The Journal of Biological Chemistry

A number of mutant Chinese hamster ovary (CHO) cell lines resistant to the cytotoxic action of n-amanitin have been isolated. The a-amanitin sensitivity of the different mutant cell lines varied widely, but correlated well with the au-amanitin sensitivity of the RNA polymerase II activity in each of these mutant cell lines. In comparison with the RNA polymerase II of wild-type cells, three mutants, Ama39, Ama6, and Amal, required respectively 2to 3-fold, 8. to lo-fold, and about 800-fold higher concentrations of a-amanitin for inhibition of their polymerase II activity. Determination of the equilibrium dissociation constants (K,) for complexes between 0[8H]methyl-demethyl-y-amanitin and RNA polymerase II indicated that differences in cu.amanitin sensitivity were reflected in differences in the ability of the enzymes to bind amanitin. Hybrids formed by fusion of mutants with cells of wild-type sensitivity contained both mutant and wild-type polymerase II activities. Thus, each of the different cu-amanitin resistance mutations was expressed co-dominantly. A test for complementation between two of these mutations by measurement of both the cY-amanitin sensitivity and the [3H [amanitin binding by RNA polymerase II in Ama x Amal hybrid cells did not reveal any wild-type RNA polymerase II activity. These data provide evidence that the mutation to cr-amanitin resistance involves structural changes in the gene coding for the a-amanitin binding subunit of RNA polymerase II. These changes appear to account for the a-amanitin-resistant phenotypes of these mutant cells.