| Project/Area Number |
61480128
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine |
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Principal Investigator |
YOSHIDA Shonen Nagoya University School of Medicine, 医学部, 助教授 (70090420)
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| Co-Investigator(Kenkyū-buntansha) |
KOIZUMI Keiko Nagoya University School of Medicine, 医学部, 講師 (00118027)
KJIMA Kiyohide Nagoya University School of Medicine, 医学部, 教授 (80073104)
IZUTA Shunji Nagoya University School of Medicine, 医学部, 助手 (50203047)
UMEKAWA Hayato Nagoya University School of Medicine ( present adress: Mie University), 生物資源学部, 助手 (90185059)
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| Project Period (FY) |
1986 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1986: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Keywords | Human ARS / Human c-myc gene / Raji cell / DNA polymerase / Topoisomerase I Cardiolipin / Phosphatidylinositol / DNAポリメラーゼα / トポイソメラーゼI / ゲルシフトアッセイ / カルジオリピン / プライマーゼ / カルシウムイオン / モノクローナル抗体 / 核マトリクス / リン脂質 |
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| Research Abstract |
I. In vitro DNA replication: An autonomousy replication sequence (ARS) at the upstream of human c-myc gene was shown to replicate in nuclear extract of Burkitt lymphoma cell line, Raji cells, in a semiconservative manner. The enzymes involved in replication were fractionated into 30-50% (NH_4)_2SO_4 precipitate and the DNA polymerase -primase complex was shown to be essential for the replication by immunoprecipitation using anti-DNA polymerase antibody and the specific inhibition by aphidicolin. Interestingly, the replication was strongly inhibited by the addition of acidic phospholipids, i.e., phosphatidylinositol and cardiolipin. These two phospholipids were also shown to interact with topoisomerase I and DNA polymererase . So, phospholipids might be involved in supressing DNA replication. II. ARS binding protein: Minimal core sequence in the ARS element (30-mer) were chemically synthesized and used for gel-shift assay and DNA-affinity chromatography. A protine, which preferentially bound to the core sequence was purified to near homogeneity. The protein migrated on SDS-PAGE at Mr. 68 KDa and reacted with antibody directed against human c-myc protein upon Western blotting. These result strongly suggeste that the binding protein is coded by the human c-myc gene. Biochemical mechanisn of action of this protein is now in progress.
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