新規in vivo可視化技術を用いた真核細胞における遺伝情報発現機構の解析

• Project/Area Number: 13GS0013
• Research Category: Grant-in-Aid for Creative Scientific Research
• Allocation Type: Single-year Grants
• Research Institution: Yokohama City University
• Principal Investigator: 古久保 哲朗 横浜市立大学, 大学院・国際総合科学研究科, 教授 (10271587)
• Co-Investigator(Kenkyū-buntansha): 白川 昌宏 京都大学, 工学研究科, 教授 (00202119)
• Project Period (FY): 2001 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥340,210,000 (Direct Cost: ¥282,640,000、Indirect Cost: ¥57,570,000); Fiscal Year 2005: ¥78,650,000 (Direct Cost: ¥60,500,000、Indirect Cost: ¥18,150,000); Fiscal Year 2004: ¥80,080,000 (Direct Cost: ¥61,600,000、Indirect Cost: ¥18,480,000); Fiscal Year 2003: ¥90,740,000 (Direct Cost: ¥69,800,000、Indirect Cost: ¥20,940,000); Fiscal Year 2002: ¥90,740,000 (Direct Cost: ¥90,740,000)
• Keywords: 転写調節 / モニタリング / 遺伝子発現 / イメージング / NMR / MRI / ポリリン酸 / レポーター遺伝子

Research Abstract

すべての生命現象は必要な遺伝情報がプログラム通りに正しく発現することにより支えられている。遺伝子発現プログラムにおいて最も重要な制御段階は「転写」であり、その制御反応に関与する蛋白質の多くはすでに同定されたと考えられるが、それらの生体内における作用機構の詳細は依然として明らかではない。本研究の目的は、生物個体における遺伝子発現を非破壊的に計測する新規手法の開発を行い、その手法を用いて真核細胞の転写制御を支える分子的基盤及びその作動原理を明らかにすることである。
無機リン酸のポリマーであるポリリン酸は、全ての生物に普遍的に存在し、^<31>P-NMRによる定量的なマイクロイメージングが可能であることから、遺伝子発現量をモニターする上で非常に有効なプローブになりうるものと考えられる。これまで我々は、出芽酵母、動物培養細胞、動物・植物個体を宿主とし、ポリリン酸合成酵素(または合成・蓄積に関与するタンパク質)を新規レポーター遺伝子として用いてプロモーター活性を効率よくモニタリングできる手法の開発を進めてきた。今年度は、^<31>P-MRI測定系の検出限界を打破するべく、T_1緩和時間の変化を^1H-MRIにより画像化する新規モニタリング手法の開発を行った。別途開発を進めてきた自動コロニースポッターと組み合わせることにより、少なくとも出芽酵母においては、極めて短時間に上流活性化配列(UAS)、及びコアプロモーター配列の決定を行うことが可能となった。

Research Products

• [Journal Article] A novel magnetic resonance-based method to measure gene expression in living cells (2006)
  • Author(s): S.Ki, F.Sugihara, K.Kasahara, H.Tochio, A.Okada-Marubayashi, S.Tomita, M.Morita, M.Ikeguchi, M.Shirakawa, T.Kokubo
  • Journal Title: Nucleic Acids Research (印刷中)
• [Journal Article] Crystal structure of SUMO-3-modified thymine-DNA glycosylase (2006)
  • Author(s): D.Baba, N.Maita, J.G.Jee, Y.Uchimura, H.Saitoh, K.Sugasawa, F.Hanaoka, H.Tochio, H.Hiroaki, M.Shirakawa
  • Journal Title: Journal of Molecular Biology (印刷中)
• [Journal Article] Resonance assignments of 30 kDa complexes of TFIID subunit TAF1 with TATA-binding protein (2005)
  • Author(s): TK.Mal, D.Liu, Y.Masutomi, L.Zheng, Y.Nakatani, T.Kokubo, M.Ikura
  • Journal Title: Journal of Biomolecular NMR 33・1 Pages: 76-76
• [Journal Article] Crystal structure of thymine DNA glycosylase conjugated to SUMO-1 (2005)
  • Author(s): D.Baba, N.Maita, J.G.Jee, Y.Uchimura, H.Saitoh, K.Sugasawa, F.Hanaoka, H.Tochio, H.Hiroaki, M.Shirakawa
  • Journal Title: Nature 435・7044 Pages: 979-982
• [Journal Article] Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae (2005)
  • Author(s): C.Auesukaree, H.Tochio, M.Shirakawa, Y.Kaneko, S.Harashima
  • Journal Title: Journal of Biological Chemistry 280・26 Pages: 25127-33
• [Journal Article] In vivo synthesis of Taf1p lacking the TAF N-terminal domain using alternative transcription or translation initiation sites. (2004)
  • Author(s): K.Kasahara, M.Kawaichi, T.Kokubo
  • Journal Title: Genes to Cells 9・8 Pages: 709-721
• [Journal Article] Autonomous function of the amino-terminal inhibitory domain of TAF1 in transcriptional regulation. (2004)
  • Author(s): S.Takahata, K.Kasahara, M.Kawaichi, T.Kokubo
  • Journal Title: Molecular and Cellular Biology 24・8 Pages: 3089-3099
• [Journal Article] Structure of the N-terminal domain of PEX1 AAA-ATPase : characterization of a putative adaptor-binding domain (2004)
  • Author(s): Shiozawa, K.他(全9名中、Shirakawa, M.第8名)
  • Journal Title: Journal of Biological Chemistry 279・48 Pages: 50060-50068
• [Journal Article] Structural basis for distinct roles of Lys63- and Lys48-linked polyubiquitin chains. (2004)
  • Author(s): Tenno, T.他(全10名中、Shirakawa, M.第10名)
  • Journal Title: Genes to Cells 9・10 Pages: 865-875
• [Journal Article] Structure of human MTH1, a Nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates. (2004)
  • Author(s): Mishima, M.他(全10名中、Shirakawa, M.第10名)
  • Journal Title: Journal of Biological Chemistry 279・32 Pages: 33806-33815
• [Journal Article] Structure of the UBA domain of Dsk2p in complex with Ubiquitin : Molecular Determinants for ubiquitin Recognition
  • Author(s): Ohno, A., 他(全9名中、Shirakawa, M.第9名)
  • Journal Title: Structure (印刷中)
• [Publications] S.Takahata, K.Kasahara, M.Kawaichi, T.Kokubo: "Autonomous function of the amino-terminal inhibitory domain of TAF1 in transcriptional regulation."Molecular and Cellular Biology. 24・8(in press). (2004)
• [Publications] A.Kobayashi, T.Kokubo, Y.Ota, S.Yokoyama: "Promoter-specific function of the TATA element in undifferentiated P19 cells."Biochem.Biophys.Res.Commun.. 310・2. 458-463 (2003)
• [Publications] S.Takahata, H.Ryu, K.Ohtsuki, K.Kasahara, M.Kawaichi, T.Kokubo: "Identification of a novel TATA element-binding protein binding region at the N terminus of the Sacchaoomyces cerevisiae TAF1 protein."Journal of Biological Chemistry. 278・46. 45888-45902 (2003)
• [Publications] H.Ohdate, C.R.Lim, T.Kokubo, K-i.Matsubara, Y.Kimata, K.Kohno: "Impairment of the DNA-binding activity of the TATA-binding protein (TBP) renders the transcriptional function of Rvb2p/Tih2p, the yeast RuvB-like protein, essential for cell growth."Journal of Biological Chemistry. 278・17. 14647-14656 (2003)
• [Publications] A.Kobayashi, T.Miyake, M.Kawaichi, T.Kokubo: "Mutations in the histone fold domain of the TAF12 gene show synthetic lethality with the TAF1 gene lacking the TAF N-terminal domain (TAND) by different mechanisms from those in the SPT15 gene encoding the TATA binding protein (TBP)."Nucleic Acids Research. 31・4. 1261-1274 (2003)
• [Publications] C.Auesukaree, T.Homma, H.Tochio, M.Shirakawa, Y.Kaneko, S.Harashima: "Intracellular phosphate serves as a signal for the regulation of the PHO pathway in Saccaromyces cerevisiae"Journal of Biological Chemistry. (in press). (2004)
• [Publications] K.Fujiwara, T.Tenno, K.Sugasawa, J.-G.Jee, I.Ohki, C.Kojima, H.Tochio, H.Hiroaki, F.Hanaoka, M.Shirakawa: "Structure of the ubiquitin-interacting motif of S5a bound to the ubiquitin-like domain of HR23B"Journal of Biological Chemistry. 279・6. 4760-4767 (2004)
• [Publications] N.Goda, T.Tenno, H.Takasu, H.Hiroaki, M.Shirakawa: "The PRESAT-vector. Asymmetric T-vector for high throughput screening of soluble protein domains for structural proteomics"Protein Science. 13・3. 652-658 (2004)
• [Publications] Z.Kato, J.-G.Jee, H.Shikano, M.Mishima, I.Ohki, H.Ohnishi, A.Li, K.Hashimoto, E.Matsukuma, K.Omoya, Y.Yamamoto, T.Yoneda, T.Hara, N.Kondo, M.Shirakawa: "The structure and binding mode of interleukin-18"Nature Structural Biology. 10・11. 966-971 (2003)
• [Publications] M.Mishima, S.Takayama, K.Sasaki, J.-G.Pee, C.Kojima, A.Isogai, M.Shirakawa: "Structure of the male determinant factor for Brassica self-incompatibility"Journal of Biological Chemistry. 278・38. 36389-36395 (2003)
• [Publications] S.Watanabe, T.Ichimura, N.Fujita, S.Tsuruzoe, I.Ohki, M.Shirakawa, M.Kawasuji, M.Nakao: "Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link transcriptional repression and DNA repair in chromatin"Proceedings of National Academy of Sciences USA. 100・22. 12859-12864 (2003)
• [Publications] Kobayashi, A., Akasaka, K., Kawaichi, M., Kokubo, T: "Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus"Nucleic Acids Research. 30・14. 3034-3044 (2002)
• [Publications] Kobayashi, A., Miyake, T., Kawaichi, M., Kokubo, T: "Mutations in the histone fold domain of the TAF12 gene show synthetic lethality with the TAF1 gene lacking the TAF N-terminal domain (TAND) by different mechanisms from those in the SPT15 gene encoding the TATA binding protein (TBP)"Nucleic Acids Research. 31・4. 1261-1274 (2003)
• [Publications] Takahashi, M., Maraboeuf, F., Sakai, Y., Yakushiji, H., Mishima, M., 他: "Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein"Journal of Molecular Biology. 319. 129-139 (2002)
• [Publications] Sasaki, Y., Furuichi, M., Takahashi, M., Mishima, M., Iwai, S., 他: "A molecular basis for the selective recognition of 2-hydroxy-dATP and 8-oxo-dGTP by human MTH1"Journal of Biological Chemistry. 277. 8579-8587 (2002)
• [Publications] Kouno, T., Miura, K., Kanematsu, T., Shirakawa, M., Hirata, M., 他: "1H, 13C and 15N resonance assignments of GABARAP, GABAA receptor associated protein"Journal of Biomolecular NMR. 22. 97-98 (2002)
• [Publications] Yoshihiro Tsukihashi: "Requiement for yeast TAF145 function in transcriptional activation of the RPS5 promoter that depends on both core promoter structure and upstream activating sequences"Journal of Biological Chemistry. 276・28. 25715-25726 (2001)