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2007 Fiscal Year Final Research Report Summary

Host for highly efficient gene targeting in filamentous fungi

Research Project

Project/Area Number 18370001
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Genetics/Genome dynamics
Research InstitutionSaitama University

Principal Investigator

INOUE Hirokazu  Saitama University, Graduate School of Science and Engineering, Professor (60114203)

Co-Investigator(Kenkyū-buntansha) TANAKA Syuuitu  Saitama University, Graduate School of Science and Engineering, Associate Professor (90202431)
HATAKEYAMA Shin  Saitama University, Material and Life Research Center, Lecturer (00396665)
GOMI Katsuya  Tohoku University, Agriculture, Professor (60302197)
ABE Keietsu  Tohoku University, Agriculture, Associate Professor (50312624)
Project Period (FY) 2006 – 2007
KeywordsNonhomologous end joining / Gene targeting / KU70 / KU80 / Ligase IV / MRX / Neurospora / Aspergillus
Research Abstract

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.

  • Research Products

    (34 results)

All 2008 2007 2006 Other

All Journal Article (13 results) (of which Peer Reviewed: 10 results) Presentation (20 results) Remarks (1 results)

  • [Journal Article] A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improve efficiency of gene targeting in Aspergillus oryzae2008

    • Author(s)
      O. Mizutani et. al.
    • Journal Title

      Fungal Genetics and Biology 45

      Pages: 878-889

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] The Neurospora crassa UVS-3 epistasis group encodes homologues of the ATR/ATRIP checkpoint control system.2008

    • Author(s)
      Y. Kazama et. al.
    • Journal Title

      DNA Repair 7

      Pages: 213-229

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] A defect of LigD (human Lig4 homolog) for nonhomologous end Joining significantly improves efficiency of gene-targeting in Aspergillus oryzae2008

    • Author(s)
      Mizutani, o., Y. Kudo, A,. Saito, T. Matuura, H. Inoue, K. Abe, and K. Gomi.
    • Journal Title

      Fungal Genet. Biol 45

      Pages: 878-889

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Genetic and molecular analysis of the temperature-sensitive mutant un-17 carrying a mutation in the gene encoding poly (A)-polymerse in Neurospora crassa2007

    • Author(s)
      S. Tanaka et. al.
    • Journal Title

      Genes Genet. Syst. 82

      Pages: 447-454

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Detection of physical interactions by immunoprecipitation of FLAG-and HA-tagged proteins expressed at the his-3 locus in Neurospora crassa2007

    • Author(s)
      T. Kawabata and H. Inoue
    • Journal Title

      Fungal Genet. Newslett. 54

      Pages: 5-8

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] MpkA-dependent and-independent cell wall integrity signaling in Aspergillus nidulans2007

    • Author(s)
      T. Fujioka et. al.
    • Journal Title

      Eukaryotic Cells 6

      Pages: 1497-1510

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Neurospora crassa RAD5 homologue, mus-41, inactivation results in higher sensitivity to mutagens but little effect on PCNA ubiquitilqtion in response to UV-irradiation2007

    • Author(s)
      T. Kawabata et. al.
    • Journal Title

      Current Genetics 52

      Pages: 125-135

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Nonhomologous chromosomal integration of foreign DNA is completely Dependent on MUS-53 (human Lig4 homolog) in Neurospora2007

    • Author(s)
      Ishibashi et. al.
    • Journal Title

      Proc. Natl. Acad. Sci. USA 103

      Pages: 14871-14876

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Neurospora crassa RAD5 homologue, mus-41, inactivation results in higher sensitivity to mutagens but has little effect on PCNA-ubiquitilation in response to UV-iradiation2007

    • Author(s)
      Kawabata, T., A. Kato, K. Suzuki, and H. Inoue.
    • Journal Title

      Current Genetics 52

      Pages: 125-135

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Human RAD18 is improved in S phase-specific single-strand break repair without PCNA monoubiquitination2006

    • Author(s)
      N. Shiomi et. al.
    • Journal Title

      Nucleic Acids Res. 35

      Pages: e9

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Functional analysis of an endo-1, 6-β-D-glucanase gene (neg-1) from Neurospora crassa2006

    • Author(s)
      S. Oyama et. al.
    • Journal Title

      Biosci. Biotechnol. Biochem. 70

      Pages: 1773-1775

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Growth defect and mutator phenotypes of RecQ-deficient Neurospora crassa mutants separately result from homologous recombination and nonhomologous end joining during repair of DNA double-strand breaks2006

    • Author(s)
      A. Kato and H. Inoue
    • Journal Title

      Genetics 172

      Pages: 113-125

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Nonhomologous chromosomal integration of foreign DNA is Completely dependent on MUS-53 (human Lig4 homolog) in Neurospora2006

    • Author(s)
      Ishibashi, K., K. Suzuki, Y. Ando, C. Takakura, and H. Inoue.
    • Journal Title

      Proc. Natl. Acad. Sci. USA 103

      Pages: 14871-18476

    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Genome-wide analysis of fungicide-response genes regulated via his-asp phosphorelay system and hogA MAPK cascade in Aspergillus nidulans2008

    • Author(s)
      D. Hagiwara
    • Organizer
      9th European Conference on Fungal Genetics
    • Place of Presentation
      Edinburgh, England
    • Year and Date
      20080400
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Aspergillus属糸状菌のCell wall integrity (CWI) signalingと細胞壁合成2008

    • Author(s)
      阿部啓悦
    • Organizer
      日本農芸化学会
    • Place of Presentation
      名古屋大学
    • Year and Date
      20080300
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] アカパンカビにおける遺伝子ターゲッティング2007

    • Author(s)
      高倉千裕
    • Organizer
      日本分子生物学会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      20071200
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Mitogen-activated protein kinase pathways in Aspergillus oryzae2007

    • Author(s)
      Abe, K., T. Fujioka, K. Fukukawa, D. Hagiwara, J. Marui, O. Mizutani, N. Sato, A. Yoshimi, F. Hasegawa
    • Organizer
      ESF-EMBO symposium Comparative genoraics on eukaryotic microorganisms: Eukaryotic genome evolution
    • Place of Presentation
      in Spain
    • Year and Date
      20071020-25
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Mitogen-activated protein kinase (MAPK) pathways in Aspergillus oryzae2007

    • Author(s)
      K. Abe
    • Organizer
      ESF-EMBO symposium
    • Place of Presentation
      Sant de Guixols, Spain
    • Year and Date
      20071000
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] 糸状菌ゲノム情報にもとづく抗真菌剤創薬分子基盤解析システムの構築2007

    • Author(s)
      丸井淳一朗
    • Organizer
      第6回感染症沖縄フォーラム
    • Place of Presentation
      沖縄
    • Year and Date
      20070200
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] アカパンカビにおけるPOL32ホモログの損傷乗り越え合成での役割2007

    • Author(s)
      瓜尾政博
    • Organizer
      第79回日本遺伝学会
    • Place of Presentation
      岡山大学
    • Year and Date
      2007-09-20
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] アカパンカビMUS-53(LIG4ホモログ)の遺伝子ターゲッティングにおける有用性2006

    • Author(s)
      石橋和真
    • Organizer
      日本分子生物学会2006フォーラム
    • Place of Presentation
      名古屋国際会議場
    • Year and Date
      20061200
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] 麹菌のDNAligase (lig4)遺伝子破壊による高頻度組換え宿主の造成2006

    • Author(s)
      水谷治
    • Organizer
      第6回糸状菌分子生物学コンフェレンス
    • Place of Presentation
      大阪府立大学
    • Year and Date
      20061100
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] DNA repair and Genomic Instability in Neurospora crassa2006

    • Author(s)
      H. Inoue
    • Organizer
      NEUROSPORA 2008
    • Place of Presentation
      Asilomar, California, USA
    • Year and Date
      20060300-04
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] DNA repair and genomic instability in Neurospora crassa2006

    • Author(s)
      Hirokazu Inoue
    • Organizer
      2006 Neurospora meeting
    • Place of Presentation
      in Asilomar, CA
    • Year and Date
      20060300
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] アカパンカビのポリ(A)ポリメラーゼ変異株un-172006

    • Author(s)
      田中秀逸
    • Organizer
      第78回日本遺伝学会
    • Place of Presentation
      つくば国際会議場
    • Year and Date
      2006-09-27
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] 糸状菌における遺伝子ターゲッティング機構の解析2006

    • Author(s)
      井上弘一
    • Organizer
      第78回日本遺伝学会
    • Place of Presentation
      つくば国際会議場
    • Year and Date
      2006-09-26
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] アカパンカビにおける遺伝子ターゲッティング機構の解析2006

    • Author(s)
      高倉千裕
    • Organizer
      第78回日本遺伝学会
    • Place of Presentation
      つくば国際会議場
    • Year and Date
      2006-09-26
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] アカパンカビPCNAの紫外線に応答したユビキチン化について2006

    • Author(s)
      川端剛
    • Organizer
      第78回日本遺伝学会
    • Place of Presentation
      つくば国際会議場
    • Year and Date
      2006-09-26
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Establishment of gene-targeting method in fungi2006

    • Author(s)
      Hirokazu Inoue
    • Organizer
      78 th Genetic Society of Japan
    • Year and Date
      2006-09-26
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Analysis of gene targeting mechanism in Neurospora2006

    • Author(s)
      Chihiro Takakura, Yosinori Ando, Kazuma Ishibashi, Keiichiro Suzuki, and Hirokazu Inoue
    • Organizer
      78 th Genetic Society of Japan
    • Year and Date
      2006-09-26
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] 糸状菌の効率的遺伝子破壊法:宿主としての非相同組換え欠損株2006

    • Author(s)
      井上弘一
    • Organizer
      第58回日本生物工学会
    • Place of Presentation
      大阪大学豊中キャンパス
    • Year and Date
      2006-09-12
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] 麹菌(A. oryzae)のligD遺伝子破壊による高頻度相同組換え宿主の造成2006

    • Author(s)
      工藤洋平
    • Organizer
      第58回日本生物工学会
    • Place of Presentation
      大阪大学豊中キャンパス
    • Year and Date
      2006-09-12
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Construction of host for high homologous recombination in Aspergillus oryzae2006

    • Author(s)
      Youhei Kudo, Osamu Mizutani, Tomoki Matuura, Hirokazu Inoue, Keietsu Abe, and Katsuya Gomi
    • Organizer
      58 th Society for Biotechnology in Japan
    • Year and Date
      2006-09-12
    • Description
      「研究成果報告書概要(欧文)」より
  • [Remarks] 「研究成果報告書概要(和文)」より

    • URL

      http://genet.seitai.saitama-u.ac.jp/

URL: 

Published: 2010-02-04  

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