Yuki Shirakawa

RIKEN, Вако, Saitama, Japan

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Publications (4)11.46 Total impact

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    ABSTRACT: The heavy-ion beam is widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy-ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, an evaluation at the whole-genome level or of detailed genomic rearrangements in the mutant genomes has not been performed. In this study, we comprehensively characterized the mutations in Ar- or Fe-ion irradiated Arabidopsis thaliana genomes using array comparative genomic hybridization (array-CGH) and resequencing, and subsequently used this information to investigate the mutagenic effects of the heavy-ion beams. Array-CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar-ion and Fe-ion irradiation, respectively, with deletion sizes ranging from 149 to 602,180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the Ar-ion beam induced mutants were resequenced, and total mutations; including base substitutions, duplications, in/dels, inversions, and translocations; were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiple rearranged regions. These results indicated that the heavy-ion beams led to clustered DNA damage in the chromosome, and that they have a great potential to induce complicated intrachromosomal rearrangements. Heavy-ion beams will prove useful as unique mutagens for plant breeding and establishing mutant lines. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    The Plant Journal 02/2015; 82(1):93-104. DOI:10.1111/tpj.12793 · 5.97 Impact Factor
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    ABSTRACT: Heavy-ion beams are powerful mutagens. They cause a broad spectrum of mutation phenotypes with high efficiency even at low irradiation doses and short irradiation times. These mutagenic effects are due to dense ionisation in a localised region along the ion particle path. Linear energy transfer (LET; keV·μm(-1)), which represents the degree of locally deposited energy, is an important parameter in heavy-ion mutagenesis. For high LET radiation above 290 keV∙μm(-1), however, neither the mutation frequency nor the molecular nature of the mutations has been fully characterised. In this study, we investigated the effect of Fe-ion beams with an LET of 640 keV∙μm(-1) on both the mutation frequency and the molecular nature of the mutations. Screening of well-characterised mutants (hy and gl) revealed that the mutation frequency was lower than any other ion species with low LET. We investigated the resulting mutations in the 4 identified mutants. Three mutants were examined by employing PCR-based methods, one of which had 2-bp deletion, another had 178 bp of tandemly duplication, and other one had complicated chromosomal rearrangements with variable deletions in size at breakpoints. We also detected large deletions in the other mutant by using array comparative genomic hybridisation. From the results of the analysis of the breakpoints and junctions of the detected deletions, it was revealed that the mutants harboured chromosomal rearrangements in their genomes. These results indicate that Fe-ion irradiation tends to cause complex mutations with low efficiency. We conclude that Fe-ion irradiation could be useful for inducing chromosomal rearrangements or large deletions.
    Genes & Genetic Systems 09/2013; 88(3):189-97. DOI:10.1266/ggs.88.189 · 0.93 Impact Factor
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    ABSTRACT: Heavy-ion beams are an effective mutagen for use in plant breeding and analyses of gene function. Heavyion beams have high linear energy transfer (LET) and thus they effectively induce DNA double-strand breaks. To rapidly evaluate effective LETs for inducing mutations, we investigated mutation frequencies in the M1 generation using the Arabidopsis heterozygous mutant of the ALBINO PALE GREEN 3 gene. Leaves of heterozygous plants in which intact alleles were mutated showed white sectors. We irradiated heterozygous plants with heavy-ion beams with LETs ranging from 22.5 to 61.5 keV μm−1 and doses ranging from 0 to 450 Gy. The results showed that the effect of LETs could be classified into three types: first, the LET of 22.5 keV μm−1 did not effectively induce mutations, regardless of the dose; second, LETs of 30.0 or 42.5 keV μm−1 were not effective at low doses (100–200 Gy) but were effective at 300 Gy; third, LETs of 50.0 or 61.5 keV μm−1 produced comparatively high mutation frequencies at all doses. Irradiation with 42.5–61.5 keV μm−1 reduced seed productivity at the dose providing the highest mutation frequencies (300 Gy). We concluded that an LET of 30.0 keV μm−1 was optimal for obtaining Arabidopsis thaliana mutants in the M2 generation.
    Plant Biotechnology 11/2012; 29(5):441-445. DOI:10.5511/plantbiotechnology.12.0921a · 0.87 Impact Factor
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    ABSTRACT: Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124keVμm(-1). In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVμm(-1). We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M(2) pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVμm(-1) radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0keVμm(-1) and 27% for 101-124keVμm(-1)). Therefore, the 290keVμm(-1) heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 05/2012; 735(1-2):19-31. DOI:10.1016/j.mrfmmm.2012.04.010 · 3.68 Impact Factor