Controllable Expression of an E. Coli Amidophosphoribosyltransferase (ATase) Gene in ATase-Deficient Mammalian Fibroblasts—a Basic Model for Gene Therapy
Amidophosphoribosyltransferase (ATase) (E.C.184.108.40.206) is presumably a rate-limiting enzyme in de novo purine synthetic pathway under a skillful regulatory system, although 5-phosphoribosyl 1-pyrophosphate (PRPP) synthetase is another possibility. The present study was designed to investigate the rate-limiting quality of ATase by artificially controlling expression of its activity, and to test the complementality of ATase-deficiency with a foreign ATase gene in mammalian cells. The experimental scheme, as in Fig. 1, was composed of production of a recombinant plasmid between an E. coli ATase gene and a glucocorticoid-responsive promoter and its transfection into a strain of Chinese hamster ovary fibroblasts which is deficient in ATase (CHO ade-A). Transfectants were cultured in the presence or absence of glucocorticoid, and were examined as to the complementality of de novo purine and DNA synthesis in purine-free medium with dexamethasone (DEX). The expression of E. coli ATase was also examined by northern, western analysis, and ATase activity.
KeywordsHerpes Zoster Thymidine Kinase Scintillation Counter Northern Analysis Western Analysis
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