ILLEGITIMATE RECOMBINATION BY DRUG RESISTANCE ELEMENTS

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Transposable elements play a major role in mediating DNA rearrangements within and between prokaryotic organisms. We propose to investigate the mechanism and regulation of transposition by prokaryotic insertion sequences. Our analysis will particularly emphasize transposon Tn10 and its component IS10 sequences, but will also analyze some aspects of IS26 and IS5. Seven specific projects are proposed below: (1) Biochemical analysis of IS10 transposition in vitro. (2) Genetic and physical analysis of IS10 transposase protein: isolation and characterization of general and specific classes of transposase mutants. (3) Genetic and biochemical comparison of transposition by IS26 and IS5 to that of IS10. (4) Genetic analysis of the fate of the transposon donor molecule during transposition. (5) Dissection of IS10 insertion specificity and investigation of the role of DNA melting in regional specificity of IS10 and IS26. (6) Mechanism of IS10-mediated translational control by anti-sense RNA: in vitro analysis of pairing between RNA-OUT and RNA-IN and binding of ribosomes to paired and unpaired RNAs. (7) Regulation of IS10 transposition by dam methylation: kinetics of methylation on non-replicating and replicating DNA in vivo and in vitro.