Temperature-sensitive RNA polymerase II mutations in Chinese hamster ovary cells

Abstract

Mutant Chinese hamster ovary cell lines temperature-sensitive (TS) for growth and containing TS mutations in RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) have been isolated. Wild-type cells were treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and a population of cells possessing mutations in RNA polymerase II was initially selected by isolating alpha-amanitin-resistant clones at 34 degrees . Of 168 such alpha-amanitin-resistant isolates screened for temperature sensitivity, nine were TS for growth at 39.5 degrees . By examining the behavior of the alpha-amanitin resistance of these TS cell lines in somatic cell hybrids, the TS mutation in a number of them was shown to be in RNA polymerase II. Hybrid cells obtained by the fusion of the TS and alpha-amanitin-resistant cells with cells possessing alpha-amanitin-sensitive polymerase II grew at both 34 degrees and 39.5 degrees ; the TS mutations were recessive. At 34 degrees all the hybrids were alpha-amanitin-resistant and possessed a mixture of alpha-amanitin-resistant and sensitive polymerase II. At 39.5 degrees the alpha-amanitin-resistant polymerase II activities in hybrids of four of the TS cell lines were lost; these four lines were alpha-amanitin-sensitive and possessed only alpha-amanitin-sensitive polymerase II. Temperature-insensitive revertants of two of these mutants were isolated. Reversion of the TS phenotype for mutants TsAma(R)-1 and TsAma(R)-8 was accompanied by an alteration in the level of alpha-amanitin resistance of the RNA polymerase II activities in the revertant cells. Together these data provide convincing evidence that TS mutations in RNA polymerase II can be coselected with alpha-amanitin resistance.