業績情報

• A mutation in DNA polymerase γ harbours a shortened lifespan and high sensitivity to mutagens in the filamentous fungus Neurospora crassa.　　　　　　　　　　　　　　　
  Ryouhei Yoshihara; Yuzuki Shimakura; Satoshi Kitamura; Katsuya Satoh; Manami Sato; Taketo Aono; Yu Akiyama; Shin Hatakeyama; Shuuitsu Tanaka
  Genetics, 2024年11月, ［査読有り］, ［国際誌］
  Hyphal elongation is the vegetative growth of filamentous fungi, and many species continuously elongate their hyphal tips over long periods. The details of the mechanisms for maintaining continuous growth are not yet clear. A novel short lifespan mutant of N. crassa that ceases hyphal elongation early was screened and analyzed to better understand the mechanisms for maintaining hyphal elongation in filamentous fungi. The mutant strain also exhibited high sensitivity to mutagens such as hydroxyurea and ultraviolet radiation. Based on these observations, we named the novel mutant "mutagen sensitive and short lifespan 1 (ms1)". The mutation responsible for the short lifespan and mutagen sensitivity in the ms1 strain was identified in DNA polymerase γ (mip-1:NCU00276). This mutation changed the amino acid at position 814 in the polymerase domain from leucine to arginine (MIP-1 L814R). A dosage analysis by next generation sequencing (NGS) reads suggested that mitochondrial DNA (mtDNA) sequences are decreased non-uniformly throughout the genome of the ms1 strain. This observation was confirmed by quantitative PCR for three representative loci and restriction fragment length polymorphisms in purified mtDNA. Direct repeat-mediated deletions, which had been reported previously, were not detected in the mitochondrial genome by our whole-genome sequencing analysis. These results imply the presence of novel mechanisms to induce the non-uniform decrease in the mitochondrial genome by DNA polymerase γ mutation. Some potential reasons for the non-uniform distribution of the mitochondrial genome are discussed in relation to the molecular functions of DNA polymerase γ.
  英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1093/genetics/iyae201
  DOI ID：10.1093/genetics/iyae201, PubMed ID：39611774
• Near-complete de novo assembly of Tricholoma bakamatsutake chromosomes revealed the structural divergence and differentiation of Tricholoma genomes
  Hiroyuki Ichida; Hitoshi Murata; Shin Hatakeyama; Akiyoshi Yamada; Akira Ohta
  G3: Genes, Genomes, Genetics, 巻：13, 号：11, 2023年09月, ［査読有り］
  Abstract
  
  Tricholoma bakamatsutake, which is an edible ectomycorrhizal fungus associated with Fagaceae trees, may have diverged before the other species in Tricholoma section Caligata. We generated a highly contiguous whole-genome sequence for T. bakamatsutake SF-Tf05 isolated in an Oak (Quercus salicina) forest in Japan. The assembly of high-fidelity long reads, with a median read length of 12.3 kb, resulted in 13 chromosome-sized contigs comprising 142,068,211 bases with an average guanine and cytosine (GC) content of 43.94%. The 13 chromosomes were predicted to encode 11,060 genes. A contig (122,566 bases) presumably containing the whole circular mitochondrial genome was also recovered. The chromosome-wide comparison of T. bakamatsutake and Tricholoma matsutake (TMA_r1.0) indicated that the basic number of chromosomes (13) was conserved, but the structures of the corresponding chromosomes diverged, with multiple inversions and translocations. Gene conservation and cluster analyses revealed at least 3 phylogenetic clades in Tricholoma section Caligata. Specifically, all T. bakamatsutake strains belonged to the “bakamatsutake” clade, which is most proximal to the “caligatum” clade consisting of Tricholoma caligatum and Tricholoma fulvocastaneum. The constructed highly contiguous nearly telomere-to-telomere genome sequence of a T. bakamatsutake isolate will serve as a fundamental resource for future research on the evolution and differentiation of Tricholoma species.
  Oxford University Press (OUP), 研究論文（学術雑誌）
  DOI：https://doi.org/10.1093/g3journal/jkad198
  DOI ID：10.1093/g3journal/jkad198, eISSN：2160-1836
• DNA interstrand crosslink repair by XPF-ERCC1 homologue confers ultraviolet resistance in Neurospora crassa.　　　　　　　　　　　　　　　
  Kotaro Tsukada; Shin Hatakeyama; Shuuitsu Tanaka
  Fungal genetics and biology : FG & B, 巻：164, 開始ページ：103752, 終了ページ：103752, 2023年01月, ［査読有り］, ［国際誌］
  Ultraviolet (UV) light is a mutagen that causes DNA damage. Some UV-sensitive Neurospora crassa strains have been reported to exhibit a partial photoreactivation defect (PPD) phenotype, and the possible cause of this has been unknown for more than half a century. In this study, in the process of elucidating the possible causes of a PPD phenotype, we discovered that the XPF homologue MUS-38 is involved in repairing the UV-induced DNA interstrand crosslink (ICL) in N. crassa. Furthermore, the sensitivity of the Δmus-38 and Δmus-44 strains to ICL agents was significantly higher than that of other nucleotide excision repair (NER)-related gene knockout (KO) strains, indicating that the MUS-38/MUS-44 complex is involved in an NER-independent ICL repair mechanism. Based on reports concerning the mammalian homologues XPF and ERCC1 we obtained separation-of-function mutants defective only in NER in mus-38 and mus-44. Additionally, the photoreactivation ability of these mutants was significantly higher than that of the KO strains. These results indicate that the PPD phenotype is caused by a defect in the repair-ability of ICL induced by UV and that an NER-independent ICL repair by MUS-38 and MUS-44 confers resistance to UV in N. crassa.
  英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.fgb.2022.103752
  DOI ID：10.1016/j.fgb.2022.103752, PubMed ID：36435348
• Two high-mobility group domains of MHG1 are necessary to maintain mtDNA in Neurospora crassa　　　　　　　　　　　　　　　
  Hayami Seike; Keisuke Ishimori; Asagi Watanabe; Mao Kiryu; Shin Hatakeyama; Shuuitsu Tanaka; Ryouhei Yoshihara
  Fungal Biology, 巻：126, 号：11-12, 開始ページ：826, 終了ページ：833, 2022年11月, ［査読有り］
  Elsevier {BV}, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.funbio.2022.11.001
  DOI ID：10.1016/j.funbio.2022.11.001, ISSN：1878-6146, ORCID：124588814
• A partial photoreactivation defect phenotype is not due to unrepaired ultraviolet-induced pyrimidine dimers in ultraviolet-sensitive mutants of Neurospora crassa　　　　　　　　　　　　　　　
  Kotaro Tsukada; Ryouhei Yoshihara; Shin Hatakeyama; Akihiko Ichiishi; Shuuitsu Tanaka
  GENES & GENETIC SYSTEMS, 巻：95, 号：6, 開始ページ：281, 終了ページ：289, 2020年12月, ［査読有り］
  Photoreactivation is a mechanism in which photolyase directly repairs either cyclobutane pyrimidine dimers (CPDs) or (6-4) photoproducts [(6-4) PPs] caused by ultraviolet (UV) light. In the filamentous fungus Neurospora crassa, some UV-sensitive mutants such as mus-44 have been reported to exhibit a partial photoreactivation defect (PPD) phenotype, but its mechanism has not been elucidated for a long time. In this study, the N. crassa CPD photolyase PHR was overexpressed in the Delta mus-44 strain, but photoreactivation ability was not increased. Furthermore, Escherichia coli CPD photolyase or Arabidopsis thaliana (6-4) PP photolyase was also introduced into Delta mus-44; however, the PPD phenotype was not complemented. These results suggested that the PPD phenotype in N. crassa is not caused by residual unrepaired pyrimidine dimers, which are the main type of DNA damage caused by UV irradiation. Finally, we revealed that Delta mus-44, but not the Delta mus-43 strain, which does not show the PPD phenotype, displayed higher sensitivity with increasing dose rate of UV. Moreover,Delta mus-44 was also sensitive to an interstrand crosslinking agent. This indicates that the high dose of UV in our experimental condition induces DNA damage other than pyrimidine dimers, and that such damage is a likely cause of the PPD phenotype.
  GENETICS SOC JAPAN, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1266/ggs.20-00022
  DOI ID：10.1266/ggs.20-00022, ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000657317200001
• The msh1 gene is responsible for short life span mutant natural death and functions to maintain mitochondrial DNA integrity　　　　　　　　　　　　　　　
  Mitsuyoshi Endo; Takato Yokoi; Suguru Hatazawa; Yuna Kojima; Shiena Takahama; Ryouhei Yoshihara; Shuuitsu Tanaka; Shin Hatakeyama
  FUNGAL GENETICS AND BIOLOGY, 巻：144, 2020年11月, ［査読有り］
  Wild-type filamentous fungus Neurospora crassa continues to grow its hyphae for a very lengthy period of time (>2 years), whereas mutations at the natural death (nd) locus shorten life span (approximately 20 days). By positional cloning based on heat augmented mutagen sensitivity of the nd strain, we identified a nonsense mutation in the msh1 gene, an eukaryotic homolog of bacterial MutS, and this mutation resulted in encoding nonfunctional polypeptide. By tagging with GFP, subcellular localization of the MSH1 protein in the mitochondria was observed, and knock out of the msh1 gene caused severe growth deficiency accompanying mitochondrial DNA (mtDNA) aberrations such as large-scale mtDNA deletions and rearrangements as seen in the nd strain. These results suggested that MSH1 may maintain mtDNA integrity. Thus, loss of function compromises mtDNA, leading to the acceleration of cellular aging.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.fgb.2020.103465
  DOI ID：10.1016/j.fgb.2020.103465, ISSN：1087-1845, eISSN：1096-0937, Web of Science ID：WOS:000599899100004
• Selection and Characterization of Mutants Defective in DNA Methylation in Neurospora crassa　　　　　　　　　　　　　　　
  Andrew D. Klocko; Calvin A. Summers; Marissa L. Glover; Robert Parrish; William K. Storck; Kevin J. McNaught; Nicole D. Moss; Kirsten Gotting; Aurelian Stewart; Ariel M. Morrison; Laurel Payne; Shin Hatakeyama; Eric U. Selker
  GENETICS, 巻：216, 号：3, 開始ページ：671, 終了ページ：688, 2020年11月, ［査読有り］
  DNA methylation, a prototypical epigenetic modification implicated in gene silencing, occurs in many eukaryotes and plays a significant role in the etiology of diseases such as cancer. The filamentous fungus Neurospora crassa places DNA methylation at regions of constitutive heterochromatin such as in centromeres and in other A:T-rich regions of the genome, but this modification is dispensable for normal growth and development. This and other features render N. crassa an excellent model to genetically dissect elements of the DNA methylation pathway. We implemented a forward genetic selection on a massive scale, utilizing two engineered antibiotic-resistance genes silenced by DNA methylation, to isolate mutants defective in methylation (dim). Hundreds of potential mutants were characterized, yielding a rich collection of informative alleles of 11 genes important for DNA methylation, most of which were already reported. In parallel, we characterized the pairwise interactions in nuclei of the DCDC, the only histone H3 lysine 9 methyltransferase complex in Neurospora, including those between the DIM-5 catalytic subunit and other complex members. We also dissected the N- and C-termini of the key protein DIM-7, required for DIM-5 histone methyltransferase localization and activation. Lastly, we identified two alleles of a novel gene, dim-10 - a homolog of Clr5 in Schizosaccharomyces pombe - that is not essential for DNA methylation, but is necessary for repression of the antibiotic-resistance genes used in the selection, which suggests that both DIM-10 and DNA methylation promote silencing of constitutive heterochromatin.
  GENETICS SOCIETY AMERICA, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1534/genetics.120.303471
  DOI ID：10.1534/genetics.120.303471, ISSN：0016-6731, eISSN：1943-2631, Web of Science ID：WOS:000591372800008
• Analysis of localization of cell-cycle regulators in Neurospora crassa　　　　　　　　　　　　　　　
  Kazuki Kuwabara; Ryouhei Yoshihara; Shin Hatakeyama; Shuuitsu Tanaka
  FUNGAL BIOLOGY, 巻：124, 号：7, 開始ページ：613, 終了ページ：618, 2020年07月, ［査読有り］
  Most fungi are multinucleated organisms. In some fungi, they have asynchronous nuclei in the same cytoplasm. We analyzed a cell-cycle regulation mechanism using a model fungus Neurospora crassa, which can make heterokaryon cells. G1/S cyclin CLN-1 and cyclin-dependent kinase CDC-2 were tagged with different fluorescence in different strains and expressed. By forming a heterokaryon strain of these, two different fluorescence-tagged proteins were expressed in the same cytoplasm. CDC-2 was localized in all nuclei, whereas CLN-1 was not detected in most of the nuclei and was dispersed in the cytoplasm with small granular clusters. This indicates that in multinucleated fungi, cell-cycle regulators, similar to other proteins, are shared around the nuclei regardless of different cell-cycle stages. Moreover, each nucleus can select and use a special cell-cycle regulator only when it is necessary. Fungal nuclei may have a novel pickup mechanism of necessary proteins from their cytoplasm at the point of use. (C) 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
  ELSEVIER SCI LTD, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.funbio.2020.02.017
  DOI ID：10.1016/j.funbio.2020.02.017, ISSN：1878-6146, eISSN：1878-6162, Web of Science ID：WOS:000541532100001
• LET dependence on killing effect and mutagenicity in the model filamentous fungus Neurospora crassa　　　　　　　　　　　　　　　
  Liqiu Ma; Yusuke Kazama; Tomonari Hirano; Ryouhei Morita; Shuuitsu Tanaka; Tomoko Abe; Shin Hatakeyama
  INTERNATIONAL JOURNAL OF RADIATION BIOLOGY, 巻：94, 号：12, 開始ページ：1125, 終了ページ：1133, 2018年, ［査読有り］
  Purpose: To assess the unique biological effects of different forms of ionizing radiation causing DNA double-strand breaks (DSBs), we compared the killing effect, mutagenesis frequency, and mutation type spectrum using the model filamentous fungus Neurospora.Materials and methods: Asexual spores of wild-type Neurospora and two DSB repair-deficient strains [one homologous recombination- and the other non-homologous end-joining (NHEJ) pathway-deficient] were irradiated with argon (Ar)-ion beams, ferrous (Fe)-ion beams, or X-rays. Relative biological effectiveness (RBE), forward mutation frequencies at the ad-3 loci, and mutation spectra at the ad-3B gene were determined.Results: The canonical NHEJ (cNHEJ)-deficient strain showed resistance to higher X-ray doses, while other strains showed dose-dependent sensitivity. In contrast, the killing effects of Ar-ion and Fe-ion beam irradiation were dose-dependent in all strains tested. The rank order of RBE was Ar-ion > Fe-ion > C-ion. Deletion mutations were the most common, but deletion size incremented with the increasing value of linear energy transfer (LET).Conclusions: We found marked differences in killing effect of a cNHEJ-deficient mutant between X-ray and high-LET ion beam irradiations (Ar and Fe). The mutation spectra also differed between irradiation types. These differences may be due to the physical properties of each radiation and the repair mechanism of induced damage in Neurospora crassa. These results may guide the choice of irradiation beam to kill or mutagenize fungi for agricultural applications or further research.
  TAYLOR & FRANCIS LTD, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1080/09553002.2019.1524940
  DOI ID：10.1080/09553002.2019.1524940, ISSN：0955-3002, eISSN：1362-3095, Web of Science ID：WOS:000455198200006
• Phenotypic analysis of newly isolated short-lifespan Neurospora crassa mutant deficient in a high mobility group box protein　　　　　　　　　　　　　　　
  Ryouhei Yoshihara; ZhengHao Li; Keisuke Ishimori; Kazuki Kuwabara; Shin Hatakeyama; Shuuitsu Tanaka
  FUNGAL GENETICS AND BIOLOGY, 巻：105, 開始ページ：28, 終了ページ：36, 2017年08月, ［査読有り］
  To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2 weeks of cultivation, despite that of the wild-type continuing for over 2 years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhgl(KO) strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhgl(KO) strain than in the wild-type. mtDNA aberration was also observed in the mhgl(KO) strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhgl(KO) strain.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.fgb.2017.06.001
  DOI ID：10.1016/j.fgb.2017.06.001, ISSN：1087-1845, eISSN：1096-0937, Web of Science ID：WOS:000406827300004
• BASIC STUDY ON THE DECAYED BEHAVIOR OF WASTE WOODY SAMPLES CAUSED BY THREE WHITE-ROT FUNGI　　　　　　　　　　　　　　　
  Qingyue Wang; Hideharu Takahashi; Sayuri Kawamura; Shin Hatakeyama
  ENERGY AND SUSTAINABILITY VIII, 巻：224, 開始ページ：267, 終了ページ：277, 2017年
  In this study, we investigated the decayed behavior of waste woody samples caused by three kinds of white-rot fungi known as Coriolus consors, Coriolus hirsutus and Ischnoderma resinosum, which were selected to prepare and develop the decayed woody samples from original woody biomasses. After 8 weeks of decaying, the weights of all decayed woody samples had decreased. From the results of analysis of the major woody holocellulose and lignin, the same ratios of weight decrement in all decayed woody samples were found when compared with their original woody sample. It seemed that these components had degraded simultaneously. The crystallinities of woody samples had increased even though woody samples were decayed. It was thought that the amorphous parts of cellulose and hemicellulose were decomposed preferentially. We tried to use these decayed woody samples as samples for the liquefaction experiment. It is observed that the decayed woody samples may be liquefied to be the same as their original woody samples basing on the molecular weight distribution, chemical structure and other chemical parameters of the liquefied contents.
  WIT PRESS, 英語, 研究論文（国際会議プロシーディングス）
  DOI：https://doi.org/10.2495/ESUS170251
  DOI ID：10.2495/ESUS170251, ISSN：1743-3541, Web of Science ID：WOS:000450031900026
• Function of Neurospora crassa MSH1 in the maintenance of mitochondria DNA　　　　　　　　　　　　　　　
  Yokoi Takato; Yuna Kojima; Shuitsu Tanaka; Shin Hatakeyama
  GENES & GENETIC SYSTEMS, 巻：91, 号：6, 開始ページ：367, 終了ページ：367, 2016年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000405886000194
• Analysis of the natural death mutant of Neurospora crassa　　　　　　　　　　　　　　　
  Mitsuyoshi Endo; Syuuitsu Tanaka; Shin Hatakeyama
  GENES & GENETIC SYSTEMS, 巻：89, 号：6, 開始ページ：340, 終了ページ：340, 2014年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000357360600247
• The type of mutations induced by carbon-ion-beam irradiation of the filamentous fungus Neurospora crassa　　　　　　　　　　　　　　　
  Liqiu Ma; Yusuke Kazama; Hirokazu Inoue; Tomoko Abe; Shin Hatakeyama; Shuuitsu Tanaka
  FUNGAL BIOLOGY, 巻：117, 号：4, 開始ページ：227, 終了ページ：238, 2013年04月, ［査読有り］
  Heavy-ion beams are known to cause great damage to cellular components and are particularly renowned for their ability to generate DNA double-strand breaks (DSBs). To gain insight into the mutagenic effect of carbon-ion beams and how such damage is repaired by the cell, Neurospora crassa mutants deficient in one of three components involved in the repair of DSBs, nonhomologous end-joining (NHEJ), homologous recombination repair (HR), and the Mre11-Rad50-Xrs2 (MRX) complex, were irradiated with a carbon-ion beam and killing effect, mutation frequencies, and the type of mutation incurred by survivors were analysed. The sensitivity of the NHEJ-deficient strain (mus-52) was higher than that of the wild-type and the HR-deficient (mei-3) strains at low doses of radiation, but was little changed as the level increased. As a result both the wild-type and HR-deficient strains were more sensitive than the NHEJ-deficient strain at high radiation levels. In addition, the frequency of forward mutation at the adenine-3 (ad-3) loci of the NHEJ-deficient mutant was lower than that of the wild-type strain at all levels, while the mutation frequency of the HR-deficient strain was consistently similar to 3-fold higher than the wild-type. From the comparison of mutation type of each strain, deletions were frequently observed in wild-type strain, whilst base substitution and deletion in the mus-52 and mei-3 strains. These mutations resulting from carbon-ion-beam irradiation depend on the mechanism invoked to cope with DSBs. Furthermore, in wild-type cells, these mechanisms likely compete to repair DSBs. (c) 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
  ELSEVIER SCI LTD, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.funbio.2013.01.002
  DOI ID：10.1016/j.funbio.2013.01.002, ISSN：1878-6146, Web of Science ID：WOS:000318961600001
• A uvs-5 Strain Is Deficient for a Mitofusin Gene Homologue, fzo1, Involved in Maintenance of Long Life Span in Neurospora crassa　　　　　　　　　　　　　　　
  Kiminori Kurashima; Michael Chae; Hirokazu Inoue; Shin Hatakeyama; Shuuitsu Tanaka
  EUKARYOTIC CELL, 巻：12, 号：2, 開始ページ：233, 終了ページ：243, 2013年02月
  Mitochondria are highly dynamic organelles that continuously fuse and divide. To maintain mitochondria, cells establish an equilibrium of fusion and fission events, which are mediated by dynamin-like GTPases. We previously showed that an mus-10 strain, a mutagen-sensitive strain of the filamentous fungus Neurospora crassa, is defective in an F-box protein that is essential for the maintenance of mitochondrial DNA (mtDNA), long life span, and mitochondrial morphology. Similarly, a uvs-5 mutant accumulates deletions within its mtDNA, has a shortened life span, and harbors fragmented mitochondria, the latter of which is indicative of an imbalance between mitochondrial fission and fusion. Since the uvs-5 mutation maps very close to the locus of fzo1, encoding a mitofusin homologue thought to mediate mitochondrial outer membrane fusion, we determined the sequence of the fzo1 gene in the uvs-5 mutant. A single amino acid substitution (Q368R) was found in the GTPase domain of the FZO1 protein. Expression of wild-type FZO1 in the uvs-5 strain rescued the mutant phenotypes, while expression of a mutant FZO1 protein did not. Moreover, when knock-in of the Q368R mutation was performed on a wild-type strain, the resulting mutant displayed phenotypes identical to those of the uvs-5 mutant. Therefore, we concluded that the previously unidentified uvs-5 gene is fzo1. Furthermore, we used immunoprecipitation analysis to show that the FZO1 protein interacts with MUS-10, which suggests that these two proteins may function together to maintain mitochondrial morphology.
  AMER SOC MICROBIOLOGY, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1128/EC.00226-12
  DOI ID：10.1128/EC.00226-12, ISSN：1535-9778, eISSN：1535-9786, Web of Science ID：WOS:000314213000009
• Morphological change of mitochondria contribute mutagen hypersensitivity and lifespan in Neurospora crassa　　　　　　　　　　　　　　　
  Kiminori Kurashima; Michael Chae; Hirokazu Inoue; Shin Hatakeyama; Shuuitsu Tanaka
  GENES & GENETIC SYSTEMS, 巻：87, 号：6, 開始ページ：407, 終了ページ：407, 2012年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000323535700139
• Killing effect and mutagenesis of Fe-ion beam irradiation in Neurospora crassa　　　　　　　　　　　　　　　
  Ma Liqiu; Yusuke Kazama; Tomoko Abe; Shuuitsu Tanaka; Shin Hatakeyama
  GENES & GENETIC SYSTEMS, 巻：87, 号：6, 開始ページ：425, 終了ページ：425, 2012年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000323535700209
• Neurospora mrc1 homologue is involved in replication stability and is required for normal cell growth and chromosome integrity in mus-9 and mus-21 mutants　　　　　　　　　　　　　　　
  Michiyoshi Wakabayashi; Nobuyuki Saijyou; Shin Hatakeyama; Hirokazu Inoue; Shuuitsu Tanaka
  FUNGAL GENETICS AND BIOLOGY, 巻：49, 号：4, 開始ページ：263, 終了ページ：270, 2012年04月
  Stalled replication forks easily collapse and such structures can induce DNA strand Breaks or toxic recombination products. Therefore, factors involved in stabilization of replication should be important for genome integrity. In our previous study, loss of both ATM and ATR homologues was shown to cause growth defects and chromosome instability in Neurospora crassa. To elucidate the relationships between these defects and replication instability, we focused on one of the viable replication factors, mrc1. We identified an mrc1 homologue from the N. crassa genome database. The mrc1 disruptant was sensitive to the replication inhibitor hydroxyurea (HU) and delayed restart of the cell cycle from HU treatment. Importantly. HU treatment induced histone H2A phosphorylation in the mrc1 mutant but not in I he wild type. Furthermore, the HU-induced H2A phosphorylation was completely dependent on the AT: VI homologue mus-21, and dysfunction of mus-21 increased HU sensitivity of the mrc1 mutant. These results indicate that Neurospora mrc1 is important for stabilization of replication forks and that loss of mrc1 causes activation of the DNA damage checkpoint. Unexpectedly, loss of mrc1 did not affect cell growth, but the deletion of mrc1 reduced hyphal growth speed and conidia viability in the mus-9 and mus-21 mutants. The mrc1 mus-9 and mrc1 mus-21 double mutants also showed accumulation of micronuclei, which is a typical marker of chromosome instability. These results imply that activation of the checkpoint pathway can protect cells from instability of DNA replication caused by loss of mrc1. (C) 2012 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.fgb.2012.02.007
  DOI ID：10.1016/j.fgb.2012.02.007, ISSN：1087-1845, eISSN：1096-0937, Web of Science ID：WOS:000302763500001
• Rapid evaluation of effective linear energy transfer in heavy-ion mutagenesis of Arabidopsis thaliana　　　　　　　　　　　　　　　
  Yusuke Kazama; Liqiu Ma; Tomonari Hirano; Sumie Ohbu; Yuki Shirakawa; Shin Hatakeyama; Shuuitsu Tanaka; Tomoko Abe
  PLANT BIOTECHNOLOGY, 巻：29, 号：5, 開始ページ：441, 終了ページ：445, 2012年
  Heavy-ion beams are an effective mutagen for use in plant breeding and analyses of gene function. Heavy-ion 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 M-1 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 mu m(-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 mu m(-1) did not effectively induce mutations, regardless of the dose; second, LETs of 30.0 or 42.5 keV mu m(-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 mu m(-1) produced comparatively high mutation frequencies at all doses. Irradiation with 42.5-61.5 keV mu m(-1) reduced seed productivity at the dose providing the highest mutation frequencies (300 Gy). We concluded that an LET of 30.0 keV mu m(-1) was optimal for obtaining Arabidopsis thaliana mutants in the M-2 generation.
  JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.5511/plantbiotechnology.12.0921a
  DOI ID：10.5511/plantbiotechnology.12.0921a, ISSN：1342-4580, Web of Science ID：WOS:000315161100003
• Mutagen sensitive strains and their morphological abnormalities of mitochondria in Neurospora crassa　　　　　　　　　　　　　　　
  Kiminori Kurashima; Michael Chae; Shuuitsu Tanaka; Shin Hatakeyama
  GENES & GENETIC SYSTEMS, 巻：86, 号：6, 開始ページ：426, 終了ページ：426, 2011年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000303700800173
• C-, Fe-, Ar-ion beams-induced killing effects and forward mutations in the ad-3 region of Neurospora crassa　　　　　　　　　　　　　　　
  Ma Liqiu; Yusuke Kazama; Tomoko Abe; Syuuitsu Tanaka; Shin Hatakeyama
  GENES & GENETIC SYSTEMS, 巻：86, 号：6, 開始ページ：426, 終了ページ：426, 2011年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000303700800174
• Analysis of DNA integration in Neurospora crassa　　　　　　　　　　　　　　　
  Junko Funayama; Tomoyuki Murakami; Shin Hatakeyama; Syuithu Tanaka
  GENES & GENETIC SYSTEMS, 巻：85, 号：6, 開始ページ：432, 終了ページ：432, 2010年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000290243200144
• The role of delta-lyase dependent base exsion repair in vivo　　　　　　　　　　　　　　　
  Hikaru Hashimoto; Shin Hatakeyama; Shuuitu Tanaka
  GENES & GENETIC SYSTEMS, 巻：85, 号：6, 開始ページ：436, 終了ページ：436, 2010年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000290243200158
• ATM and ATR homologes of Neurospora crassa are essential for normal cell growth and maintenance of chromosome integrity　　　　　　　　　　　　　　　
  Michiyoshi Wakabayashi; Chizu Ishii; Shin Hatakeyama; Hirokazu Inoue; Shuuitsu Tanaka
  FUNGAL GENETICS AND BIOLOGY, 巻：47, 号：10, 開始ページ：809, 終了ページ：817, 2010年10月
  Genome integrity is maintained by many cellular mechanisms in eukaryotes. One such mechanism functions during the cell cycle and is known as the DNA damage checkpoint. In the filamentous fungus Neurospora crassa, mus-9 and mus-21 are homologes of two key factors of the mammalian DNA damage checkpoint, AIR and ATM, respectively. We previously showed that mus-9 and mus-21 mutants are sensitive to DNA damage and that each mutant shows a characteristic growth defect: conidia from the mus-9 mutant have reduced viability and the mus-21 mutant exhibits slow hyphal growth. However, the relationship between these two genes has not been determined because strains carrying both mus-9 and mus-21 mutations could not be obtained. To facilitate analysis of a strain deficient in both mus-9 and mus-21, we introduced a specific mutation to the kinase domain of MUS-9 to generate a temperature-sensitive mus-9 allele (mus-9(ts)) which shows increased mutagen sensitivity at 37 degrees C. Then we crossed this strain with a mus-21 mutant to obtain a mus-9(ts) mus-21 double mutant. Growth of the mus-9(ts) mus-21 double mutant did not progress at the restrictive temperature (37 degrees C). Even at the permissive temperature (25 degrees C), this strain exhibited a higher mutagen sensitivity than that of the mus-9 and mus-21 single mutants, as well as slow hyphal growth and low viability of conidia. These results indicate that the mus-9(ts) mutation causes hypomorphic phenotypes in the mus-21 mutant and that these two genes regulate different pathways. Interestingly, we observed accumulation of micronuclei in the conidia of this double mutant, and such micronuclei were likely to correlate with spontaneous DSBs. Our results suggest that both mus-9 and mus-21 pathways are involved in DNA damage response, normal growth and maintenance of chromosome integrity, and that at least one of the pathways must be functional for survival. (C) 2010 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.fgb.2010.05.010
  DOI ID：10.1016/j.fgb.2010.05.010, ISSN：1087-1845, eISSN：1096-0937, Web of Science ID：WOS:000281999900003
• Deletion of a Novel F-Box Protein, MUS-10, in Neurospora crassa Leads to Altered Mitochondrial Morphology, Instability of mtDNA and Senescence　　　　　　　　　　　　　　　
  Akihiro Kato; Kiminori Kurashima; Michael Chae; Satoshi Sawada; Shin Hatakeyama; Shuuitsu Tanaka; Hirokazu Inoue
  GENETICS, 巻：185, 号：4, 開始ページ：1257, 終了ページ：1269, 2010年08月
  While mitochondria are renowned for their role in energy production, they also perform several other integral functions within the cell. Thus, it is not surprising that mitochondrial dysfunction can negatively impact cell viability. Although mitochondria have received an increasing amount of attention in recent years, there is still relatively little information about how proper maintenance of mitochondria and its genomes is achieved. The Neurospora crassa mus-10 mutant was first identified through its increased sensitivity to methyl methanesulfonate (MMS) and was thus believed to be defective in some aspect of DNA repair. Here, we report that mus-10 harbors fragmented mitochondria and that it accumulates deletions in its mitochondrial DNA (mtDNA), suggesting that the mus-10 gene product is involved in mitochondrial maintenance. Interestingly, mus-10 begins to senesce shortly after deletions are visualized in its mtDNA. To uncover the function of MUS-10, we used a gene rescue approach to clone the mus-10 gene and discovered that it encodes a novel F-box protein. We show that MUS-10 interacts with a core component of the Skp, Cullin, F-box containing (SCF) complex, SCON-3, and that its F-box domain is essential for its function in vivo. Thus, we provide evidence that MUS-10 is part of an E3 ubiquitin ligase complex involved in maintaining the integrity of mitochondria and may function to prevent cellular senescence.
  GENETICS SOCIETY AMERICA, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1534/genetics.110.117200
  DOI ID：10.1534/genetics.110.117200, ISSN：0016-6731, eISSN：1943-2631, Web of Science ID：WOS:000281907700011
• High efficient gene targeting on the AGAMOUS gene in an Arabidopsis AtLIG4 mutant　　　　　　　　　　　　　　　
  Shuuitsu Tanaka; Chizu Ishii; Shin Hatakeyama; Hirokazu Inoue
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 巻：396, 号：2, 開始ページ：289, 終了ページ：293, 2010年05月
  Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell. (C) 2010 Elsevier Inc. All rights reserved.
  ACADEMIC PRESS INC ELSEVIER SCIENCE, 英語, 研究論文（学術雑誌）
  DOI：https://doi.org/10.1016/j.bbrc.2010.04.082
  DOI ID：10.1016/j.bbrc.2010.04.082, ISSN：0006-291X, Web of Science ID：WOS:000278658000019
• The role of Neurospora crassa pnk gene in repair of alkylated DNA damage　　　　　　　　　　　　　　　
  Hikaru Hashimoto; Shin Hatakeyama; Hirokazu Inoue; Shuuitsu Tanaka
  GENES & GENETIC SYSTEMS, 巻：84, 号：6, 開始ページ：438, 終了ページ：438, 2009年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000277071300025
• Research of DNA Damage repair pathways induced by ion beam　　　　　　　　　　　　　　　
  Liqiu Ma; Shuuitsu Tanaka; Hirokazu Inoue; Yusuke Kazama; Hiroyuki Ichida; Tomoko Abe; Shin Hatakeyama
  GENES & GENETIC SYSTEMS, 巻：84, 号：6, 開始ページ：462, 終了ページ：462, 2009年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000277071300118
• Two mechanisms related to genome stabilization of Neurospora crassa　　　　　　　　　　　　　　　
  Wakabayashi Michiyosh; Nobuyuki Saijyo; Shin Hatakeyama; Hirokazu Inoue; Shuuitsu Tnaka
  GENES & GENETIC SYSTEMS, 巻：84, 号：6, 開始ページ：437, 終了ページ：437, 2009年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000277071300024
• Neurospora crassa mus-16 gene encodes Saccharomyces cerevisiae Rtt109 homolog　　　　　　　　　　　　　　　
  Shin Hatakeyama; Inamori Atsuko; Yokoyama Mika; Han Yunxia; Tanaka Shuuitsu; Inoue Hirokazu
  GENES & GENETIC SYSTEMS, 巻：83, 号：6, 開始ページ：480, 終了ページ：480, 2008年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000265228400011
• Novel gene of which mutation causes the hyphal growth defect, encodes the F-box protein in Neurospora　　　　　　　　　　　　　　　
  Kiminori Kurashima; Akihiro Kato; Satoshi Sawada; Shin Hatakeyama; Shuuitsu Tanaka; Hirokazu Inoue
  GENES & GENETIC SYSTEMS, 巻：83, 号：6, 開始ページ：480, 終了ページ：480, 2008年12月
  GENETICS SOC JAPAN, 英語
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000265228400012

• 菌類の事典　　　　　　　　　　　　　　　
  日本菌学会, ［分担執筆］, 遺伝的組換え、RIP
  朝倉書店, 2013年10月
  日本語, 総ページ数：xv, 717p, 図版16p
  CiNii Books：http://ci.nii.ac.jp/ncid/BB13962148
  ISBN：9784254171471, CiNii Books ID：BB13962148
• Neurospora: Genomics and Molecular Biology　　　　　　　　　　　　　　　
  Durgadas P. Kasbekar; Kevin Mccluskey, ［分担執筆］
  Caister Academic Press, 2013年01月
  総ページ数：294
  ISBN：1908230126
• 再生医療工学の技術 (CMCテクニカルライブラリー)　　　　　　　　　　　　　　　
  筏, 義人
  シーエムシー出版, 2007年09月
  日本語, 総ページ数：251
  CiNii Books：http://ci.nii.ac.jp/ncid/BA83168881
  ISBN：4882319373, ASIN：4882319373, EAN：9784882319375, CiNii Books ID：BA83168881
• アカパンカビにおける除去修復系の解析　　　　　　　　　　　　　　　
  畠山, 晋
  畠山晋, 1998年03月
  日本語, 総ページ数：1冊
  CiNii Books：http://ci.nii.ac.jp/ncid/BA40730749
  CiNii Books ID：BA40730749

• 2005年04月 - 現在
  微生物学, 埼玉大学
• 2006年04月 - 2020年03月
  遺伝学, 東京電機大学
• 2005年09月 - 2020年03月
  応用微生物学, 東京電機大学

• 2010年10月 - 現在, アメリカ微生物学会
• 2005年04月 - 現在, 糸状菌分子生物学コンファレンス
• 2005年04月 - 現在, 米国遺伝学会
• 2005年04月 - 現在, 日本遺伝学会

• 早期に細胞死をもたらす突然変異のミトコンドリアDNA欠失生成のメカニズム　　　　　　　　　　　　　　　
  日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 2008年 - 2010年
  井上 弘一; 田中 秀逸; 畠山 晋, 埼玉大学
  配分額（総額）：4810000, 配分額（直接経費）：3700000, 配分額（間接経費）：1110000
  アカパンカビmus-10変異株はDNAのアルキル化剤であるMMSに対する高感受性株として単離された。変異原に高感受性を示すことからmus-10はDNA修復に関わることが予想されたが、その詳細は明らかではない。解析を進める中でこの株は数回の植継ぎで成長を停止し、それに伴ってミトコンドリアDNAに欠失が生じることが分かった。また、ミトコンドリア形態は野生株が管状であるのに対しmus-10変異株では粒状であった。原因遺伝子はF-box ドメインをもつタンパク質をコードしており、MUS-10タンパク質の機能にF-boxドメインは必須であることからユビキチン/プロテアソーム系を介したタンパク質分解経路で働くことが示唆された(Kato et al. 2010)。
  MUS-10の機能をさらに解析するため、ミトコンドリア形態に注目し研究を行った。mus-10変異株ではミトコンドリアが断片化していたため、ミトコンドリアの融合がうまくいかない、もしくは、分裂が亢進していることが考えられる。ミトコンドリアの分裂に必須な遺伝子であるfis-1とmus-10の二重欠損株を作製した結果、二重欠損株ではミトコンドリア形態が野生株と似た形状を示しただけでなく、変異原高感受性や生育の早期停止もみられなくなった。このことはMUS-10タンパク質がミトコンドリア形態の維持に機能することにより、変異原高感受性や短寿命を防いでいることを示唆する。MUS-10はタンパク質分解経路で働き、分解される標的タンパク質と結合することが予想される。そのため、MUS-10と既知のミトコンドリア形態制御因子との結合をみた結果、ミトコンドリアの融合に関わるFZO-1と結合することが明らかとなった。FZO-1がMUS-10の分解の標的となっているかを調べるため、二重欠損株の作製を試みたが、fzo-1欠損株を作製することが出来ず、fzo-1は生育に必須である可能性が高い。また、mus-10変異株においてFZO-1を構成的に発現させることを試みた。
  MUS-10がFZO-1の分解に働くのであれば、mus-10変異株ではFZO-1が蓄積しており、構成的に発現させることにより表現型の悪化がみられることが予想される。しかし、構成的なFZO-1の発現によりmus-10のミトコンドリア形態の異常、変異原高感受性、生育の早期停止がみられなくなった。以上より、MUS-10のミトコンドリア形態維持機構にはより複雑な仕組みがあることが示唆される。
  課題番号：20570001
• 効率的遺伝子ターゲッティングのための糸状菌における宿主の開発　　　　　　　　　　　　　　　
  日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 2006年 - 2007年
  井上 弘一; 田中 秀逸; 畠山 晋; 五味 勝也; 阿部 啓悦, 埼玉大学
  配分額（総額）：9420000, 配分額（直接経費）：8400000, 配分額（間接経費）：1020000
  多くの生物でゲノム解読が進み、コンピューターによる遺伝子のアノテーションが行なわれているが、遺伝子の機能を知るには知りたい遺伝子を破壊して突然変異株を得ることが最も確実な方法である。そこで、多くの生物では本来生物が持っている相同組換え機構を利用し、細胞外から破壊したい遺伝子と相同部分を持つDNAを導入し、相同部分での組み換えにより遺伝子を破壊する方法が試みられてきた。しかしながら、多くの場合DNAは相同部分以外にランダムにゲノムに挿入されてしまう。我々は、ランダムに組み込まれる際に必要なタンパク質を作っている遺伝子(KU70およびKU80)を破壊することで100%相同部分に組み込むことに成功した。本研究では(1)KU以外の非相同組換えに関わる遺伝子を破壊し、ターゲット効率を更に上げること、(2)アカパンカビでこれまで試みてきた方法が他の糸状菌で同じ様に使えるかどうかの2点にしぼって研究を行った。その結果(1)KU以外でヒトのLig4ホモログ遺伝子やXRCC4ホモログ遺伝子を破壊しても高い率でターゲットができることを確認した。また(2)コウジ菌でもアカパンカビと同様に、LigD破壊株で効率のターゲットができることがあきらかになった。これからはこの方法を使用し自由に細胞内の遺伝子の改変ができるため、これらの結果の意味するところは極めて大きい。キノコについても同じ様に実験が行なわれたが、予想のできなかった困難のため、さらにここしばらくの実験が必要である。しかしながら基本的にはこの方法が有効であると思われるため引き続き実験を行なって行く予定である。現在、アカパンカビおよびコウジカビではこの方法をもとにしたシステマティックな遺伝子破壊が行なわれている。
  課題番号：18370001
• 担子菌の標的遺伝子を高効率かつ高選択性をもって破壊する方法の開発　　　　　　　　　　　　　　　
  日本学術振興会, 科学研究費助成事業 萌芽研究, 萌芽研究, 2005年 - 2006年
  畠山 晋; 井上 弘一, 埼玉大学
  配分額（総額）：3400000, 配分額（直接経費）：3400000
  1.Phanerochaete chrysosporiumのおける非相同末端結合関連遺伝子のクローニング
  平成17年度において、非相同末端結合に関わる遺伝子をゲノムデータベースを用いて探索したところ、K70、K80、Lig4のホモログの存在が明らかとなった。この遺伝子を含むゲノムDNAのクローニングをPCR法によって行なった。現在、下で述べるマーカーをこの遺伝子の読み枠に挿入し、相同的組み換えによる遺伝子破壊のためのコンストラクトを作製している。
  2.遺伝子導入ベクターの開発
  Phanerochaete chrysosporiumにおいて構成的に発現している、GPD遺伝子(glyceraldehyde-3-phosphate dehydrogenase)のプロモーター領域とターミネーター領域をクローニングし、これらによってハイグロマイシン耐性遺伝子の構造遺伝子を挟み込んだコンストラクトを作製した。このDNA断片をマーカー遺伝子として用い、遺伝子破壊のためのマーカー、および、遺伝子導入方法の最適化のための形質転換実験に用いた。
  3.遺伝子導入方法の検討
  3-1 コロニー形成培地の検討
  Phanerochaete chrysosporiumの遺伝子導入を確認するために、容易に観察できるコロニーを形成できる培地の組成を検討した。その結果として、糸状菌の最小培地として用いられるVogelの培地をベースにした培地が良好なコロニーを形成した。さらにソルボースとトリプトンの濃度を最適化した。また、薬剤耐性の選択において用いるハイグロマイシンの濃度も最適化し、形質転換体を選択するための培地条件の最適化に成功した。
  3-2 電気穿孔法による遺伝子導入の最適化
  担子菌の遺伝子導入において一般的ではない電気穿孔法の条件を最適化した。アカパンカビにおける電気穿孔法の条件から、電圧、抵抗を段階的に変化させて遺伝子導入の最適条件を探索した。また、Phanerochaete chrysosporiumの分生子の発芽時間と菌糸細胞壁の再生時間がリンクするという予想のもとに復帰培養に要する時間の検討も行なった。その結果として、電気穿孔法による遺伝子導入から復帰培養についての最適時間を決定することができた。
  課題番号：17651110