JBC, Vol. 251, Issue 9, 2729-2734, May, 1976
Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines
C. J. Ingles, A. Guialis, J. Lam and L. Siminovitch
A number of mutant Chinese hamster ovary (CHO) cell lines resistant to the
cytotoxic action of alpha-amanitin have been isolated. The alpha-amanitin
sensitivity of the different mutant cell lines varied widely, but
correlated well with the alpha-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 2- to 3-fold, 8- to 10-fold, and about 800-fold
higher concentrations of alpha-amanitin for inhibition of their polymerase
II activity. Determination of the equilibrium dissociation constants (KD)
for complexes between 0-[3H]methyl-demethyl-gamma-amanitin and RNA
polymearse II indicated that differences in alpha-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 alpha-amanitin resistance mutations was expressed
co-dominantly. A test for complementation between two of these mutations by
measurement of both the alpha-amanitin sensitivity and the [3H]amanitin
binding by RNA polymerase II in Ama6 X Amal hybrid cells did not reveal any
wild-type RNA polymerase II activity. These data provide evidence that the
mutation to alpha-amanitin resistance involves structural changes in the
gene coding for the alpha-amanitin binding subunit of RNA polymerase II.
These changes appear to account for the alpha-amanitin-resistant phenotypes
of these mutant cells.
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