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The Three Faces of Riboviral Spontaneous Mutation: Spectrum, Mode of Genome Replication, and Mutation Rate

Figure 1

Schematic representation of the experimental system used to isolate RT mutants.

A. E. coli NR16205 cells were transformed with pQßm100, which constitutively expresses low levels of Qß (−) strands. Some ß subunits of the Qß replicase are also transcribed from pQßm100 and, once translated, they replicate the (−) strands yielding (+) strands, which are infectious (i.e., they produce Qß virions). Transformants were plated with NR16205 cells and incubated overnight to generate wt Qß plaques, which were individually isolated. B. E. coli RTH cells, which carry pQßRT that expresses RT constitutively, were infected with wt Qß. After one cycle of infection (75 min), chloroform was added to the experimental cultures to prevent further infections. Samples of the resulting lysates were plated with RTH cells, rendering Qß plaques that were independently harvested in 96-well plates. The use of RTH cells during these steps assures that any RT mutant generated during the replication of wt Qß would be as able to produce a plaque as its wild-type siblings. C. Using a replica plater, the isolated Qß plaques were tested for the RT phenotype (impaired growth on non-complementing NR16205 cells but normal growth on RTH cells). Putative RT mutants were identified and sequenced, after which up to four sub-isolates per verified RT mutant were isolated and sequenced.

Figure 1

doi: https://doi.org/10.1371/journal.pgen.1002832.g001