| Project/Area Number |
63480407
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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 |
Morphological basic dentistry
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| Research Institution | Showa University |
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Principal Investigator |
YAMAMOTO Ayako Showa University School of Dentistry Associate Professor, 歯学部, 助教授 (20085773)
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| Co-Investigator(Kenkyū-buntansha) |
GOTO Nobuichi Showa University School of Dentistry Professor, 歯学部, 教授 (10077175)
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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 |
¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | Bacteroides gingivalis / Collagenase / Collagen / Periodontal disease / Oral bacteria / Agarose / Gene cloning / 最近コラゲナーゼ / 遺伝子 |
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| Research Abstract |
Collagenases produced by several oral bacteria appear to play an important role to both establish and progress periodontal diseases. In order to determine the function of the bacterial collagenases in degradation of periodontal tissue, we intended to clone the bacterial genes encoding collagenase.
First, we developed a simple method for detecting collagenase activities of bacteria. Since the conventional isotope-labeled-collagen-digestion-assay method is not convenient to screen collagen-producing bacteria, we therefore, developed a method using collagen-agarose- plates. In order to avoid heat denaturation, we sterilized collagen with a microwave oven and used low melting point agarose instead of agar to make plates. By using the plates, we tested the collagenase activities of 16 species (total 53 strains) of oral bacteria and found that only Bacteroides gingivalis strains had the enzyme activities.
We examined a total of 31 B. gingivalis strains for the presence of plassid and found that none of the strains carried plasmid. We, therefore, concluded that the genes encoding collagenase are located on the chromosome. Then we extracted partially-digested Sau3A I fragments of four to nine kilobase pairs and cloned then into the BamHI-site of the cloning vector pUC13 by which Escherichia coli JM103 was transformed. Approximately 2.500 transformants were tested for seven genetic markers of the donor: collagenases trypsin-like protease, glycylprolyl protease, abilities to digest gelatin and skin milk. and kanamycin- resistance and formation of black pigment. We detected none of the activities in the strains tested.
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