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Regulation and Molecular Mechanisms of Antigen Receptor Gene Rearrangements

Research Project

Project/Area Number 07101001
Research Category

Grant-in-Aid for Specially Promoted Research

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionThe University of Tokyo

Principal Investigator

SAKANO Hitoshi  Graduate School of Science, The University of Tokyo, Professor, 大学院・理学系研究科, 教授 (90262154)

Co-Investigator(Kenkyū-buntansha) TSUBOI Akio  Graduate School of Science, The University of Tokyo, Research Associate, 大学院・理学系研究科, 助手 (20163868)
NAGAWA Fumikiyo  Graduate School of Science, The University of Tokyo, Lecturer, 大学院・理学系研究科, 講師 (10241233)
Project Period (FY) 1995 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥249,000,000 (Direct Cost: ¥249,000,000)
Fiscal Year 1999: ¥43,000,000 (Direct Cost: ¥43,000,000)
Fiscal Year 1998: ¥46,000,000 (Direct Cost: ¥46,000,000)
Fiscal Year 1997: ¥46,000,000 (Direct Cost: ¥46,000,000)
Fiscal Year 1996: ¥55,000,000 (Direct Cost: ¥55,000,000)
Fiscal Year 1995: ¥59,000,000 (Direct Cost: ¥59,000,000)
KeywordsV(D)J joining / RSS signals / RAG proteins / RAG / RSS complex / footprinting / 3'-phosphorylation / transposition / 12 / 23 rule / 抗原受容体遺伝子 / 免疫グロブリン遺伝子 / 組み換えシグナル配列 / リコンビナーゼ / リンパ細胞の分化 / 組み換え制御因子 / トランスジェニックマウス / 臭覚受容体遺伝子 / V(D)J結合 / エンハンサー / 対立形質排除 / 体細胞突然変異 / エンハンサーエレメント / 組み換え抑制因子
Research Abstract

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the recombination-activating genes (RAGs). Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin homeodomain of the RAG1 protein and adjacent major and minor grooves of the 9mer DNA.
When two RSSs are cleaved in V(D)J joining, the signal-end (SE) complex is formed with RAG1 and RAG2 proteins in the presence of DNA bending protein HMG1. Using magnetic beads, we have purified the SE complex and analyzed the SE DNA by DNaseI footprinting. It was found that the 7mer rather than the 9mer plays a primary role in the SE complex formation. It was also found that the 3'-OH of the 7mer is phosphorylated in the SE complex in both 12- and 23-RSS's after the cleavage. The 3'-phosphate is transferred from carrier DNA or RNA by trans-esterification after nicking of the carrier. In vitro assays demonstrated that the 3'-phosphorylation prevents the transposition of the SE complex. These results indicate that the 3'-phosphorylation may have an active role in preventing harmful transposition of an excision product of V(D)J joining.
We have shown that the PU.1 binding motif (GAGGAA) in the 3'-enhancer of the IgィイD2κィエD2 gene is responsible for the negative regulation of VィイD2κィエD2-JィイD2κィエD2 joining. In the transcriptional activation, the PU.1 motif acts in a positive manner cooperatively with the PIP and BSAP motifs, which are located in the same enhancer core region. Interestingly, base substitutions in either the PIP or the BSAP motif did not affect the B/T or pro-B/pre-B specificity of VィイD2κィエD2-JィイD2κィエD2 joining. Thus, the PU.1 motif alone appears to regulate both temporal and tissue-specific rearrangements of the IgィイD2κィエD2 gene.

Report

(6 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • 1996 Annual Research Report
  • 1995 Annual Research Report
  • Research Products

    (72 results)

All Other

All Publications (72 results)

  • [Publications] Hiramatsu,R.,et al.: "The 3'-enhancer region determines the B/T specificity and proB/preB specificity of immunoglobulin V_k-J_k joining."Cell. 83. 1113-1123 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Asai,H.,et al.: "Genomic structure and transcription of a murine odorant receptor gene:differential initiation of transcription in the olfactory and testicular cells"Biochem.Biophys.Res.Comm.. 221. 240-247 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hayashi,R.,et al.: "The PU.1 binding site is a cis-element that regulates pro-B/pre-B specificity of V_k-J_k jouning."J.Immunol.. 159. 4145-4149 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagawa,F.,et al.: "Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination"Mol.Cell.Biol.. 18. 655-663 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Touhara,K.,et al.: "Functional identification and reconstitution of an odorant receptor in single olfactory neurons"Proc.Natl.Acad.Sci.USA. 96. 4040-4045 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsuboi,A.,et al.: "Olfactory neurons expressing closely linked and honologous odorant receptor genes ten* to project their axons to neighboring glomeruli on the olfactory bulb"J.Neurosci.. 19. 8409-8419 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsuboi,A.,et al.: "YAC transgenic approach to clarify the mechanisms of odorant receptor gene expression and specific projection of olfactory neurons"Chem.Senses. 24. 591 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hashimoto,S.,et al.: "Prf,a novel Ers family protein that binds to the PU.1 binding motif,is specifically expressed in restricted stages of B cell development"Int.Immunol.. 11. 1423-1429 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagaoka,H.,et al.: "Requirement of Ras for pre-B cell survival"J.Exp.Med.. 191(in press). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Yoshida,T.,et al.: "The DNA bending protein,HMG1,is required for correct cleavage of 23 bp-recombination signal sequences by RAG proteins in vitro"Int.Immunol.. 12(in press). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Serizawa,S.,et al.: "Odorant receptor gene expression and specific projection of olfactory neurons in YAC transgenic mice"Science. (submitted). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagawa,F.,et al.: "Footprint analysis of signal-end DNA isolated from the post-cleavage complex of V(D)J recombination:3'-end phosphorylation by RAG1/RAG2 proteins"Cell. (submitted). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 名川文清、他: "中枢神経系における多様性とその分子遺伝学的基礎"蛋白質・核酸・酵素. 40. 633-642 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 坪井昭夫、他: "Receptor editingとB細胞トレランス"臨床免疫. 27. 125-128 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 坂野 仁: "V(D)J結合を負に制御するDNAエレメント"実験医学. 13. 1371-1377 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 平松隆司、他: "リンパ球の分化と遺伝子再構成の制御"細胞工学. 15. 229-235 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 坂野 仁: "リンパ球の分化と遺伝子再構成の制御"生化学. 68. 1470-1474 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 石黒啓一郎、他: "抗原レセプター遺伝子におけるV(D)J組み換えの素過程とその制御"実験医学. 15. 1246-1253 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 橋本修一、他: "抗体遺伝子組み換えの制御機構"細胞工学. 17. 48-56 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 名川文清、他: "V(D)J組み換えの素過程 -RAG/RSS複合体のフットプリント解析ー"蛋白質・核酸・酵素. 43. 101-110 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 坪井昭夫、他: "嗅覚受容体遺伝子の発現と嗅球への投射"実験医学. 18(発表予定). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 坂野 仁: "医科分子生物学"南光堂. 11 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 石黒啓一郎、他: "標準免疫学3"医学書院. 22 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 石黒啓一郎、他: "生体防御・免疫と感染症"共立出版(発表予定). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hiramatsu, R. et al.: "The 3' enhancer region determines the B/T specificity and pro-B/pre-B specificity of immunoglobulin VィイD2κィエD2-JィイD2κィエD2 joining"Cell. 83. 1113-1123 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Asai, H. et al.: "Genomic structure and transcription of a murine odorant receptor gene : Differential initiation of transcription in the olfactory and testicular cells"Biochem. Biophys. Res. Commun.. 221. 240-247 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hayashi, R. et al.: "The PU.1 binding site is a cis-element that regulates proB/preB specificity of VィイD2κィエD2-JィイD2κィエD2 joining"J. Immunol.. 159. 4145-4149 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagawa, F. et al.: "Footprint-analysis of the RAG recombination signal sequence complex for V(D)J type recombination"Mol. Cell. Biol.. 18. 655-663 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Touhara, K. et al.: "Functional identification and reconstitution of an odorant receptor in single olfactory neurons"Proc. Natl. Acad. Sci. USA. 96. 4040-4045 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsuboi, A. et al.: "Olfactory neurons expressing closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb"J. Neurosci.. 19. 8409-8418 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsuboi, A. et al.: "YAC transgenic approach to clarify the mechanisms of odorant receptor gene expression and specific projection of olfactory neurons"Chem. Senses. 24. 591 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hashimoto, S. et al.: "Prf, a novel Ets family protein that binds to the PU.1 binding motif, is specifically expressed in restricted stages of B cell development"Int. Immunol.. 11. 1423-1429 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagaoka, H. et al.: "Requirement of Ras for pre-B cell survival"J. Exp. Med.. 191, (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Yoshida, T. et al.: "The DNA bending protein, HMG1, is required for correct cleavage of 23 bp-recombination signal sequences by RAG proteins in vitro"Int. Immunol.. 12,(in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Serizawa, S. et al.: "Mutually exclusive expression of an odorant receptor gene in YAC transgenic mice"Science. (submitted).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagawa, F. et al.: "Footprint analysis of signal-end DNA isolated from the post-cleavage complex of V(D)J recombination : 3'-end phosphorylation by RAG1/RAG2 proteins"Cell. (submitted).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagawa, F. et al.: "Genetic basis of diversity in the mammalian olfactory system (in Japanese)"Protein, Nucleic Acid, Enzyme. 40. 633-642 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsuboi, A. et al.: "Receptor editing and B-cell tolerance (in Japanese)"Clinic. Immunol.. 27. 125-128 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Sakano, H.: "PU.1 binding motif is responsible for the negative regulation of Vκ-Jκ joining (in Japanese)"Exp. Med.. 13. 1371-1377 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hiramatzu, T. et al.: "Regulation of Ig gene rearrangement during B-lymphocyte development (in Japanese)"Cell Technol.. 15. 229-235 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Sakano, H.: "Developmental regulation of Ig gene rearrangement (in Japanese)"Biochemistry. 68. 1470-1474 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Ishiguro, K. et al.: "Molecular mechanisms of V(D)J joining in antigen receptor genes (in Japanese)"Exp. Med.. 15. 1246-1253 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hashimoto, S. et al.: "Regulation of immunoglobulin gene rearrangement (in Japanese)"Cell Tech.. 17. 48-56 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Nagawa, F. et al.: "Footprint analysis of the RAG/RSS complex (in Japanese)"Protein, Nucleic Acid, Enzyme. 43. 101-110 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsuboi, A. et al.: "Regulation of odorant receptor gene expression (in Japanese)"Exp. Med.. 18, (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Sakano, H.: "Regulation of antigen receptor genes (in Japanese)"Med. Mol. Biol., Nankodo, 11. (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Ishiguro, K. et al.: "Antigen receptors and their genes (in Japanese)"Standard Immunol. 3, Igaku-shoin, 22. (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Ishiguro, K. et al.: "Organization and reorganization of antigen receptor genes (in Japanese)"Immunol. Infect. Diseases, Kyoritsu-shuppan, (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Touhara K., et al.: "Functional identification and reconstitution of an odorant receptor in single olfactory neurons"Proc. Natl. Acad. Sci. USA. 96. 4040-4045 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsuboi A., et al.: "Olfactory neurons expressing closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb"J. Neurosci.. 19. 8409-8419 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsuhoi A., et al.: "YAC transgenic approach to clarify the mechanisms of odorant receptor gene expression and specific projection of olfactory neurons"Chem. Senses. 24. 591 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Hashimoto, S., et al.: "Prf, a novel Ets family protein that binds to the PU.1 binding motif, is specifically expressed in restricted stages of B cell development"Int. Immunol.. 11. 1423-1429 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Nagaoka H., et al.: "Requirement of Ras for pre-B cell survival"J. Exp. Med.. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Yoshida T., et al.: "The DNA bending protein, HMGI, is required for correct cleavage of 23 bp-recombination signal sequences by RAG"Int. Immunol.. 12(in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 石黒啓一郎、坂野仁: "「生体防御:免疫と感染症」免疫系における多様性獲得の分子基盤"共立出版(発表予定). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 坪井昭夫、坂野仁: "「発生・神経研究の最前線2000」嗅覚器官形成・再生の分子的基盤"実験医学(発表予定). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 名川文清・石黒啓一郎・坂野 仁: "V(D)J組み換えの素過程-RAG/RSS複合体のフットプリント解析-" 蛋白質・核酸・酵素. 43. 101-110 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Touharu et al.: "Functional identification and reconstitution of an odorant receptor in single olfactory neurons." Proc.Natl.Acad.Sci.USA. 96. in press (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hayashi,R., et al.: "The PU.1 binding site is a cis-element that regulates proB/preB specificity of Vκ-Jκ joining." J.Immunol.159. 4145-4149 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Nagawa,F., et al: "Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination." Mol.Cell.Biol.18. 655-663 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] 石黒啓一郎、他: "抗原レセプター遺伝子におけるV(D)J組み換えの素過程とその制御." 実験医学. 15. 1246-1253 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 石黒啓一郎、坂野仁: "標準免疫学3 抗原受容体の構造と遺伝子" 医学書院(谷口克、宮坂昌之編), 22 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Hisae Asai: "Genomic Structure and Transcription of a Murine Odorant Receptor Gene:Differential Initiation of Transcription in the Olfactory and Testicular Cells" Biochem.Biophys.Res.Commun.221. 240-247 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 名川文清: "鋤鼻嗅覚受容体" 蛋白質・核酸・酵素. 41. 2101-2106 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 坂野仁: "医科分子生物学「抗原受容体遺伝子の発現制御」" 南江堂(村松正実、谷口維紹編), 12 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Ryuji Hiramatsu: "The 3′ Enhancer Region Determines the B/T Specificity and Pro-B/Pre-B Specificity of Immunoglobulin Vκ-Jκ Joining" Cell. 83. 1113-1123 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 坂野 仁: "V(D)J結合を負に制御するDNAエレメント" 実験医学. 13. 1371-1377 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 坪井 昭夫: "Receptor editingとB細胞トレランス" 臨床免疫. 27. 125-128 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 名川 文清: "中枢神経系における多様性とその分子遺伝学的基礎" 蛋白・核酸・酵素. 40. 633-642 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 平松 隆司: "リンパ球の分化と遺伝子再構成の制御" 細胞工学. 15. 229-235 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Hitoshi Sakano: "Encyclopedia in Molecular Biology,“Recombinase"" Academic Press,London(ed.J.C.Kendrew), 3 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 坂野 仁: "医科分子生物学「抗原レセプター遺伝子の発現と制御」" 南江堂(村松正美、谷口維紹編)(印刷中), 12

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2018-03-28  

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