1995 Fiscal Year Annual Research Report

酵母を用いた蛋白間相互作用因子のクローニング法の確立

Research Project

Project/Area Number	06557010
Research Institution	Nagoya City University Medical School
Principal Investigator	岡本尚名古屋市立大学, 医学部, 教授 (40146600)
Co-Investigator(Kenkyū-buntansha)	久下周佐東京大学, 医科学研究所, 助手 (50186376) 加藤宏幸北海道大学, 薬学部, 助教授 (60185866)
Keywords	蛋白・蛋白相互作用 / two-hybrid system / NFkB / Tat / PTB
Research Abstract	蛋白分子間相互作用の解析は、生命現象の分子レベルでの解明に必要かつ不可欠のものである。申請者らは、酵母を用いた人為的な分子遺伝学的検出系を用いて、蛋白間相互作用をもとに未知の蛋白分子をコードする遺伝子を効率よくクローニングする方法を確立した。研究対象は、AIDSの原因ウイルスであるHIVの増殖に必須な2種類の転写因子NFkBとTatおよびPTB(polypyrimidine tract binding protein)とし、酵母のtwo-hybrid実験系によりこれら既存の蛋白分子と結合する未知の蛋白分子の同定と遺伝子クローニングを行ってきた。NFkB(岡本)又はTat(加藤)とDNA結合能を持つGal4 subdomain(Gal4 db)の融合蛋白を発現するレポーター酵母にCDNAをLiAC法によって移入することによって行った。また、PTB結合蛋白のクローニングにはSRFのDNA結合領域(SRF-db)を利用した(久下)。これらの酵母細胞を用いてHuman HeLa S3 Match-maker CDNA libraryをスクリーニングした。この操作を繰り返しそれぞれ107以上のCDNAクローンにつきスクリーニングした結果、NFkB結合蛋白(岡本)、Tat結合蛋白(加藤)およびPTB結合蛋白(久下)のCDNAクローンを分離した。現在、これらの遺伝子につき、相互作用の特異性を確認すると共に、塩基配列決定、など基礎的情報を調べているところである。

Research Products
(16 results)

All Other

All Publications (16 results)

[Publications] Okamoto.T.: "Regulation of HIV replication by a host transcription factor NFkB." Jap J Med Science Bio. 47. 120-121 (1994)
[Publications] Sachi,Y.,Hirota,K.,Masutani,H.,Toda,K.,Okamoto,T.Takigawa,M.,and Yodoi,J.: "Iduction of ADF/TRX by Oxidative Stress in Keratinocytes and Lumhoid cells" Immunology Lett.44. 189-193 (1995)
[Publications] Scheffer,U.,Okamoto,T.,Forrest,J.M.S.,Muller,W.E.G.,and Schroder,H.C.: "Interaction of 68-kDa TAR-binding Protein and other cellular proteins with protein-RNA stem-loop.(in press)." (1995)
[Publications] Yang,J.P.,Merin,J.p.,Nakano,T.,Kato,T.,Kitade,Y.,Okamoto,T.: "Inhibition of the DNA-binding activity of NF-kB by gold compounds in vitro." FEBS Lett,. 361. 89-96 (1995)
[Publications] Yamada,K.,Mrozumi,H.,and Okamoto,T.: "LTR-directed homologous recombination of full-length HIV-1 provirus clone in recA(-) bacteria." Arch,Virol.140. 1007-1014 (1995)
[Publications] Kira,T.,J.P,Merin.,Baba,M.,Shigeta,S.,and Okamoto,T.: "Anti-Tat MTT assay:ANovel Anti-HIV Drug Screening System Using the Viral logic of Replication." AIDS Res.Hum.Retrovir. 11. 1359-1566 (1996)
[Publications] Tozawa,K.,Sakurada,S.,Kohri,k.,and Okamoto,T.:"Effects of Anti-NFkB Reagents in Biocking Adhesion of Human Cancer Cells to Vascular Endothelial Cells." Cancer Res.55. 4162-4167 (1995)
[Publications] Hohashi,N.,Hayashi,T.,Fesaki,N.,Takeuchi,M.,Higurashi,M.,Okamoto,T.,Semba,K.,and Yamamoto,T.: "The Protein tyrosine kinase Fyn activates transcription from the HIV promoter via activation of NFkB-like DNA-binding proteins." Int.Immunol.11. 1851-1859 (1995)
[Publications] Yokomizo,A.,Ono M.,Nanri,H.,Mariko,Y.,Ohga,T.,Wada M.,Okamoto,T.,Yodoi,J.,Kuwano,M.,and Kohno,K.: "Cellular levels of thioredoxin associated with drug sensitivity to cisplatin,mitomycin C,doxocubicin," Cancer Res.55;4293-4296,1955.55. 4293-4296 (1995)
[Publications] Iida,T.,Suzumori,K.,Ikuta,K.,Tenemura,M,Yagami,Y.,Okamoto.T.,and Hata,A.: "Identification of a Gly^<862> to Ser substitution in the type 1 collagen gene from a single sperm.(in press)," Hum.Reprod.(1996)
[Publications] Okamoto,T.,Shinnsaku,S.,Jian-Ping Yang.,and Jocelyn P.Merin.: "Regulation of NF-kB and Disease Control:Identification of a Novel Serine Kinase and Thioredoxin as Effectors for Signal Transduction Pathway for NF-kB Activation.Current Topics in Cellular Regulation.(in press)" (1996)
[Publications] Chiba,H.,Muramatsu,M.,Nomoto,A.and Kato,H.: "Two human homologue of Saccharomvces corevialae SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid reseptor." Nucleic Acids Res.,. 22. 1815-1820 (1994)
[Publications] Shusuke KUGE and Nicolas JONES.: "YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides." EMBO J.13. 655-664 (1994)
[Publications] Okamoto.T.and Hayashi,T.:Redox regulation of HIV gene expression.Proceedings for the Conference on the Development of Antioxidant and Chelating Drugs for the Treatment of Enviroment-associated Diseases(in press)., (1995)
[Publications] Okamoto.T.:Regulatory Proteins of Human Immmodeficiency Virus and Therapy.Anti-AIDS Drug Development: Challenge Strategies and Prospects(Eds.,Mohan and Baba)Harwood Publishers Inc., 11 (1995)
[Publications] Okamoto,T.:Redox regulation of NF-kB and disease control (a review article)Current Topics in Cellular Regulation (eds.by Stadtman,E.R.,and Chock,p.B.),Academic Press,(in press)., (1996)

1995 Fiscal Year Annual Research Report

酵母を用いた蛋白間相互作用因子のクローニング法の確立

Principal Investigator

岡本 尚 名古屋市立大学, 医学部, 教授 (40146600)

Research Products

[Publications] Okamoto.T.: "Regulation of HIV replication by a host transcription factor NFkB." Jap J Med Science Bio. 47. 120-121 (1994)

[Publications] Sachi,Y.,Hirota,K.,Masutani,H.,Toda,K.,Okamoto,T.Takigawa,M.,and Yodoi,J.: "Iduction of ADF/TRX by Oxidative Stress in Keratinocytes and Lumhoid cells" Immunology Lett.44. 189-193 (1995)

[Publications] Scheffer,U.,Okamoto,T.,Forrest,J.M.S.,Muller,W.E.G.,and Schroder,H.C.: "Interaction of 68-kDa TAR-binding Protein and other cellular proteins with protein-RNA stem-loop.(in press)." (1995)

[Publications] Yang,J.P.,Merin,J.p.,Nakano,T.,Kato,T.,Kitade,Y.,Okamoto,T.: "Inhibition of the DNA-binding activity of NF-kB by gold compounds in vitro." FEBS Lett,. 361. 89-96 (1995)

[Publications] Yamada,K.,Mrozumi,H.,and Okamoto,T.: "LTR-directed homologous recombination of full-length HIV-1 provirus clone in recA(-) bacteria." Arch,Virol.140. 1007-1014 (1995)

[Publications] Kira,T.,J.P,Merin.,Baba,M.,Shigeta,S.,and Okamoto,T.: "Anti-Tat MTT assay:ANovel Anti-HIV Drug Screening System Using the Viral logic of Replication." AIDS Res.Hum.Retrovir. 11. 1359-1566 (1996)

[Publications] Tozawa,K.,Sakurada,S.,Kohri,k.,and Okamoto,T.:"Effects of Anti-NFkB Reagents in Biocking Adhesion of Human Cancer Cells to Vascular Endothelial Cells." Cancer Res.55. 4162-4167 (1995)

[Publications] Hohashi,N.,Hayashi,T.,Fesaki,N.,Takeuchi,M.,Higurashi,M.,Okamoto,T.,Semba,K.,and Yamamoto,T.: "The Protein tyrosine kinase Fyn activates transcription from the HIV promoter via activation of NFkB-like DNA-binding proteins." Int.Immunol.11. 1851-1859 (1995)

[Publications] Yokomizo,A.,Ono M.,Nanri,H.,Mariko,Y.,Ohga,T.,Wada M.,Okamoto,T.,Yodoi,J.,Kuwano,M.,and Kohno,K.: "Cellular levels of thioredoxin associated with drug sensitivity to cisplatin,mitomycin C,doxocubicin," Cancer Res.55;4293-4296,1955.55. 4293-4296 (1995)

[Publications] Iida,T.,Suzumori,K.,Ikuta,K.,Tenemura,M,Yagami,Y.,Okamoto.T.,and Hata,A.: "Identification of a Gly^<862> to Ser substitution in the type 1 collagen gene from a single sperm.(in press)," Hum.Reprod.(1996)

[Publications] Okamoto,T.,Shinnsaku,S.,Jian-Ping Yang.,and Jocelyn P.Merin.: "Regulation of NF-kB and Disease Control:Identification of a Novel Serine Kinase and Thioredoxin as Effectors for Signal Transduction Pathway for NF-kB Activation.Current Topics in Cellular Regulation.(in press)" (1996)

[Publications] Chiba,H.,Muramatsu,M.,Nomoto,A.and Kato,H.: "Two human homologue of Saccharomvces corevialae SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid reseptor." Nucleic Acids Res.,. 22. 1815-1820 (1994)

[Publications] Shusuke KUGE and Nicolas JONES.: "YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides." EMBO J.13. 655-664 (1994)

[Publications] Okamoto.T.and Hayashi,T.:Redox regulation of HIV gene expression.Proceedings for the Conference on the Development of Antioxidant and Chelating Drugs for the Treatment of Enviroment-associated Diseases(in press)., (1995)

[Publications] Okamoto.T.:Regulatory Proteins of Human Immmodeficiency Virus and Therapy.Anti-AIDS Drug Development: Challenge Strategies and Prospects(Eds.,Mohan and Baba)Harwood Publishers Inc., 11 (1995)

[Publications] Okamoto,T.:Redox regulation of NF-kB and disease control (a review article)Current Topics in Cellular Regulation (eds.by Stadtman,E.R.,and Chock,p.B.),Academic Press,(in press)., (1996)

岡本尚名古屋市立大学, 医学部, 教授 (40146600)