| Project/Area Number |
04670395
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
内科学一般
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| Research Institution | Keio University School of Medicine |
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Principal Investigator |
MIMORI Tsuneyo Keio University, Dpt.of Medicine, Assistant Professor, 医学部, 講師 (10157589)
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| Co-Investigator(Kenkyū-buntansha) |
OHOSONE Yasuo Keio University, Dpt.of Medicine, Associate Physician, 医学部, 助手 (20160492)
HAMA Nobuaki Keio University, Dpt.of Medicine, Associate Physician, 医学部, 助手 (70228526)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
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| Keywords | Autoantibody / Ku antigen / DNA-terminal binding protein / DNA-dependent protein kinase / gene polymorphism / autoimmune disease / collagen disease / overlap syndrome / DNA依存性プロテインキナーゼ / オクタマー配列 / DNA依存性蛋白キナーゼ / 自己抗原 / PCR / PCR-SSCP |
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| Research Abstract |
Anti-Ku autoantibodies in patients with PSS-PM overlap syndrome recognize a 70kD/80kD protein (p70/p80) heterodimer which selectively binds to terminal region of dsDNA.I this project, I have investigated 1) genomic structure and polymorphism of epitope regions of the Ku antigen, 2) structure of DNA that binds to the Ku antigen, and 3) association between the Ku antigen and DNA-dependent protein kinase (DNA-PK).
Structure of genomic DNAs that encode for epitopes of p70 (aa 545-609) and p80 (aa 696-732) was examined by single-strand conformation polymorphism (SSCP) after those DNA regions were amplified by polymerase chain reaction (PCR). Although there was no difference in genomic epitope structure between normals and patients, one patient with Xeroderma pigmentosum and one normal subject showed polymorphic structures of the p70 gene. p70 appeared to be ecoded by at least two independent genes. These results suggest a presence of a gene family encoding the Ku antigen.
DNAs that were extra
… More
cted from immunoprecipitates between anti-Ku antobodies and the Ku antigen from HeLa cells were subcloned into M13 vector and their nucleotide sequences were determined. When 30 DNA clones were examined (mean length 196bp), the octamer-like sequence [ATTT (G/T) (C/T) (A/T) T] and the transferrin receptor element-like sequence [GAAGTNA (C/G)] appeared at 37 and 18 binds specific DNA sequences as well as DNA termini.
Anti-Ku antibodies precipitated a 350kD protein besides of p70/p80, when HeLa cells were extracted in an isotonic buffer as antigen source. This protein was identified as the catalytic subunit p350 of DNA-PK,since the 350kD protein precipitated with anti-Ku was recognized by a hyperimmune rabbit serum to DNA-PK p350. Binding between the Ku and p350 required the presence of dsDNA and was dissociated reversibly by 0.5M NaCl. Enzymatic activity of DNA-PK required the presence of both Ku and DNA.These results indicate that the Ku antigen binds p350 to form the DNA-PK holoenzyme and acts as an activation subunit of DNA-PK. Less
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