| Project/Area Number |
63570521
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
内分泌・代謝学
|
|---|
| Research Institution | Institute of Clinical Medicine, The University of Tsukuba |
|---|
Principal Investigator |
ITAKURA Mitsuo Univ. of Tsukuba, Inst. of Clin. Med., Assist. Prof., 臨床医学系, 講師 (60134227)
|
|---|
| Project Period (FY) |
1988 – 1989
|
| Project Status |
Completed (Fiscal Year 1989)
|
| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥1,600,000 (Direct Cost: ¥1,600,000)
|
|---|
| Keywords | Recombinant DNA / Purine metabolism / Molecular biology / Glucocorticoid / Promoter / Gene therapy / Cell Proliferation / Enzyme induction / 遺伝子 / 代謝調節 / 調節発現 / プロモーター |
|---|
| Research Abstract |
[AIM OF THE STUDY] The regulatory mechanism of amidophosphoribosyltransferase (ATase), the rate-limiting enzyme of de novo purine synthetic pathway, was studied by the molecular biology approach.
[METHODS] First, the recombinant plasmid DNA of E.coli ATase and glucocorticoid-dependent mouse mammary tumor virus (MMTV) LTR was transferred to mammalian fibroblasts (CHO ade-A) which are deficient only for ATase. The effect of the modulation of E. coli ATase gene expression bk glucocorticoid on the rate of de novo purine synthesis was studied. Second, the effect of insulin on the rate of de novo purine synthesis and the expression of ATase was studied in primary cultured rat hepatodytes. Third, amalian ATase was purified from rat liver and its native and denatured subunit molecular weights were examined.
[RESULTS] The successful transfer and transcription of E. coli ATase gene to a single band of about 3.8kb was shown by Southern and Northern Analysis. The enzyme activity of the extract was increased with glucocorticoid-dependency from 4.4 to 18.3% of the wild type fibroblasts. Western Analysis showed two bands of 57 and 7OkD in E. coli, and a glucocorticoid- dependent 72kD band in the transformants. The rate of DNA or de novo purine synthesis was not increased, nor the purine-independent growth capacity was acquired. Insulin selectively induced ATase enzyme protein in primary cultured rat hepatocytes. ATase purified from rat liver was a heterotetrameric protein with a molecular weight of 240 kD and two subunit molecular weights of 62 and 57 kD.
[CONCLUSION AND DISCUSSION] The artificial expression of E. coli ATase in purine-auxotrophic mammalian cells did not complement the purine-dependent growth in spite of the demonstrable enzyme activity in the cell extract, probably because of the limited enzyme activity or of the two molecular species of ATase subunits. The molecular cloning of mammalian ATase is under way.
|
