Regulation of RNA polymerase II activity in alpha-amanitin-resistant CHO hybrid cells

Abstract

CHO hybrid cell lines obtained by fusing cells of wild-type sensitivity to alpha-amanitin with mutant cells containing RNA polymerase II activity resistant to alpha-amanitin have both sensitive (wild-type) and resistant forms of RNA polymerase II. When these hybrids were grown in medium containing alpha-amanitin, the sensitive form of polymerase II was inactivated, and the activity resistant to alpha-amanitin increased proportionally. The total polymerase II activity level therefore remained constant. This regulation of RNA polymerase II activity occurred independently of that of RNA polymerase I and was similar to that observed previously in the alpha-amanitin-resistant rat myoblast mutant clone Ama102 (Somers, Pearson, and Ingles, 1975a). A sensitive radioimmunoassay was developed to quantitate the total mass of RNA polymerase II enzyme. Under conditions of regulation of the enzymatic activity when hybrids grown in alpha-amanitin exhibited a 2-3 fold increase in the activity of the alpha-amanitin-resistant enzyme, no major change in the enzyme mass was detected immunologically. However, quantitation of the alpha-amanitin-inactivated polymerase II of wild-type sensitivity by 3H-amanitin binding indicated that the loss of its enzymic activity was accompanied by a loss of 3H-amanitin binding capacity in the cell lysates. All these results taken together indicate that a mechanism for regulating the intracellular level of RNA polymerase II exists and that it involves changes in the concentration of enzyme.