| Project/Area Number |
14572093
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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 |
医薬分子機能学
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
NEGISHI Kazuo Okayama University, Advanced Science Res. Ctr., Associate Professor, 自然生命科学研究支援センター, 助教授 (70116490)
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| Co-Investigator(Kenkyū-buntansha) |
OTSUKA Chie Okayama University, Advanced Science Res. Ctr., Research Associate, 自然生命科学研究支援センター, 助手 (60253001)
TOYODA Tetsuya Kurume University, School of Medicine, Professor, 医学部, 教授 (00197972)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | nucleotide analogs / error catastrophe / replicational error / ambiguous base-pairing / RNA virus / retrovirus / RNA polymerase / HPLC / PTP / KTP / エラーカタストロフィー / ポリオウイルス / C型肝炎ウイルス / supF / 両義的な取組み |
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| Research Abstract |
1. Synthesis of nucleotide analogs.
We have established the methods for synthesis of N^4-hydroxycytidine triphosphate (N^4-OhCTP),N^4-methoxycytidine (N^4-MoCTP), and N^4-methycytidine (N^4-McCTP), and obtained pure materials. With these pure, samples, their incorporation into RNA catalyzed by T3 RNA polymerase was studied. RNA synthesis took place if N^4-OhCTP, or N^4-MoCTP was present in place of UTP, but not when they were added in place of CTP. This results shows that they behave only as UTP analogs in spite of their ambiguity in bass-pairing. In contrast, N^4-MeCTP behaved as CTP analog only. Incorporation properties of isoGTP, one of typical oxidation products from nucleotide, was studied using a commercial sample. It shows no incorporation by the RNA polymerase.
2.Properties of the analogs in templates in the reversetranscription.
Above finding enabled us to make RNA whose U or C was totally replaced with the analogs. Thus we have prepared RNA with analogs, and they were used as a template for reverse transcription with AMV reversetranscriptase. Resulting cDNA was amplified and analyzed with a DNA sequencer. The analysis indicated that N^4-hydroxycytidine was read as C and T almost equally. It suggests that this analog may cause mutation in a reversetranscription process.
3. Anti-HIV virus activities of the analogs.
Some analogs ware found to be very hopeful. Further study is continuing.
4. Effects on RNA viruses and their polymerase.
Action of analog triphosphates to Polio RNA polymerase was studied.
5. Use of chitosan-triphosphate complexes.
We have found that chitosan-triphosphate complex made by a reported method was not stable enough. Improvement of the protocol is currently underway.
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