Evolution in vitro: Sequence and phenotype of a mutant RNA resistant to ethidium bromide

Abstract

Ethidium bromide inhibits the in vitro replication of MDV-1 RNA (a small replicating RNA molecule) by reducing the rate of chain elongation. In a serial transfer experiment, in the presence of ethidium, a mutant RNA was selected that was more resistant to ethidium inhibition than is the wild-type MDV-1 RNA.The complete nucleotide sequence of the mutant RNA was determined and three nucleotides in the mutant sequence were found to be different from those in the wild type. The mixture of mutant and wild-type RNAs present in successive transfers was also sequenced. Each of the three point mutations occurred at a different time. These results show that the mutant RNA did not arise from a pre-existing strand present in the wild-type population, but rather, occurred de novo in the course of the experiment.It is probable that the chemical basis of resistance is the elimination of ethidium binding sites due to the specific alterations in the nucleotide sequence, since the mutant RNA was found to bind less ethidium than the wild-type molecules.