1992 Fiscal Year Final Research Report Summary
Construction of promoter-detection plasmid in animal cells by using the metapyrocatechase gene
| Project/Area Number |
03557013
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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 |
General medical chemistry
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| Research Institution | Yamaguchi University |
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Principal Investigator |
NAKAZAWA Atsushi Yamaguchi Univ.Sch.Med. Prof., 医学部, 教授 (90025594)
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| Co-Investigator(Kenkyū-buntansha) |
KUMAHARA Hiromi Ube Industries Ube Inst.Director, 宇部研究所, 課長
NOMA Takafumi Yamaguchi Univ.Sch.Med. Assist.Prof., 医学部, 講師 (40189428)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Promoter / Vector / Cultured cells / Catechol / C230 |
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| Research Abstract |
To test the ability of expression of the metapyrocatechase (C230) gene in the animal cells, we first constructed two plasmids. In one plasmid, the C230 gene was put under the control of the promoter and enhancer of SV40 early genes, while in the other, it was controlled by the promoter and enhancer of the human elongation factor gene. Neither of these plasmid showed production of active C230 in HeLa and CHO cells. Next, we made a gene fusion in which the N-terminal two residues of chicken adenylate kinase was ligated to the N-terminal truncated C230. The C230 gene of the above two plasmids was replaced by the gene fusion, and the plasmid products were checked for their expression in the animal cells. However, the results were negative. Since the transcription of these plasmids were thought to be active in the animal cells, failure in the expression of the C230 gene is probably due to a barrier in the process after the translation.
During the course of this study, we obtained a plasmid pKS230, in which the C230 gene is put under the control of the lac promoter. The restriction sites for HincII and EcoRV can be used as a cloning site for foreign DNA, since insertion to these sites make the plasmid inactive to produce the active C230. The inserted plasmid is easily detected by yellow color development after spraying a catechol solution. Therefore, this plasmid can be used a less expensive cloning vector comparing with the lac promoter vector that uses X-Gal as a detector.
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