Reversed genetics: a new approach to the elucidation of structure--function relationship

Methods for generating point mutations at predetermined sites of RNA or DNA genomes have been developed. With Qbeta RNA as a template, minus strands were synthesized in vitro in a stepwise, substrate-controlled reaction. The nucleotide analogue N4-hydorxyCMP was introduced in the desired position, the minus strands were completed with the four standard triphosphates and used as templates to synthesize plus strands; about 30% of the progeny plus strands showed a base transition at the position corresponding to the nucleotide analogue. Two mutant RNAs with extracistronic nucleotide substitutions have been generated; one of these was viable, albeit with a reduced propagation rate, while the other was non-infectious. Furthermore, mutants with changes at the initiation codon of the coat cistron were prepared. An analysis of ribosome binding to such mutant RNAs revealed the importance of the A-U-G region for the formation of the initiation complex. With a similar approach applied to the beta-globin complementary DNA (CDNA) plasmid PbetaG, point mutations have been introduced at the positions corresponding to amino acids 121 to 123.