| Project/Area Number |
15510059
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Risk sciences of radiation/Chemicals
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| Research Institution | OSAKA PREFECTURE UNIVERSITY |
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Principal Investigator |
KUBO Kihei OSAKA PREFECTURE UNIVERSITY, AGRICULTURE AND BIOLOGICAL SCIENCES, PROFESSOR, 農学生命科学研究科, 教授 (40117619)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUYAMA Satoshi OSAKA PREFECTURE UNIVERSITY, AGRICULTURE AND BIOLOGICAL SCIENCES, ASSOCIATE PROFESSOR, 農学生命科学研究科, 助教授 (10254442)
UI Masahiro OSAKA PREFECTURE UNIVERSITY, AGRICULTURE AND BIOLOGICAL SCIENCES, RESEARCH ASSOCIATE, 農学生命科学研究科, 助手 (10336810)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | DNA REPAIR / REACTIVE OXYGEN / GLYCOSYLASE / BASE DAMAGE / AP SITES / THYMINE GLYCOL / BASE EXCISION REPAIR / MAMMALIAN / マウス / グルコシラーゼ |
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| Research Abstract |
Reactive oxygen species induced by ionizing radiation and intracellular metabolic process attack DNA bases to create various oxidative damages, such as thymine glycol(TG). These damaged bases are mainly repaired by base excision repair(BER) pathway. The novel monofunctional thymine glycol(TG) activity was extracted from isolated nuclei of mouse stomach and spleen after removing mNTH1 and major AP endonuclease by the high-salt treatment. The western blot analysis revealed that the extract prepared did not include a detectable amount of mNTH1 protein. The nuclear extract was further purified either by the combination of UNO-S1 and UNO-Q1 column or by hydrophobic, hydroxyapatite and UNO-S1 column chromatography. The optimum pH and salt condition of the target activity were 100 mM KCl and pH 8.0, respectively. The activity was resistant to 0.58 mM EDTA. After SDS-PAGE and 2D-electrophesis analyses, we found several candidate protein spots or bands ; 2 major (61,56 kDa) with 6 minor candidates (70,53,37,33,27,25 kDa). We analyzed them with MALDI-FTICR Mass Spectrometry. No known TG-DNA glycosylases (mNth1 and mNei1) were included in the candidate proteins analyzed by Mascot Search. XP_140547, BAB_32018, XP_359164 (NCBI protein data base) were identified in the analysis. These proteins were PCR-cloned and the pET-expression systems were constructed.
To analyze the action of the glycosylase, a novel AP site detection method was developed using fluorescein-conjugated ARP (FARP-1). The intracellular AP sites were successfully and quantitatively detected by the method. By the aid of this new method, intranuclear BER reaction was successfully monitored after MMS treatment of HeLa cells.
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