Development of a Method for Long-Range DNA Sequencing
| Project/Area Number |
63880031
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Research Field |
分子遺伝学・分子生理学
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| Research Institution | Kyoto University |
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Principal Investigator |
SUGISAKI Hiroyuki Institute for Chemical Research, Kyoto Univ. Instructor, 化学研究所, 助手 (60135598)
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| Co-Investigator(Kenkyū-buntansha) |
AOYAMA Takashi Institute for Chemical Research, Kyoto Univ. Instructor, 化学研究所, 助手 (80202498)
TAKANAMI Mituru Institute for Chemical Research, Kyoto Univ. Professor, 化学研究所, 教授 (10027013)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥10,100,000 (Direct Cost: ¥10,100,000)
Fiscal Year 1990: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1989: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1988: ¥5,200,000 (Direct Cost: ¥5,200,000)
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| Keywords | Type IIS Restriction-Modification System / DNA Methylase / Endonuclease / Gene Structure. / DNA Sequencing / Sequence Recognition / II型制限酵素 / HgaI制限修飾係 / 5ーメチルシトシンメチラ-ゼ / 非対称塩基配列の認識 / 8塩基対認識の制限酵素 / 放線菌 / 蛋白質デザイン / FokI制限修飾系 / アデニンメチラ-ゼ / 活性ドメイン / DNAシークエンシング / Taq DNAポリメラーゼ / 制限酵素 / 修飾酵素 / ドメイン構造 |
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| Research Abstract |
FokI Restriction-Modification System
A DNA fragment that carried the genes coding for FokI endonuclease and methylase was cloned from the chromosomal DNA of Flavobacterium okeanokoites, and the coding regions were assigned to the nucleotide sequence. Analysis of the gene products expressed in E. Coli cells by gel filtration and sodium dodecyl sulfateーpolyacrylamide gel electrophoresis indicated that the molecular weights of both enzymes coincided well with the values estimated from the nucleotide sequences, and that the monomeric formes were catalytically active. No significant similarity was found between the sequences of the two enzymes.
Based on the findings that the FokI methylase consists of 647 amino acid residues and containing two copies of the segment specific for adenine methylase, Asp-Pro-Pro-Tyr, at amino acid positions 218-221 and 548-551, the role of these copies in the methylation reaction was investigated by introduction of a mutation into each segment. The results of ana
… More
lysis showed that different strands were modified in an asymmetric way by the introduction of mutations into one of the two segments, and that the segments at the N-terminal and C-terminal moieties participated in modification of the strands carrying 5'-GGATG-3' and 3'-CCTAC-5', respectively. We concluded that FokI methylase contained two functional domains each of which was responsible for modification of different stands in the target DNA.
HgaI Restriction-Modification System
A DNA fragment that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structually similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicated that the two genes are involved in modification of the HgaI restriction-modification system. Analysis of the species and positions of modified bases by Maxam-Gilbert method demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA. Less
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Report
(4 results)
Research Products
(14 results)
