saccharomyces cerevisiae.

The role of the RAD57 gene in double-strand gap (DSG) repair has been examined. The repair of a linearized plasmid, bearing a DSG, has been analyzed in a rad57-1 mutant of Saccharomyces cerevisiae. For effective rejoining of the ends of plasmid DNA in the rad57 mutant the sequence of chromosomal DNA homologous to the DSG region is required. However, DSG repair (restoration of plasmid circularity) in rad57 cells is not accompanied by the recovery of DSGs. The DSG repair, which depends on an homologous chromosomal DNA sequence, requires the cohesive ends of DSGs. The non-cohesive-ended DSGs are repaired in rad57 cells by a pathway independent of the homologous recombination between chromosomal and plasmid DNA. We presume that the rad57-1 mutation is connected with the inhibition of DNA repair synthesis, required for filling the DSG. This situation produces a condition of "homology-dependent ligation", the alternative minor mechanism of recombinational DSG repair, that takes place in mutant cells. A molecular model for "homology-dependent ligation" in rad57 cells is proposed.

Genetic control of plasmid DNA double-strand gap repair in yeast, Saccharomyces cerevisiae.

The repair of double-strand gaps (DSGs) in the plasmid DNA of radiosensitive mutants of Saccharomyces cerevisiae has been analyzed. The proportion of repair events that resulted in complete plasmid DNA DSG recovery was close to 100% in Rad+ cells. Mutation rad55 does not influence the efficiency and preciseness of DSG repair. The mutant rad57, which is capable of recombinational DNA DSB repair, resulted in no DSG recovery. Mutation rad53 substantially inhibits the efficiency of DSG repair but does not influence the precision of repair. Plasmid DNA DSG repair is completely blocked by mutations rad50 and rad54.

Two pathways of DNA double-strand break repair in G1 cells of Saccharomyces cerevisiae.

G1 cells of the diploid yeast Saccharomyces cerevisiae are known to be capable of a slow repair of DNA double-strand breaks (DSB) during holding the cells in a non-nutrient medium (Luchnik et al., 1977; Frankenberg-Schwager et al., 1980). In the present paper, S. cerevisiae cells gamma-irradiated in the G1 phase of the cell cycle are shown to be capable of fast repair of DNA DSB; this process is completed within 30-40 min of holding the cells in water at 28 degrees C. For this reason, the kinetics of DNA DSB repair during holding the cells in a non-nutrient medium are biphasic, i.e., the first, 'fast' phase is completed within 30-40 min, whereas the second, 'slow' phase is completed within 48 h. Mutations rad51, rad52, rad54 and rad55 inhibit the fast repair of DNA DSB, whereas mutations rad50, rad53 and rad57 do not significantly influence this process. It has been shown that the observed fast and slow repair of DNA DSB in the G1 diploid cells of S. cerevisiae are separate pathways of DNA DSB repair in yeast.

Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae.

We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae. The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid. The frequency of "lithium" transformation of Rad+ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene. This effect is due to recombinational repair of the plasmid DNA. Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA. The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14-18 h of incubating cells on selective medium.

DNA topological linking numbers in malignantly transformed Syrian hamster cells.

The topological linking numbers in closed superhelical loops of nuclear DNA were measured as the density of DNA topological turns ('titratable superhelical turns') per unit length of DNA by means of sedimentation of superhelical DNA (in nucleoids) in gradients of 15-30% sucrose containing 1.95 M NaCl and various concentrations of ethidium bromide. In four malignantly transformed Syrian hamster cell lines (three SV40-transformed and one spontaneous) the density of DNA topological turns was equal to or higher than the density of DNA topological turns in early passage embryonal Syrian hamster cells (/delta/ greater than or equal to 0.076) and, in contrast to normal cells, the malignant ones did not decrease the density of their DNA topological turns upon cultivation. It is proposed that the persistence of high densities of DNA topological turns in malignant cells is responsible for activation of transcription of a number of genes during malignant transformation.

Decrease in the number of DNA topological turns during Friend erythroleukemia differentiation.

Murine erythroleukemia cells were induced to undergo erythroid differentiation by growing in presence of dimethylsulfoxide, butyric acid or actinomycin D. topological linking numbers in closed loops of nuclear DNA were measured by means of centrifugation of nucleoids containing superhelical DNA in sucrose gradients containing varying concentrations of ethidium bromide. All cells were grown to G1 stage of the cell cycle. It was found that the mean density of the DNA topological linking number decreases from 0.076 turns per 10 nucleotide pairs in non-differentiated cells to 0.062 turns in the cells induced to differentiate. This decrease in topological linking number of DNA loops is quite sufficient for the change in the DNA double helix secondary structure which in turn may be responsible for coordinate switch in transcription of genes which control cellular differentiation (Luchnik, 1980a, b).

Repair of DNA double-strand breaks requires two homologous DNA duplexes.

DNA repair and cell survival in haploid and its diploid derivative strains of Saccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in the G1 stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.