Abstract
To test the rate-limiting quality of ATase for de novo purine synthesis and the complementality of a prokaryotic ATase for ATase-deficiency in CHO fibroblasts (CHO-ade) as a basic model for gene therapy, a recombinant between E.coli ATase and a glucocorticoid-responsive MMTV promoter was made. Stable transfectants of CHO-ade with this recombinant, cloned by G-418 cotransfection, were examined for dexamethasone (DEX)-dependent ATase expression at 10−6 M. The average ATase activity in 9 transfectants with DEX reached 18.3% of that in a wild type strain of CHOK1, while it was only 4.4% without DEX. Western analysis was performed with anti-E.coli ATase antibody. An E.coli extract showed two bands of 57 and 70K, of which 57K corresponds to a size of purified ATase and a reading frame of 1515bp of cloned DNA. A CHO-ade extract showed no band irrespective of DEX. Extracts of several lines of transfectants showed only one DEX-responsive band of 70K with a good correlation to ATase activity. Based on these data it is suggested that E.coli ATase is bound with other components resulting in a 70K protein both in E.coli and CHO fibroblasts. The complementality of E.coli ATase for ATase-deficiency of CHO-ade is under way. The recombinant technology affords a valuable means in elucidation of purine synthesis regulation.
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Itakura, M., Yamaoka, T., Yoshikawa, H. et al. 64 CONTROLLABLE EXPRESSION OF E.COLI AMIDOPHOSPHORIBOSYLTRANSFERASE (ATase) GENE IN ATase-DEFICIENT MAMMALIAN FIBROBLASTS-A BASIC MODEL FOR GENE THERAPY. Pediatr Res 24, 121 (1988). https://doi.org/10.1203/00006450-198807000-00088
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DOI: https://doi.org/10.1203/00006450-198807000-00088