In vitro site-directed mutagenesis: generation and properties of an infectious extracistronic mutant of bacteriophage Qbeta.
An infectious extracistronic mutant of phage Qbeta has been prepared by site-directed mutagenesis. Qbeta RNA minus strands containing the mutagenic base analog N4-hydroxy-CMP instead of UMP at position 39 from the 5' end were synthesized in vitro and used as template for Qbeta replicase to synthesize one generation of plus strands. E. coli spheroplasts were infected with the newly synthesized plus strands and phage recovered from single plaques. RNA sequence analysis revealed that four out of the eighteen phage clones analyzed contained RNA with an A leads to G transition at position 40 from the 3' end (which corresponds to position 39 of the minus strand). Thus, the viability of phage Qbeta does not depend on a unique nucleotide sequence in the 3'-extracistronic RNA segment. Upon in vivo propagation of mutant 40, spontaneous true revertants arose with high frequency and overgrew the parental clone within about 10 passages, indicating a selective disadvantage of the extracistronic mutant. Replication of mixtures of wild type and mutant RNA in vitro resulted in a decrease of the proportion of mutated RNA in the progeny plus strands. The fact that Qbeta RNA containing an A leads to G transition in nucleotide--40 of Qbeta RNA is less efficiently replicated in vitro may explain the selective disadvantage of the mutant phage in vivo. The preparation of an infectious mutated RNA by site-directed mutagenesis shows that the method is suitable to produce specific nucleotide exchanges without impairing the biological competence of the RNA.