Vadim M. Glaser

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

SummaryWe 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.

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.

Decrease in the number of DNA topological turns during friend erythroleukemia differentiation

SummaryMurine 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).

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

SummaryThe 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.

DNA topological linking numbers in malignantly transformed Syrian hamster cells

SummaryThe 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 (|δ|≧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.

Lake Baikal: biomonitoring of pulp and paper mill waste water

The quality and biological effects of the waste waters released by Baikalsk Pulp and Paper Mill were monitored chemically and biologically. In 1:5 dilution, the treated waste water induced monooxygenase activity in the liver of grayling and bullhead in standardised laboratory conditions. The Ames test was used to monitor mutagenicity of waste waters of the mill and tissue extracts of fish, sponge, mollusc and plankton collected near the mill and of seals collected from the middle part of the lake. Mutagens were found in waste water produced during pulp chlorination before treatment and in a few samples of fully treated waste water during 10 years (1982–1993). Bottom sediments near the mill showed some mutagenicity. Mutagens were not found in tissues of most aquatic animals studied with the exception of zooplankton, roach and seals collected in Lake Baikal close to the discharge outlet of the mill.

Mutagenicity of bleached and unbleached effluents from Baikalsk pulp and paper mill at Lake Baikal, Russia

The mutagenicity of bleached pulp mill effluents was compared to the mutagenicity of unbleached waste waters. Mutagenicity was assessed with the Ames test, using metabolic activation systems isolated from the liver of rats and fish. Liver extracts from fish caught in polluted areas, and from fish experimentally exposed to waste water, were also investigated. Pulp mill effluents taken after chlorination showed mutagenic activity. The activity diminished during the waste water treatment. Tissue extracts from fish exposed to various concentrations of treated bleached and unbleached pulp mill effluents showed only slight mutagenic activity in a few samples. In the case of bleached pulp mill effluents monooxygenase activities were elevated in those samples where slight mutagenicity was observed. In the case of unbleached effluents no such correlation was found.

Accumulation of Mutagenic Xenobiotics in Fresh Water (Lake Baikal) and Marine (Hornoya Island) Ecosystems

Extracts from tissues of a wide range of aquatic organisms (plants, plankton, decapods, molluscs, fish, Baikal seals, and fish-eating birds and their eggs) from Lake Baikal and from the Selenga River estuary and tissue extracts of birds breeding on Hornoya Island (northern Norway) were assayed for mutagenicity using the Ames Salmonella/microsome test. The activities of cytochrome P-450 and the enzymes of phase II of detoxification were also studied in the liver of fish and birds. Evidence was found for the accumulation of mutagens in the food chain. The relationship of bioaccumulation to the levels of enzyme activities possessing both detoxification and activation functions is discussed in the cases of fish and birds. The accumulation of mutagens was found to depend on the activity and the level of induction of the enzymes providing the detoxification and metabolic activation in the livers of fish and birds.

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

Abstract 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.

Gene clpP2Encoding Peptidase in the Synechocystissp. PCC 6803 Cyanobacterium Controls Cell Sensitivity to Photoinhibition

A homozygous insertion mutant with the inactivated clpP2gene, which encodes the proteolytic subunit of ATP-dependent peptidase, was obtained in the unicellular cyanobacterium Synechocystissp. PCC 6803. The mutant cannot grow under photoautotrophic conditions, but cells grown under heterotrophic conditions in a glucose-containing medium have active photosystems Iand II(PS Iand PS II). The loss of capacity for photoautotrophic growth is determined by a high sensitivity of mutant cells to the inactivating effect of light. Their incubation under light with an intensity above 10 μE m–2s–1inhibits cell growth in culture and causes degradation of photosynthetic pigments. It is proposed that the ClpP2 peptidase is involved in the protection of Synechocystis6803 cells from photoinhibition.