Recently we have found a genetic element (msDNA element) encoding a reverse transcriptase (RT) and a branched, covalently linked RNA-DNA compound (msDNA-RNA compound) in the E. coli B chromosome. This genetic element is not present in E. coli K12 strains. The msDNA-RNA compound is synthesized by a novel reverse transcription mechanism using the 2'OH of a specific internal guanine as a primer. In order to understand the nature of the msDNA element and a possible role of reverse transcription in the evolution of procaryotes, we plan to characterize the msDNA element and the process of reverse transcription in E. coli B by genetic and biochemical methods. We will determine precisely its location and length, the structure of the msDNA element-chromosome junctions, and its functional components by comparative studies of the restriction sites and nucleotide sequences of E. coli B and E. coli K12 chromosomes in the vicinity of the msDNA element. These experiments should show similarities and differences between the msDNA element of E. coli B and retroelements in eucaryotes. We will test the hypothesis that the msDNA element is a retrotransposon by assaying transpositional activity after putting a selective marker into the msDNA element. We plan to purify the E. coli B RT and characterize its template-primer specificity, which would determine whether or not RT copies cellular RNA. We also plan to develop a simple system to study mutants in msDNA synthesis and to screen for RT inhibitors by taking advantage of the antisense nature of msDNA.
