Performance information

• 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, Nov. 2024, [Reviewed], ［International magazine］
  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.
  English, Scientific journal
  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, Volume：13, Number：11, Sep. 2023, [Reviewed]
  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), Scientific journal
  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, Volume：164, First page：103752, Last page：103752, Jan. 2023, [Reviewed], ［International magazine］
  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.
  English, Scientific journal
  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, Volume：126, Number：11-12, First page：826, Last page：833, Nov. 2022, [Reviewed]
  Elsevier {BV}, English, Scientific journal
  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, Volume：95, Number：6, First page：281, Last page：289, Dec. 2020, [Reviewed]
  English, Scientific journal
  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, Volume：144, Nov. 2020, [Reviewed]
  English, Scientific journal
  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, Volume：216, Number：3, First page：671, Last page：688, Nov. 2020, [Reviewed]
  English, Scientific journal
  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, Volume：124, Number：7, First page：613, Last page：618, Jul. 2020, [Reviewed]
  English, Scientific journal
  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, Volume：94, Number：12, First page：1125, Last page：1133, 2018, [Reviewed]
  English, Scientific journal
  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, Volume：105, First page：28, Last page：36, Aug. 2017, [Reviewed]
  English, Scientific journal
  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, Volume：224, First page：267, Last page：277, 2017
  English, International conference proceedings
  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, Volume：91, Number：6, First page：367, Last page：367, Dec. 2016
  English
  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, Volume：89, Number：6, First page：340, Last page：340, Dec. 2014
  English
  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, Volume：117, Number：4, First page：227, Last page：238, Apr. 2013, [Reviewed]
  English, Scientific journal
  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, Volume：12, Number：2, First page：233, Last page：243, Feb. 2013
  English, Scientific journal
  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, Volume：87, Number：6, First page：407, Last page：407, Dec. 2012
  English
  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, Volume：87, Number：6, First page：425, Last page：425, Dec. 2012
  English
  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, Volume：49, Number：4, First page：263, Last page：270, Apr. 2012
  English, Scientific journal
  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, Volume：29, Number：5, First page：441, Last page：445, 2012
  English, Scientific journal
  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, Volume：86, Number：6, First page：426, Last page：426, Dec. 2011
  English
  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, Volume：86, Number：6, First page：426, Last page：426, Dec. 2011
  English
  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, Volume：85, Number：6, First page：432, Last page：432, Dec. 2010
  English
  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, Volume：85, Number：6, First page：436, Last page：436, Dec. 2010
  English
  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, Volume：47, Number：10, First page：809, Last page：817, Oct. 2010
  English, Scientific journal
  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, Volume：185, Number：4, First page：1257, Last page：1269, Aug. 2010
  English, Scientific journal
  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, Volume：396, Number：2, First page：289, Last page：293, May 2010
  English, Scientific journal
  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, Volume：84, Number：6, First page：438, Last page：438, Dec. 2009
  English
  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, Volume：84, Number：6, First page：462, Last page：462, Dec. 2009
  English
  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, Volume：84, Number：6, First page：437, Last page：437, Dec. 2009
  English
  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, Volume：83, Number：6, First page：480, Last page：480, Dec. 2008
  English
  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, Volume：83, Number：6, First page：480, Last page：480, Dec. 2008
  English
  ISSN：1341-7568, eISSN：1880-5779, Web of Science ID：WOS:000265228400012

• 菌類の事典　　　　　　　　　　　　　　　
  日本菌学会, [Contributor]
  Oct. 2013
  Japanese, Total pages：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, [Contributor], Mutagen response and repair
  Caister Academic Press, Jan. 2013
  Total pages：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
  Sep. 2007
  Japanese, Total pages：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
  Mar. 1998
  Japanese, Total pages：1冊
  CiNii Books：http://ci.nii.ac.jp/ncid/BA40730749
  CiNii Books ID：BA40730749

• Apr. 2005 - Present
  Microbiology, Saitama University
• Apr. 2006 - Mar. 2020
  Genetics, Tokyo Denki University
• Sep. 2005 - Mar. 2020
  Applied microbiology, Tokyo Denki University

• Oct. 2010 - Present, American Society for Microbiology
• Apr. 2005 - Present
• Apr. 2005 - Present, Genetic Society of America
• Apr. 2005 - Present, Genetic Society of Japan

• Mechanism of mtDNA deletion in short lived mutant of Neurospora.　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), 2008 - 2010
  INOUE Hirokazu; TANAKA Shuuitsu; HATAKEYAMA Shin, Saitama University
  Grant amount(Total)：4810000, Direct funding：3700000, Indirect funding：1110000
  The mus-10 mutant was isolated which showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in The mus-10 mutant was isolated which showed highly sensitivity to alkylating agent methylmethane sulfonate (MMS). It had been forecasted that mus-10 gene belonged to the some DNA repair pathway, because of it sensitivity to mutagen. This mutant have other unique characteristics ; unable to grow after several times sequential inoculation, or stop growing after 2 to 3 weeks culture. Furthermore, these phenotypes are accompanied the deletion of mitochondrial DNA and fragmented mitochondrial feature comparing to the normal (tubular) shape in wild type strain. The responsible gene of mus-10 was cloned by complementation of its MMS sensitivity. This gene encodes the F-box domain containing polypeptide, and deletion of F-box domain showed identical phenotype with mus-10 mutant. Since F-box protein is known as a counterpart of SCF (Skp-Cullin-F-box) comlex, which have a role for the degradation of some target protein via ubiquitination following degrading in proteasome.
  To uncover the mus-10 gene function, we focused the feature of mitochondria. We examined whether 1) mitochondrial fusion is inhibited, or 2) mitochondrial fission is stimulated in mus-10 mutant. Double mutation of mus-10 and fis-1, which was essential for mitochondrial fission, showed quite resemble feature of mitochondria with wild type strain. And also this double mutant suppressed sensitivity to mutagen and short life span. These results suggested that MUS-10 protein prevent from the mutagen sensitivity and short life span according to maintain a mitochondrial feature. MUS-10 protein was considered to have a function of degradation of some target protein. So MUS-10 protein should be bound to that protein. Considering MUS-10 protein was correlated to maintenance of mitochondria feature, one candidate FZO-1 arose which had functions in the mitochondrial fusion. Using immunoprecipitation mthod, we could show that MUS-10 protein bound to FZO-1. Next, we tried to make fzo-1 knock out strain, but couldn't. The fzo-1 gene thought to be essential gene and fusion of mitochondria was important for maintenance of life span in Neurospora. Further, we forecasted that constitutive expression of FZO-1 protein in mus-10 mutant might show eviler phenotype than the mus-10 single mutant, because FZO-1, target of MUS-10, might be accumulated in that strain by escaping degradation. However, any evil phenotypes were not observed. Above these results, it was suggested that there were complex mechanism to maintain the mitochondrial feature.
  Grant number：20570001
• Host for highly efficient gene targeting in filamentous fungi　　　　　　　　　　　　　　　
  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), 2006 - 2007
  INOUE Hirokazu; TANAKA Syuuitu; HATAKEYAMA Shin; GOMI Katsuya; ABE Keietsu, Saitama University
  Grant amount(Total)：9420000, Direct funding：8400000, Indirect funding：1020000
  To investigate gene function, most desirable method is gene targeting. However, gene targeting frequency is extremely low in many organisms. To raise the frequency, some trials have been carried out, but conclusive technique was not found.
  Double strand DNA breaks are repaired by two different recombination mechanisms: homologous recombination and nonhomologous end joining. From our study in Neurospora crassa, we speculated that homologous recombination frequency is increased if function of nonhomologous end joining is blocked. We disrupted ku70 and ku80 homolog genes of Neurospora and used them as a host in transformation experiments. In this study, high homologous integration of DNA was observed (Ninomiya, et. al. 2004).
  To develop more convenient host, we disrupted other Neurospora genes involving in nonhomologous end joining; Ligase IV and XRCC4 homolog genes. These mutants showed high targeting frequency even if homologous length of introduced DNA is short. We also tested targeting frequency in MRX-defective strains. MRX is complex of Mre11-Rad50-Xrs2 and functions in double strand breaks repair. Many transformants were from homologous integration, though transformation frequency was low.
  To know whether this system works in other organisms, Aspergillus oryzae was selected for test. Mutant of LigD ( human Lig4 homolog) was constructed in A. oryzae. Targeting experiments using ligD mutant as a host indicated high homologous integration.
  In many other fungi, similar experiments have been coducted by researchers all over the world and they confirmed high targeting efficiency by using ku mutants. as we presented.
  However, our trials in basidiomycetes did not reached to final step since there were unexpected problems in transformation, selection marker, so on. We will continue these experiments for construction of more convenient targeting system.
  Grant number：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　　　　　　　　　　　　　　　
  2005 - 2006
  Grant amount(Total)：3400000, Direct funding：3400000
  Grant number：17651110