Characterization of molecular mechanism for transcriptional induction of PCB degradation system
| Project/Area Number |
16380058
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | Nagaoka University of Technology |
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Principal Investigator |
FUKUDA Masao Nagaoka Univ.Technol., Dept.Bioeng., Prof., 工学部, 教授 (20134512)
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| Co-Investigator(Kenkyū-buntansha) |
MASAI Eiji Nagaoka Univ.Technol., Dept.Bioeng., Asoc.Prof., 工学部, 助教授 (20272867)
SENDA Toshiya Adv.Indust.Sci.Technol., Biol.Info.Res.Ctr., G.L., 生物情報解析研究センター, 主任研究員 (30272868)
宮内 啓介 長岡技術科学大学, 工学部, 助手 (20324014)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥15,500,000 (Direct Cost: ¥15,500,000)
Fiscal Year 2006: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2005: ¥8,500,000 (Direct Cost: ¥8,500,000)
Fiscal Year 2004: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Keywords | PCB / transcriptional regulation / two-component regulatory system / biodegradation |
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| Research Abstract |
Characterization of transcriptional regulatory mechanism by BphST for induction of PCB degradation enzymes was performed to improve PCB degradation activity of Rhodococcus sp. RHA1. BphS and BphT are suggested to constitute two-component regulatory system. BphS is proposed to phosphorylate BphT in the presence of an inducing substance such as biphenyl and ethylbenzene, and phosphorylated BphT is estimated to activate target promoters. In this study, the following projects were performed. (1) The transcriptional start in etbA4 promoter was determined. The consensus sequence shared by the promoters under the control of BphST was found thirty-two bases upstream from the start, suggesting this consensus appears to be the target for specific BphT binding. (2) His- and Trx-tags sequences were fused to the N-terminal of BphT1 gene to express HisBphT1 and TrxBphT1 in a rhodococcal host. Both fusions exhibited biphenyl-induced transcriptional activation of bphA1 promoter. We succeeded in detecting the mobility retardation of bphT1 and bphA1 promoter sequence complex in agarose gel electrophoresis. This retardation was specific to bphA1 promoter sequence. Gel-filtration chromatography suggested that BphT1 appears to form a dimmer. (3) Deletion of BphS1 N-terminal was attempted using exonucleasse III to obtain an N-terminal deletion derivative of bphS1, which constitutively activates transcription of bphA1 promoter. No such derivatives were obatained, suggesting the possibility of interaction between N-and C-terminals of BphS.
We couldn't obtain the progressive results with BphS, probably because BphS protein is huge in size (190 kDa) and appears to have interaction with cell membrane. But we succeeded in purification of BphT and indication of bphT binding to the target promoter consensus in gel-shift assay.
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Report
(4 results)
Research Products
(9 results)
