Induced Gene Mutation

Abstract:The radiomimetic agent hydrogen peroxide is known to produce DNA strand breaks, chromosome damage and cell death. It has also been identified as one of the cytotoxic agents formed during certain drug metabolism and by phagocytic cells in the respiratory burst. Our laboratory recently identified the ultimate reactive species responsible for the DNA-damaging and cytotoxic effect of H2O2 as being hydroxyl radical. This was achieved by the use of the specific iron chelator o-phenanthroline, which prevents the occurrence of a Fenton reaction between H2O2 and chromatin bound ferrous ions. In this paper we show that H2O2 is able to induce mutation at the HGPRT locus in V79 cells and morphological transformation of C3H/10T1/2 cells. o-Phenanthroline abolishes both effects, indicating that hydroxyl radical is directly involved in mutation and carcinogenesis.

Abstract:诱导基因突变在μ检查taMouse after an intraperitoneal injection of 7, 8-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg in a collaborative study participated by four laboratories. Although the DMBA dose used was lower than the level that has been reported to induce micronucleated erythrocytes maximally in several mouse strains, a killing effect appeared after day 9 of the post-treatment interval. Mutations in lacZ transgene were detected by the positive selection assay following in vitro packaging of phage lambda from the genomic DNA of the transgenic animals that survived. The mutant induction was evaluated in the bone marrow, liver, skin, colon, kidney, thymus, and testis 7 to 28 days after the treatment. In the bone marrow, the mutant frequency reached a maximum, approximately a 30-fold increase, 14 days after the treatment and the increased frequency persisted at least up to day 28 of the post-treatment. Induction of mutants was detected in the liver, colon, thymus, and skin to lesser extents. Marginal responses were obtained in the kidney and testis. The slight increases in the mutant frequencies in the kidney and testis observed in some laboratories were within laboratory-to-laboratory or animal-to-animal variations. In contrast to the gene mutation induction in the bone marrow, the frequency of micronucleated reticulocytes increased transiently 3 days after the treatment and returned to a control level before day 8 of the post-treatment. It was suggested that DMBA induced gene mutation is fixed in stem cells depending on cell proliferation while DNA damages responsible for chromosome breakage are not transmitted to progeny cells.

Abstract:Aminoacridines引起移码突变photodynamically active, depending on whether visible light is absent or present. Therefore, a test system which allows to compare quantitatively the genetic effects of aminoacridines irradiated or unirradiated by visible light ought to be susceptible to the different DNA alterations which can be induced by these substances. For this reason in most experiments mitotic gene conversion and only in some selected experiments reverse mutation was chosen as the indicator of genetic activity. In contrast to mutation systems mitotic gene conversion has never shown a response specific to only some types of mutagens. The three aminoacridine derivatives used-acridine orange (AO), proflavine (PF), and acridine yellow (AY)—were successful in the induction of convertants at two different loci. No locus-specificity could be observed. The time-dependent induction of convertants proceeds quickly but soon reaches—especially after treatment without light—a saturation point. The dose/effect-curve after treatment in the dark has a slope increasing with increasing concentration. Irradiation with visible light results in a dose/effect-curve consisting of three parts. At first the increase of convertants is nearly linear extending one (AY) to three (AO) orders of magnitude. After that a saturation effect begins at the point at which an effectiveness of the acridines in the dark is apparent. At high concentrations an induction of convertants can again be observed which is nearly the same as that after treatment in the dark. To determine whether the dose/effect-curves obtained for gene conversion refer to similar curves for gene mutations after treatment with AO at the same locus not only gene conversions but also reverse mutations were scored for. AO-treatment in the dark is ineffective in inducing reverse mutations. Irradiation with visible light results in a dose/effect-curve beeing parallel only in its first part to the dose/effect-curve obtained for gene conversion, while in its second part a mutation frequency decline can be observed. Comparing the dose/effect-curves of AO resulting from the induction of gene conversion and gene mutation, and taking into account that no mutants can be induced by AO-treatment in the dark, the increase in convertants at high acridine-concentrations can be explained as an addition of light-dependent and light-independent effects. That means, in mutation systems at low concentrations of aminoacridines irradiation with visible light should cause transitions, transversions and microlesions, at intermediate concentrations frameshift lesions should begin to appear, and at very high concentrations nearly exclusively frameshift lesions should occur. The dose/effect-curves of aminoacridines compared with those of other mutagens are very complex. The dose/effect-curves of the mutagens of other type of action tested are linear in a double logarithmic scale, and parallel for induced gene conversion and induced gene mutation. These results indicate that the gene conversion ability of a given compound depends on its mutagenic property. That means, many mutagens may exert specific genetic effects not directly but mainly in indirect ways by leading to DNA damage, a situation for repair synthesis resulting as well in mutations as recombinations.

Abstract:Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.

Abstract:Toxicants of different classes were analysed for capacity to induce gene mutation and mitotic non-disjunction in Aspergillus nidulans, using selective and permissive tests, respectively. Ethanol, Amphotericin B and MIcanozole, all affecting membrane integrity, induced only non-disjunction, emphasizing the role of the membrane in mitosis. Benomyl and isopropyl-3-chlorophenyl carbamate (CIPC), two pesticides which interfere with spindle system, induced only non-disjunction. Conversely, mitomycin C markedly increased mutation rate but not ono-disjunction and scarcely affected the viability. The comparative analysis of these two different genetic damages should prove useful in evaluating hazards of drugs.