The recent discovery of reverse transcriptase (RT) in the prokaryotes opens an exciting new biological system to study the origin of RT, and its roles and functions in the prokaryotes. RT in myxobacteria appears to be as old as other genomic genes, indicating that this enzyme existed before the eukaryotes evolved. On the other hand, our data suggest that RTs in Escherichia coli were acquired during the course of their evolution. In both cases, RT is associated with a genetic system (retron), which functions to produce a peculiar branched RNA-linked msDNA. In this proposal, we will use E. coli as a model system to elucidate their functions and roles in the cells. For this purpose: (1) We will first focus our efforts on further biochemical characterization of extensively heterogeneous retrons from independent clinical E. coli isolates. We will determine the entire genetic structure of these retrons including the DNA sequence and how they are integrated in the E. coli genome. (2) In order to elucidate the roles and functions of the retron, we will attempt to determine how the msDNA molecules exist in the cells. For this purpose, the protein-msDNA complex will be isolated and biochemically characterized, and its possible role in the production of cDNA in the cells and its ability to function as a transposable element will be examined. (3) We will also attempt to purify RTs and other components required for the synthesis of msDNA in order to elucidate the precise molecular events leading to the production of msDNA molecules. (4) In order to elucidate how many more natural E. coli isolates for the existence of retrons and to establish their phylogenetic relationship. We will also examine the existence of retrons or the production of msDNA in other prokaryotes, in particular archaebacteria. (5) We will attempt to create artificial retrons in E. coli which may be used for cDNA synthesis in vivo, for gene amplification, for the production of stable ribozymes or as an easy screening method to detect drugs against RT such as AZT.
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