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Journal ArticleDOI

Effect of heavy ion irradiation on DNA DSB repair in Methanosarcina barkeri.

TL;DR: DNA double strand break repairing ability of mesophilic archaea Methanosarcina barkeri (DSM 804) was studied using (7)Li, (12)C and (16)O heavy ions and compared with that of (60)Co gamma-rays.
About: This article is published in Anaerobe.The article was published on 2003-02-01. It has received 5 citations till now. The article focuses on the topics: Methanosarcina barkeri.
Citations
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Journal ArticleDOI
TL;DR: In this paper, high-density DNA microarrays were used to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage.
Abstract: In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.

125 citations

Journal ArticleDOI
TL;DR: After desiccation M. barkeri has the innate capability to survive extended periods of exposure to air and lethal temperatures, and the protection mechanisms are likely adequate to maintain cell viability during periodic exposure events.
Abstract: Viable methanogens have been detected in dry, aerobic environments such as dry reservoir sediment, dry rice paddies and aerobic desert soils, which suggests that methanogens have mechanisms for long-term survival in a desiccated state. In this study, we quantified the survival rates of the methanogenic archaeon Methanosarcina barkeri after desiccation under conditions equivalent to the driest environments on Earth and subsequent exposure to different stress factors. There was no significant loss of viability after desiccation for 28 days for cells grown with either hydrogen or the methylotrophic substrates, but recovery was affected by growth phase, with cells desiccated during the stationary phase of growth having a higher rate of recovery after desiccation. Synthesis of methanosarcinal extracellular polysaccharide (EPS) significantly increased the viability of desiccated cells under both anaerobic and aerobic conditions compared with that of non-EPS-synthesizing cells. Desiccated M. barkeri exposed to air at room temperature did not lose significant viability after 28 days, and exposure of M. barkeri to air after desiccation appeared to improve the recovery of viable cells compared with that of desiccated cells that were never exposed to air. Desiccated M. barkeri was more resistant to higher temperatures, and although resistance to oxidative conditions such as ozone and ionizing radiation was not as robust as in other desiccation-resistant microorganisms, the protection mechanisms are likely adequate to maintain cell viability during periodic exposure events. The results of this study demonstrate that after desiccation M. barkeri has the innate capability to survive extended periods of exposure to air and lethal temperatures.

42 citations

Journal ArticleDOI
TL;DR: In this paper, the effects of low-dose carbon ion irradiation on reproductive system of mice were investigated by measuring DNA double-strand breaks (DNA DSBs) and oxidative stress parameters including malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and testis weight and sperm count at 12h, 21d and 35d after irradiation in mouse testis.

9 citations

Journal ArticleDOI
TL;DR: The strain was one of the members of the group of uncultivated bacterium as evident from phylogenetic analysis, thus indicating the successful cultivation of an as yet uncultivable novel microbe and also the non-specific growth of microbes in prescribed medium.
Abstract: Problem statement: This study was an attempt to isolate anaerobic microbes with potential for DNA double strand break repair using methanogen specific medium (DSMZ 120) from East Calcutta Wetland in India. It also intended to verify the specificity of the medium for isolation of the desired family of microbe. Approach: Culture based technique was used to obtain the pure isolate that was further characterized in details. For double strand break repair studies, isolate was irradiated with different doses of 60Co gamma rays and its subsequent repair was observed using pulse field gel electrophoresis and asymmetric field inversion gel electrophoresis. Inhibitor was used to predict the mechanism of repair. Results: In this study we isolated and characterized a metal sensitive anaerobic microbial strain obtained using methanogen specific medium (DSMZ 120) from East Calcutta Wetland in India. The strain was one of the members of the group of uncultivated bacterium as evident from phylogenetic analysis, thus indicating the successful cultivation of an as yet uncultivable novel microbe (GenBank Acc. No. FJ 930097) and also the non-specific growth of microbes in prescribed medium. It was a Gram positive Bacilli, member of Fermicutes with optimum growth at 25°C and pH-7. The growth curve analysis showed a lag phase up to 24 h, log phase from 24-48 h, an early stationary phase from 96 h onwards. The strain could repair the DNA double strand break caused by irradiation with 60Co γ rays. The dose profile study revealed maximum repair at 60 Grays and thereafter a drop in repair ability with increase in irradiation dose. The time required for repair showed an essential incubation period of 4 h. The DNA polymerase inhibitor, Arabinose CTP inhibited the repair indicating the involvement of polymerase in the repair process and thus pointing towards homologous recombination as the underlying mechanism. Conclusion: In this study we were able to cultivate an as yet uncultivable anaerobic bacterial isolate and predict the growth conditions for the isolate. On irradiation with 60Co γ rays the isolate showed maximum repair following 60 Gray damage. DNA polymerase inhibitor arabinose CTP inhibited the repair mechanism completely. This indicated that DNA polymerase took active part in repair process and thus the mechanism was that of homologous recombination repair.

1 citations


Cites background from "Effect of heavy ion irradiation on ..."

  • ...It is at par wi th the finding of Raychaudhuri et al.([2]) in case of methanogen....

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  • ...Methanogens are closer to bacteria in their morphol ogy while being closer to their eukaryotic counterpart in he information processing systems ([2])....

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  • ...Prediction of the mechanism of repair by using inhibitor: Arabinosine CTP, a polymerase inhibitor had been used to check the involvement of polymerase in the repair process as recombination repair would be affected by polymerase inhibitor while NHEJ would not be affected....

    [...]

  • ...In bacteria and yeast homologous recombination is the process for repairing the DNA double strand break, while vertebrates mostly repai r the double strand breaks in DNA by Non-Homologous End Joining (NHEJ) ([2,3])....

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  • ...It is at par with the finding of Raychaudhuri et al.[2] in case of methanogen....

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References
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Journal ArticleDOI
TL;DR: It is suggested that a proportion of the radiation-induced mutants suffer extensive genetic damage, and that some forms of this damage may be induced with high efficiency by radiations of high linear energy transfer.
Abstract: SummaryInactivation and mutation to thioguanine-resistance of V79 hamster cells were studied after irradiation with accelerated helium, boron or nitrogen ions covering a range of linear energy transfer from 28 to 470 keV µm−1. For all radiation qualities a dose-dependent increase in mutant frequency was found for doses giving surviving fractions greater than about 0·20. The effectiveness per unit dose for both inactivation and mutation induction increased with the linear energy transfer of the radiation to a maximum in the range 90–200 keV µm−1. However, the maximum mutagenic effectiveness relative to γ-rays was about two or more times that for inactivation. It is suggested that a proportion of the radiation-induced mutants suffer extensive genetic damage, and that some forms of this damage may be induced with high efficiency by radiations of high linear energy transfer.

258 citations

Journal ArticleDOI
TL;DR: The ability to disaggregate and grow Methanosarcina spp.
Abstract: The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.

192 citations

Journal ArticleDOI
TL;DR: Recent advances in several areas of the radiobiology of these densely ionizing radiations, also known as heavy ions are reviewed, including novel applications of pulsed-field gel electrophoresis, fluorescence in situ hybridization, linkage analysis, and studies of gene expression and protein expression.
Abstract: Shortly after the discovery of polonium and radium by Marie Curie and her husband and colleague, Pierre Curie, it was learned that exposure to these alpha-particle emitters produced deleterious biological effects. The mechanisms underlying the increased biological effectiveness of densely ionizing radiations, including alpha particles, neutrons and highly energetic heavy charged particles, remain an active area of investigation. In this paper, we review recent advances in several areas of the radiobiology of these densely ionizing radiations, also known as heavy ions. Advances are described in the areas of DNA damage and repair, chromosome aberrations, mutagenesis, neoplastic transformation in vitro, genomic instability, normal tissue radiobiology and carcinogenesis in vivo. We focus on technical innovations, including novel applications of pulsed-field gel electrophoresis, fluorescence in situ hybridization (FISH), linkage analysis, and studies of gene expression and protein expression. We also highlight the use of new cellular and animal systems, including those with defined DNA repair deficiencies, as well as epithelial cell model systems to assess neoplastic transformation both in vitro and in vivo. The studies reviewed herein have had a substantial impact on our understanding of the genotoxic effects of heavy ions as well as their distinct effects on tissue homeostasis. The use of these radiations in cancer therapy is also discussed. The use of both heavy-ion and proton therapy is on the upswing in several centers around the world, due to their unique energy deposition characteristics that enhance the therapeutic effect and help reduce damage to normal tissue.

170 citations

Journal ArticleDOI
TL;DR: Overall, the above results indicate that AFIGE is a sensitive method for detecting DSBs in DNA.
Abstract: A new method is described for detecting DNA double-strand breaks (DSBs) that utilizes asymmetric field inversion gel electrophoresis (AFIGE). DNA purified from cells in agarose plugs is subjected to AFIGE and DNA breakage quantitated by the fraction of DNA released from the plug. To test the specificity of the method for DNA DSBs, purified DNA in agarose plugs was treated for increasing times with restriction endonuclease, XhoI. After an initial time period, the fraction of DNA released increased in direct proportion to time. This correlates with the expected response for a randomly broken DNA molecule. In contrast, treatment with the single-strand breaking agent, hydrogen peroxide, over a 1000-fold range produced no release of DNA from the plug. Thus the assay appears to be specific for DNA DSBs and was used to measure DNA breaks induced by gamma radiation. Purified DNA, irradiated in agarose plugs, exhibited a log-linear dose response up to doses that release greater than 90% DNA from the plug. When live cells were irradiated in agarose, a similar linear dose response was observed up to 40 Gy and a significant signal as low as 2.5 Gy. Also in live cells, a threefold lower percentage of DNA was released from the plug over the same dose range. However, less DNA per gray is released at doses above 40 Gy and may reflect a crosslinking effect produced by the irradiation of DNA in live cells. DNA which was "pulse-labeled" was used to test the effect of DNA replication on the ability of AFIGE to detect DNA DSBs. Replicating DNA irradiated in the cell or after purification exhibited a reduced rate of release from the plug per dose of irradiation. Overall, the above results indicate that AFIGE is a sensitive method for detecting DSBs in DNA.

152 citations