1996 Fiscal Year Final Research Report Summary
Structural and Functional Diversity of Eukaryotic Vacuolar ATPases : Identification of Superfamily in Mammalian Cells and Studies on the Physiological Roles of Vacuolar ATPases by Functional and Genetical Disruption
| Project/Area Number |
07672360
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Okayama University |
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Principal Investigator |
NOUMI Takato Okayama University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (90189374)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Vacuolar ATPase / H^+-ATPase / Trnasgenic mouse / Gene knockout / Papillomavirus / Lysosomal pH |
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| Research Abstract |
(1) Transformation of Rat Fibroblasts by E5 Onco-protein from Bovine Pappiloma Virus
The gene for E5 onco-protein was cloned under the control of SV40 promoter from bovine pappiloma virus genome. The expression plasmid was transfected into the rat fibroblast and stable transformants were isolated. The oncogenic transformation was stimulated by PDGF or co-transfection with PDGF-R and proteolipid cDNAs. The intra-lysosomal pH was revealed to be elevated in these transformants. Currently the molecular mechanism for cell transformaiton by E5 through PDGF-R and proteolipid is under the investigation.
(2) Establishment of E5 Transgenic Mouse
The E5-expression vector was injected into fertilized egges from mice. Three lines of transgenic mouse were established, and leukemia was developed in one of these lines. The origin of leukemia cells and alteration of acidification in the endomembranous organelles are being investigated.
(3) Molecular Cloning and Genomic Structures of Genes for Mouse Proteolipid
The genomic library from mouse was screened and genes encoding proteolipid were isolated. The genome structure including 3 exons were determined by DNA sequenceing. Two possible pseudo-genes were also isolated and their genome structures were determined. Expression of proteolipid gene in ES cells were confirmed by Northern blot analysis. The targeting vector for disruption of the proteolipid gene was constructed and transfected into ES cells by electroporation. Six homologous recombinants were isolated, which grew and developed normally, Chimaera mice were established by injecting these homologous recombinants into eight-cell stage mouse embryos. We further isolated F1 hetero-knockout mice by breeding chimaera with wild-typd. These hetero-knockout mice are growing normally. In the near future, F2 homo-knockout mice will be produced.
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[Publications] Noumi, T., Inoue, H., Shimomura, T., Takimoto, N., Tsuchiaya, T., and Kanazawa, H.: "pH-dependent growth retardation by enhancement of a Na^+/H^+ antiporter activity of Escherichia coli : An application to isolation of antiporter defective mutants" J.Bacteriol.(in press). (1997)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Moritani, C., Sawada, K., Takemoto, K., Shin, Y., Nemoto, S., Noumi, T., and Kanazawa, H.: "Interactions of the F1-ATPase subunits from Escherichia coli detected by the yeast two-hybrid system" Biochim.Biophys.Acta. 1274. 67-72 (1996)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Shin, Y., Sawada, K., Miyanaga, M., Moritani, C., Noumi, T., Tsuchiya, T., and Kanazawa, H.: "Reconstitution of F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase at their amino termini" Biochim.Biophys.Acta. 1273. 62-70 (1996)
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[Publications] Inoue, H., Noumi, T., Tsuchiya, T., and Kanazawa, H.: "Essential aspartic acid residues, Asp-133, Asp-163, and Asp-164, in the transmembrane helices of a Na^+/H^+ antiporter (NhaA) from Escherichia coli" FEBS Lett.363. 264-268 (1995)
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「研究成果報告書概要(欧文)」より
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[Publications] Kanazawa, H., Yabuki, M., Miki, J., Fudemoto, T., Ikeda, H., Noumi, T., and Shin, Y.: "Enhancement of Escherichia coli H^+-ATPase caused by binding of monoclonal antibodies attributed to structural changes of Leu-456 and Ser-440 in the alpha subunit." Arch.Biochem.Biophys.317. 348-356 (1995)
Description
「研究成果報告書概要(欧文)」より
