AUTOCATALYTIC REPLICATION OF RECOMBINANT RNAS

The principle goal of this project is to apply recombinant RNA techniques to the study of problems of current biological interest. Recombinant RNAs consist of a heterologous sequence inserted at a selected site within a natural template for QBeta replicase. Large amounts of recombinant RNA can be synthesized autocatalytically by QBeta replicase in vitro. Two techniques for constructing recombinants will be developed. The first involves the direct ligation of heterologous RNAs within a modified MDV-1 (+) RNA cloning vector. The second employs a plasmid that serves as a template for the synthesis of perfect copies of MDV-1 RNA by Escherichia coli RNA polymerase in vitro. We plan the construction of a series of bifunctional recombinant RNAs, each retaining the biological function of the heterologous sequence and the replicability of the MDV-1 sequence. Three projects are outlined: 1) the synthesis of recombinant RNAs that will serve as substrates for the isolation and characterization of the RNA processing enzyme ribonuclease E from E. coli; 2) the construction and synthesis of recombinant substrates for the isolation of a putative prokaryotic RNA splicing enzyme from the thermaoacidophilic archaebacterium Sulfolobus solfataricus; and 3) the in vitro evolution and cloning of infectious recombinant potato spindle tuber viroid RNAs for use in determining mechanisms of viroid replication and pathogenesis.