| Project/Area Number |
63571042
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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 |
Biological pharmacy
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
NEGISHI Kazuo OKAYAMA UNIVERSITY, GENE RESEARCH CENTER, ASSOCIATE PROFESSOR, 遺伝子実験施設, 助教授 (70116490)
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| Co-Investigator(Kenkyū-buntansha) |
UMEDA Masato UNIVERSITY OF TOKYO, FACULTY OF PHARMACEUTICAL SCIENCES, RESEARCH ASSOCIATE, 薬学部, 助手 (10185069)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1988: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Bisulfite / Methoxyamine / Left-handed DNA / B-Zjunction / Monoclonal Antibody / High-salt / Chemical Modification / DNA polymerase / B型-Z型接合部 / Z型DNA / モノクローナル抗体 / 過マンガン酸カリウム / 亜硫酸塩 / 高次構造 / 5-メチルシトシン / 閉環状プラスミド |
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| Research Abstract |
Bisulfite-methoxyamine mixture attacks (deoxy) cytidines in single-stranded region of nucleic acids. We have investigated the ability of this reagent as a chemical probe for B-Z junction of DNA. First, we studied the properties of the product N^4-methoxy-5, 6-dihydro(deoxy)cytidine 6-sulfonate. This product has two diastereomers. We have found that a commercially available monoclonal antibody against A4-methoxy-5,6- dihydro(deoxy) cytidine 6-sulfonate can bind specifically to the one disstereomer. Three double stranded oligonucleotides with blunt-end were synthesized. Two oligonucleotides contained cytosines at internal positions and 3'-end or 5'-end, respectively. The third oligonucleotide had cytosines only in internal positions. These oligonucleotides were modified with bisulfite-methoxyainine and analyzed with gel electrophoresis. The results showed that both 3' and 5' cytosines were accessible by the reagent. The gel was blotted onto a nylon membrane. The analysis of the blot with the monoclonal antibody revealed that 3'cytosine was modified to one diasteromer and 5' cytosine was modified to the odier diasteromer.
Next, DNA containing Z-DNA was probed with bisulfite-methoxyamine. Alternating GC structure in pRW756 was made to Z form by supertwisting. This circular DNA was treated with bisulfite-methoxyamine. S1 nuclease digestion was employed to locate modification site. The results showed that cytosines of both junctions between oligo(GC) and B form DNA were modified. The modified cytosines were mapped in the sequence level by use of the pause of DNA polymerase action in opposite to the modified cytosines in the template. The modification of salt-induced B-Z junctions with bisulfite-methoxyamine gave similar results. These results indicates that B-Zjunction induced by supertwist and high salt have the similar structures.
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