Mutant Caenorhabditis elegans RNA polymerase II with a 20,000-fold reduced sensitivity to α-amanitin

Abstract

A double mutant ama-1(m118m526) gene results in an RNA polymerase (Rpo) II that is unusually resistant to α-amanitin. Rpo II activity in isolated Caenorhabditis elegans cell nuclei is inhibited 50% by α-amanitin at a concentration of 150 μg/ml, making this enzyme 150 times more resistant to the toxin than Rpo II from the singly mutant allele, ama-1(m-118), 20,000 times more resistant than the wild-type Rpo II, and about six times more resistant to amanitin than is Rpo III. It was determined that the SL1 spliced leader precursor is transcribed by Rpo II, and this transcript was used to measure Rpo II activity. The Rpo II activity is unstable in vitro, and the mutant strain has a temperature-sensitive sterile phenotype. The highly resistant double mutant was selected among four million progeny of the mutagenized ama-1(m118) parent by its ability to grow and reproduce in 200 μg/ml amanitin in the presence of a permeabilizing agent, Triton X-100.