1988 Fiscal Year Final Research Report Summary
Introduction of Artifitial Translocator into Plasma membrane in Cultured Human Cell-Line
| Project/Area Number |
61870016
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Jichi Medical School |
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Principal Investigator |
KAGAWA Yasuo Jichi Medical School Professor, 医学部, 教授 (30048962)
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| Co-Investigator(Kenkyū-buntansha) |
HAMAMOTO Toshiro Jichi Medical School Lecturere, 医学部, 講師 (30189625)
OHTA Shigeo Jichi Medical School Lecturere, 医学部, 講師 (00125832)
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| Project Period (FY) |
1986 – 1988
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| Keywords | Cultured Cell / Gene manipulation / Translocator / Gene expression / 遺伝病 / ミトコンドリア / 遺伝子操作 |
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| Research Abstract |
A cell is surrounded by biomembranes, which permeate specific substrates via specific translocators. Therefore, even if the cultured cell, it is hard to control the cellular conditions by changing the extra culture medium. We have attempted to control the cellular energy state by two methods. First, we have tried to isolate the mutant cell lines which are deficient in oxidative phosphorylation. Second, we have tried to incorporate an artificial tanslocator by introducing the manipulated gene; in this case, we use a fusion protein of ADP/ATP translocator with a retro virus envelop protein, which target plasma membrane. The following results were obtained by several trials.
1) To detect the ATP concentration easily, an ATP sensor was made by using thermo-stable proton translocating ATPase purified from a thermophilic bacterium.
2) The cytochrome C oxidase deficient cell line were established from a patient with mitochondrial myopathy. The cell survive could be controlled by substrate for oxidative phospholylation.
3) The pyruvate dehydrogenase deficient cell line was established from a patient with its deficiency. The defective gene were analyzed in detail.
4) The mitochondrial deficient cell were made by treating the human promyelocytic leukemia cell HL-60 with retinol. In addition, the gene expression of mitochondrial proteins were also examined by Northern blotting method.
5) The human cDNA encoding ADP/ATP translocator was cloned and sequenced.
6) The ADP/ATP translocator cDNA was fused to a retro virus envelop gene under SV40 promoter. The constructed gene was transfected into mouse fibroblast cells. The resulting transformant was unstable and could not be maintained. Therefore, neomycin resistant gene was connected directly and then transfected to HeLa cells. The experiment is just on the way.
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