Donald R. Mills

Autocatalytic replication of recombinant RNA

We demonstrate that a heterologous RNA sequence can be copied in vitro by Q beta replicase when it is inserted into a naturally occurring Q beta replicase template. A recombinant RNA was constructed by inserting decaadenylic acid between nucleotides 63 and 64 of MDV-1 (+) RNA, using phage T4 RNA ligase. The insert was located away from regions of the template known to be required for the binding of the replicase and for the initiation of product strand synthesis. To minimize the disruption of template structure, we inserted the heterologous sequence into a hairpin loop on the exterior of the molecule. Q beta replicase copied this recombinant RNA in vitro, and the complementary product strands served as templates for the synthesis of additional copies of the original recombinant RNA. The reaction was therefore autocatalytic and the amount of recombinant RNA increased exponentially. A 300-fold amplification of the recombinant RNA occurred within nine minutes. Insertion of biologically significant RNAs into the MDV-1 RNA sequence should allow them to be replicated autocatalytically.

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

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.