Guialis A, Beatty B G, Ingles C J, Crerar M M
Cell. 1977 Jan;10(1):53-60. doi: 10.1016/0092-8674(77)90139-8.
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.
通过将对α-鹅膏蕈碱具有野生型敏感性的细胞与含有对α-鹅膏蕈碱具有抗性的RNA聚合酶II活性的突变细胞融合而获得的CHO杂交细胞系，同时具有敏感型（野生型）和抗性型的RNA聚合酶II。当这些杂交细胞在含有α-鹅膏蕈碱的培养基中生长时，聚合酶II的敏感型失活，而对α-鹅膏蕈碱具有抗性的活性则成比例增加。因此，聚合酶II的总活性水平保持恒定。RNA聚合酶II活性的这种调节独立于RNA聚合酶I的调节发生，并且与先前在对α-鹅膏蕈碱具有抗性的大鼠成肌细胞突变克隆Ama102中观察到的情况相似（Somers、Pearson和Ingles，1975a）。开发了一种灵敏的放射免疫测定法来定量RNA聚合酶II酶的总质量。在酶活性调节的条件下，当在α-鹅膏蕈碱中生长的杂交细胞中对α-鹅膏蕈碱具有抗性的酶的活性增加2至3倍时，通过免疫学方法未检测到酶质量有重大变化。然而，通过3H-鹅膏蕈碱结合对野生型敏感性的α-鹅膏蕈碱失活的聚合酶II进行定量分析表明，其酶活性的丧失伴随着细胞裂解物中3H-鹅膏蕈碱结合能力的丧失。所有这些结果综合起来表明，存在一种调节RNA聚合酶II细胞内水平的机制，并且它涉及酶浓度的变化。
