Several methods for site-specific mutagenesis have been developed that have aided in the recovery of altered genes carried on small circular DNA molecules. The characterization of these mutations and the identification of their mutant phenotype affords a means for studying gene products. By introducing mutations and correlating them with alterations in gene function, it has been possible to define transcriptional promotors, RNA splicing and polyadenylation sequences, ribosome binding sites, and polypeptide domains needed for enzymatic activities. This laboratory has long been involved in projects that involve the synthesis of RNA by QB replicase from templates which have been altered in their nucleatide sequence. Now a small DNA plasmid has been developed that expresses significant quantities of the replicase subunits and a second plasmid that allows assay of replicase activity through synthesis of a suppressor tRNAs. By generating a large number of mutants in the phage coded subunit of RNA replicase one will be albe to determine the precise structural requirements for replicase activity. Dr. Mills has worked with this enzymne for twenty years and is now in the enviable position of using in vitro genetics for a structure-function analysis of this protein. Over the years he has made many significant contributions. Support is strongly recommended.
