[MIR experiment for radiation biology effect: rpsL mutation induction in B. subtilis spore].
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Bacillus subtilis↗
Biomedical subjects
F Yatagai
Publications and source records attributed to F Yatagai.
LET dependence of cell death and chromatin-break induction in normal human cells irradiated by neon-ion beams.
We investigated the LET dependence of cell death and chromatin-break induction in normal human embryo cells irradiated by accelerated neon-ion beams. Neon-ion beams were generated by the Riken Ring Cyclotron (RRC) at the Institute of Physical and Chemical Research, Japan. Chromatin breaks were measured by counting the number of chromatin fragments detected by the premature chromosome condensation (PCC) technique. The results indicated that cell death and the induction of remaining chromatin breaks showed a qualitatively similar LET dependence. The LET RBE curves for both cell death and the induction of remaining chromatin breaks had a broad peak in the LET range of 120-300 keV/microm and steeply downward trend up to 340 keV/microm. These results suggest that there is a good correlation between cell death and the induction of remaining chromatin breaks by neon-ion beams with different LET values.
Cell Death↗
Dependence of induction of interphase death of Chinese hamster ovary cells exposed to accelerated heavy ions on linear energy transfer.
Induction of interphase death was examined in Chinese hamster ovary cells exposed to accelerated heavy ions (carbon, neon, argon and iron) of various linear energy transfers (LETs) (10-2000 keV/microm). The fraction of cells that underwent interphase death was determined by observing individual cells with time-lapse photography (direct method) as well as by counting cells undergoing interphase death made visible by the addition of caffeine (indirect method). After exposure to X rays, interphase death increased linearly with dose above a threshold of about 10 Gy, whereas it increased at a higher rate without a threshold after exposure to high-LET heavy ions. The relative biological effectiveness (RBE) compared to X rays, as determined at the 50% level of induction, increased with LET, reached a maximum at an LET of approximately 230 keV/microm and then decreased with further increase in LET. The range of LET values corresponding to the maximum RBE appears to be narrower for interphase death than for reproductive death (120-230 keV/microm), as assayed using loss of colony-forming ability as a criterion. The inactivation cross section for interphase cell death reached a plateau of 5-10 microm2. This means that the probability for the induction of interphase death by traversal of a single heavy-ion track through the nucleus (size: about 130 microm2) is about 0.04-0.08.
Animals↗
The role of DNA repair on cell killing by charged particles.
It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderated RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.
Animals↗
LET dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions.
We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.
Carbon↗
A comparison of biological effects of modulated carbon-ions and fast neutrons in human osteosarcoma cells.
PURPOSE: To compare the biological effects of a 135 MeV/u carbon-ion beam and 13 MeV fast neutron beam using human osteosarcoma cells. METHODS AND MATERIALS: We have studied the clonogenic cell survival, recovery of potentially lethal damage (PLD) in plateau phase cells, and spheroid cure in multicellular spheroid after irradiation at various positions in the plateau and spread out Bragg peak (SOBP) of a 135 MeV/u carbon-ion beam and with 13 MeV neutrons. The carbon beam had a 4-cm range in water and a range filter was used to produce a 3-cm extended-peak region. The reference radiation was 137Cs gamma-rays. RESULTS: The relative biological effectiveness (RBE) values for 10% survival level of plateau phase cells for carbon-ions at the position of plateau, proximal peak, midpeak, and distal peak within the SOBP, and neutrons were 1.71, 2.48, 2.63, 3.47, and 2.29, respectively. Corresponding RBE values at 1% level were 1.64, 1.93, 2.06, 2.49, and 2.05. The extent of recovery from PLD was reduced after carbon-ions at proximal peak, midpeak, and distal peak, and neutrons, although not substantially reduced after carbon-ions at plateau. The RBE values for 50% spheroid cure level of spheroids for carbon-ions at the position of plateau, proximal peak, midproximal peak, middistal peak, and distal peak within the SOBP, and neutrons were 1.69, 1.88, 1.87, 1.94, 2.03, and 1.90, respectively. CONCLUSIONS: The biological parameters measured all indicate an approximately comparable biological effectiveness between 75-80 KeV/microns carbon-ions of the SOBP and 13 MeV neutrons in the human tumor model studied in vitro.
Carbon↗
Analysis of mutations in the human HPRT gene induced by accelerated heavy-ion irradiation.
Multiplex PCR analysis of HPRT(-) mutations in human embryo (HE) cells induced by 230 keV/microns carbon-ion irradiation showed no large deletion around the exon regions of the locus gene in contrast to the irradiations at different LETs. To identify these mutations, the sequence alterations in a cDNA of hprt gene were determined for 18 mutant clones in this study. Missing of exon 6 was the most frequent mutational event (10 clones), and missing of both exons 6 and 8 was next most frequent event (6 clones), then base substitutions (2 clones). These characteristics were not seen in a similar analysis of spontaneous mutations, which showed base substitution (5 clones), frameshift (2 clones), missing of both exons 2 and 3 (2 clones), and a single unidentified clone. Direct sequencing and restriction enzyme digestion of the genomic DNA of the mutants which showed missing of exons 6 and 8 in the cDNA, supports the possibility that they were induced by aberrant mRNA splicing.
Base Sequence↗
Differences in heavy-ion-induced DNA double-strand breaks in a mouse DNA repair-deficient mutant cell line (SL3-147) before and after chromatin proteolysis.
DNA double-strand breaks induced by X- or neon beam-irradiation in a DNA double-strand break-repair-deficient mutant cell line (SL3-147) were examined. The increase in the number of DNA double-strand breaks was dose depend after irradiation with X-rays and neon beams and was enhanced by chromatin-proteolysis treatment before irradiation. These results suggest that the induction of DNA double-strand breaks by ionizing radiation, including heavy-ions, is influenced by the chromatin structure.
Animals↗
Identification of a point mutation in the cDNA of the catalytic subunit of DNA polymerase alpha from a temperature-sensitive mouse FM3A cell line.
DNA polymerase alpha is a key enzyme in eukaryotic chromosomal DNA replication. tsFT20 is a temperature-sensitive mutant cell line derived from mouse mammary carcinoma FM3A cells, and the cells contain heat-labile DNA polymerase alpha and are arrested at the S phase at the nonpermissive temperature. We isolated cDNA of the catalytic subunit of DNA polymerase alpha from tsFT20 cells. DNA sequence analysis revealed that the cDNA from tsFT20 has a single mutation, a cytosine to thymine substitution that changes amino acid 1180 from serine to phenylalanine. We have also shown that tsFT20 cells could be rescued by transfection with the wild-type cDNA. These results demonstrate that the point mutation in the gene of DNA polymerase alpha causes the temperature-sensitive phenotype of tsFT20 cells and provide additional evidence that DNA polymerase alpha is essential for chromosomal replication in mammalian cells. We also detected mutation sites in one spontaneous and six N-methyl-N'-nitro-N-nitrosoguanidine-induced growth revertants of tsFT20 cells by single strand conformation polymorphism analyses and direct sequencing. All revertant cell lines had a second point mutation adjacent to the first mutation site in tsFT20 cells.
Amino Acid Sequence↗
[Carbon beam irradiation of monolayer cells].
This study was performed to determine the biological effect of carbon beams on mammalian cells (HeLa, RMUG), in comparison with 200 KVp X-rays. Carbon beams were generated by the Riken Ring Cyclotron. An increase in the relative biological effect (RBE) was observed in both cell lines as the LET of the carbon beam increased between 20 and 80 keV/microns. The RBE depended on the size of the irradiation dose at the higher LET region, and increasing the dose from 0.8 Gy to 3.2 Gy decreased the RBE from 2.9 to 2.7. The survival curves of cells irradiated with 20 keV/microns carbon beams showed an initial shoulder. When RMUG was irradiated at 4 Gy with 200 KVp X-rays, the recovery rate between two split doses (6 hour interval) was 1.7. RMUG irradiated with 20 keV/microns carbon beams (4 Gy) showed a recovery rate of 1.4. Recovery between split dose irradiation was observed with both X-ray and carbon beam irradiation; however, the recovery rate was lower with carbon beams. Cells irradiated with LET higher than 40 keV/microns did not show recovery between split doses.
Carbon↗
Determination of high LET cosmic particles' trajectories for space radiobiological studies.
During IML-1 mission, we carried out space experiments on radiobiological effect of a single HZE cosmic particle. In the experiment, the precise determination of the distance between the center of the particle trajectory and the individual biological objects around it is an indispensable condition. For the detection of HZE particles CR-39 track detectors were used and analyzed by the video image processing. The positions of biological objects in relation to a particle trajectory were measured by referring to the laser grid marks which were printed on the surface of CR-39 detector. We describe such an experimental method and report the applicability of this method.
Bacillus subtilis↗
Microdosimetric considerations of effects of heavy ions on E. coli K-12 mutants.
The inactivation cross sections of E. coli K-12 recombination-deficient mutants, JC1553 (recA) and AB2470 (recB), for several MeV/u alpha-particles and N ions have been successfully analyzed by Katz's target theory in which radiosensitivity parameter E0 is assumed to be LET independent and equal to D37 for gamma-rays. For E. coli K-12 wild type, AB1157 (rec+, uvr+), however, it is impossible to interpret the inactivation cross section data by an LET-independent E0-value. In the latter case, as in the case of B. subtilis spore, it is necessary to assume that the radiosensitivity of the target for the core of a heavy ion is higher than that for delta-electrons. As well as Waligorski, Hamm and Katz's dose, the dose around the trajectory of an ion based on Tabata and Ito's energy deposition algorithm for electrons has been used in the course of analysis.
Algorithms↗
Radiation-induced chromosome damage in G1 phase cells as breaks in premature chromosome condensation (PCC) and its biological meaning.
We studied radiation-induced chromosome fragmentation in the G1-phase of Syrian golden hamster embryo (SHE) cells by premature chromosome condensation (PCC). SHE cells were irradiated with 14N ions (95 MeV) and 4He ions (22 MeV) generated by the cyclotron and 137Cs gamma rays. The number of chromosome breaks in PCC (PCC break) induced by 14N ions was higher than in those by the others. High linear energy transfer (LET) radiation (heavy ions) is more effective in the induction of PCC breaks than low LET radiation (gamma-rays). 530 kev/microns 14N induced PCC 2.4 times more than 137Cs gamma rays. There is an inverse relationship between the number of chromatid deletions and the incidence of mutation or transformation. There is, however, a good correlation between the incidence of PCC breaks and frequencies of mutation and/or morphological transformation. Although over 95% of the PCC breaks induced by gamma-rays were rejoined within 8 hours of post-irradiation incubation, only 35 to 45% of PCC breaks induced by heavy ions were rejoined. These results suggest that there is a qualitative difference in the initial chromosome damage caused by heavy ions and gamma rays.
Animals↗
Resolution and characterization of polymorphic DNA by SSCP and chemical cleavage methodologies.
A variety of techniques have been developed to detect single-base changes for the two different purposes. One is the detection of mutational events without phenotypic selection, and another is the rapid and conventional identification of mutations such as the specific base changes related to activation of oncogene, genetic diseases, etc. In this study, the utility of the two methods, single strand conformation polymorphism (SSCP) and chemical cleavage, was explored using 13 E. coli lacI- mutations cloned onto M13 phase. The 167 base region encompassing mutations was amplified by PCR as dsDNA. Following denaturation, these PCR products were analyzed by non-denaturing polyacrylamide gel electrophoresis (SSCP) and the separation of the ssDNA fragment carrying the altered sequence from the original sequence was found to be dependent on the location and type of the change. Hetero duplexes of changed/original sequences were also prepared by hybridization of the above PCR products. Mismatched C and T bases were modified by hydroxylamine and osmium tetroxide, respectively, and subsequently treated with piperidine to analyze the cleaved DNA fragments on a polyacrylamide gel (Chemical Cleavage). The cleavage efficiency was also found to be influenced by the type of mismatch and its surrounding sequence. Such observed characteristics should contribute to a better appreciation for these types of mutational systems, which in turn should lead to insight into the mechanisms of mutagenesis.
Base Sequence↗
Specificity of recA441-mediated (tif-1) mutational events.
To investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli. A 2.6-fold enhancement in lacI- mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced. G:C----C:G, G:C----T:A and A:T----T:A transversion events were specifically enhanced after SOS induction. A preferential 5'-Y-Purine-3' neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue). In addition, a preference for transversion events at 5'-C/GTGG-3' sequences is also observed. Fifty events were recovered at the lacI "frameshift hotspot site" and were equally represented by 4 bp addition and deletion events. This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA+ background and is a consequence of the fourfold induction of the (-)4 event. This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI- mutations.
Amino Acids↗
Specificity of SOS mutagenesis in native M13lacI phage.
Base substitutions account for 90% of all forward mutations sequenced in unmodified M13lacI DNA grown in both UV-irradiated and nonirradiated hosts. The principal effect of SOS induction was an increase in the contribution of transversions, in particular A.T----T.A events.
Bacteriophages↗
Specificity of spontaneous mutation in the lacI gene cloned into bacteriophage M13.
We have studied the specificity of spontaneous mutation in the lacI gene of Escherichia coli cloned into bacteriophage M13. The comparison of the spectrum of 85 spontaneous mutations with that of the lacI gene carried on an E. coli F' episone revealed the following characteristics: (i) base substitution was predominant, accounting for 80% of spontaneous events compared with only 11% on the F' episome; (ii) among the base substitutions, the majority were G:C----A:T transitions (86%); (iii) not one mutation recovered on M13 corresponded to a mutation at the spontaneous hotspots seen in the F' spectrum (i.e., neither the addition or deletion of the tetramer 5'-CTGG-3' at position 620-631 nor the A:T----G:C transition at position +6 of lacO were recovered). The enhanced rate of cytosine deamination in single-stranded DNA, the unique replication mechanism and the refractory nature of single-stranded DNA to excision-repair processes present likely explanations for the observed mutational spectrum.
Bacterial Proteins↗
Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein.
The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.
Amino Acid Sequence↗