1991 Fiscal Year Final Research Report Summary
Molecular cloning of collagenase inhibitor gene from bovine genomic DNA
| Project/Area Number |
02454424
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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 |
Functional basic dentistry
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| Research Institution | Niigata University |
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Principal Investigator |
NOHARA Hiroyoshi Niigata University School of Dentistry professor, 歯学部, 教授 (60018405)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Tokuya Niigata University School of Dentistry assistant professor, 歯学部, 助教授 (50018420)
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| Project Period (FY) |
1990 – 1991
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| Keywords | collagenase inhibitor / cloning / bovine genome DNA |
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| Research Abstract |
The purpose of this study was to isolate promoter regions of bovine collagenase inhibitor genes. For this purpose, we have examined three methods which have been developed for the isolation of flanking regions of known structure qenes. With inverse polymerase chain reaction(PCR)and single-specific primer PCR methods, it was unsuccessful to isolate a target gene. However, we could isolate a flanking region of bovine Ml gene by usinq nested PCR method.
After digestion of bovine lung -DNA with EcoRl', the DPIA fragments were ligated with EcoRl cassette at both ends. The resultment DNA fragments received PCR in the presence of EdoRl cassette primer Cl and a synthetic oligonucleotide complementary with a part of bovine Ml gene which spans from 141th to 160th nucleotides. The product DNA was further amplified by using EcoRl cassette primer C_2 and another synthetic oligonucleotide complementary with a part of bovine Ml gene which spans from 10th to 29th nucleotides. From this reaction, we could isolate a DNA fragment with about 500 bp in. length. This DNA fragment could not be obtained without DNA ligation reaction. Thus, the 500 bp DNA fragment should be sandwiched between two EcoRl cassettes and a upstream region of bovine Ml gene.
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