Abstract. We investigated the effect of low-LET proton irradiation on gene mutations. DNA lesions were caused by the acute exposure of the cells to 150 MeV proton irradiation (linear energy transfer of 0.539 keV/µm) and 60 Co γ-rays at doses up to 25 Gy. To detect gene mutations, we used a forward mutation rate assay that detects the mutations inactivating the arginine permease gene (Can R mutations) and a reversion assay detecting the frameshift mutations that reverts a 4-base insertion in the LYS2 gene (lys2-Bgl). The dose responses can be described by a linear relationship for both types of gene mutations. Different radiation sources that induce mutations have the relative biological effectiveness values of 1.25 for frameshift mutations and 1.69 for forward gene mutations Can R . So, the damage induced by proton irradiation of cells appears to be more serious than the damage induced by γ-irradiation, which is indicated by an increase in the yield of not only DSBs but also gene mutations. Space radiation is hazardous to long-duration space crews because it may cause cancer and damage DNA. The problem of protection from chronic exposure to cosmic radiation, which is primarily composed of protons (95 %), in future manned missions to Mars has not yet been solved. To model the effect of cosmic radiation on living cells, we used a proton accelerator beam at JINR (Dubna) and the eukaryotic unicellular yeast Saccharomyces cerevisiae. Based on the conservation of the fundamental molecular processes, including DNA repair, in the eukaryotic cells from yeast to humans, the data gained from studying model systems, such as yeast, can be estimate to studies of higher eukaryotic systems.A comparison of different ionizing radiations showed that charged particles produced a greater number of DSBs compared to abasic and oxidized base clusters than ionizing photons, with protons generating the highest ratio of DSBs to abasic and oxidized damage [1]. DNA strand breaks induced by protons were predominantly repaired by homologous recombination and postreplication repair pathways [2]. There are two branches of postreplication repair: template switching (error-free) and translesion synthesis (error-prone). In this study, we describe the results of our investigations of the effect of low-LET proton irradiation on gene mutations.The source of protons was the Phasotron at JINR (Dubna). The dose rate was 0.55 Gy/min at the energy of 171 MeV (150 MeV at the target with the LET of 0.539 KeV/µm). The source of γ-rays was 60 Co (the dose rate of 0.7 Gy/min, the LET of 0.25 KeV/µm) at the therapeutic equipment "Rokus" (JINR, Dubna). Yeast cells were exposed to protons and γ-rays in Eppendorf tubes. It was not necessary to grow cells in the dark following the exposure to protons and γ-rays as photoreactivation does not repair strand breaks. Cells were kept in ice to prevent DSB repair.To study the kinetics of proton-induced gene mutations, several genetic yeast assays were used.
We have investigated the biological effects induced by different accelerated ions ( 4 He, 11 B, 12 C, 15 N, and 20 Ne) with different energies and linear energy transfers (LETs) and determined their relative biological effectiveness (RBE) for lethal damage and gene mutations. In particular, base pair substitution induction by ionizing radiation in haploid and diploid yeast Saccharomyces cerevisiae has been studied. We have detected the GC-AT transition in the haploid strain and the AT-TA transversion in the diploid strain. The RBE dependence on LET for lethal mutations is described by a curve with a local maximum at LET of about 100 keV/μm. It is shown that the mutation frequency increases with increasing the dose up to 1000 Gy for diploid cells irradiated by different ions. A decrease in RBE with increasing LET has been observed for diploid cells. However, for haploid cells irradiated at doses of up to 100 Gy, the curves seem to have a plateau. The RBE dependence on LET for haploid cells is different and also has a plateau. But for substitution induction in haploid cells, an ion beam with a high LET (177 keV/μm) is less mutagenic than the one with a low LET (44-127 keV/μm). Therefore, we have obtained different biological effects of accelerated ions for haploid and diploid cells.
Abstract. To study the kinetics of UV-induced gene and chromosome mutations in the yeast S. cerevisiae, several genetic assays were used. We treated yeast cells with UV light of up to 130 J/m 2 . UV irradiation induced all types of base substitutions, although transitions -in particular, GC-AT events -were predominant. Frameshift mutations were induced at the same frequency as the base pair substitution GC-AT, while forward mutations in the CAN1 gene exceeded the more expressive base pair substitutions by about an order of magnitude. Chromosome mutations were the most efficient. The kinetic of the induced gene and chromosome mutations is represented by a linear-quadratic function. Such curves have been reported for UV mutagenesis in bacteria and they have been explained by the induction of SOS error-prone repair. Similar biphasic kinetics was described for yeast in our work. These data suggest the occurrence of several factors forming the mutagenic response of eukaryotic cells to UV light.Key words: UV irradiation, frameshift mutation, base pair substitution, forward mutation, rearrangement of chromosome and plasmid DNA, yeast Ultraviolet (UV) light has strong genotoxic effectsit can induce DNA damage, mutations, and, in the worst case, cancerogenesis. Several human genetic disorders, including xeroderma pigmentosum and Cockayne syndrome, are characterized by a defect in UV lesion repair. Investigations of the mechanisms of mutagenesis are continued and our knowledge is made more profound. To study the kinetics of the UVinduced gene and structural mutations on the bases of the model of the yeast S. cerevisiae, several new interesting genetic assays were used. They included a forward mutation rate assay that detects mutations inactivating the arginine permease gene (Can R mutations), frameshift reversion assays [1] detecting mutation that reverts a 4-base insertion in the LYS2 gene (lys2ΔBgl), and a collection of six isogenic trp5-strains, specifically diagnostic for all possible base-pair substitutions [2]. Assays for intrachromosome homologous recombination (HR repair) based on the 5'trancated lys2 sequence and the LEU2 gene integrated into chromosome II as a direct repeat with the lys2:HS-D allele which is 658 bp-insertion in BamH1 site of 3'-termini of LYS2 gene are shown in [3]. The [YCpL2]-plasmid assay was used to detect the extent of deletions including two or more genes which arise during NHEJ repair [4].Mutant strains were grown nonselectively in YEPD. Selective growth was on a synthetic complete medium containing 2 % glucose (SM) [5] and lacking the appropriate nutrient [6]. Canavanine-resistant mutants in the forward mutation assay were identified on SM-Arg plates supplemented with 60 μg/ml canavanine. All growth was at 30 o C. Overnight cultures (~2x10 8 cells/ml) were grown in 5 ml YEPD. Cells were resuspended in water and plated on YEPD and appropriate SM-based selective media to assess cell survival and mutagenesis, respectively. Within 1 hr of plating, cells were exposed to 254 nm UV light and va...
Targeting sonic hedgehog pathway in combination with proton radiation or gamma irradiation decreases viability of glioma cell lines .
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