MOLECULAR MECHANISM OF DNA TRANSPOSITION

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The focus of the proposed program is the continuing styd of the molecular mechanisms for transposition of the transposable elements IS1 and IS50 (and other recombination events mediated by the elements). These elements participate in the formation of compund transposons like Tn9 (IS1) and Tn5 (IS50). A model system has been developed and used to study transposition, cointegrate formation (plasmid fusion) and other recombination events mediated by the elements. This system uses an F-derived plasmid containing no resident transposable elements as one component, and various non-conjugative, pBR322-derived plasmids as the other element. The system is highly manipulable, easily portable into different genetic backgrounds, and permits precise analysis of the recombination products. The studies carried out so far in this program have established that cointegrates are not intermediates in the transposition of Tn9 or Tn5, in contrast to the major pathway for transposition of the Tn3-like elements. The precise nature of the molecular pathways are not yet clear, however. The goals of the proposed program will be: 1) to understand the molecular basis of the strong modulation of transposition activity by external transcription that we have discovered, 2) to understand the quantitative effects of differing activities for the flanking elements of the compound transposons on the spectrum of recombination products (we have developed a simple quantitative model that can be tested using our system), 3) to understand the source of the strong target specificity and orientation effect that we have observed for IS1, 4) to understand the roles of the structural components of IS1 in the transposition process (the cloned end sequences, and the six largest, overlapping open reading frames within the element) by cloning and site-directed mutagenesis methods, 5) to analyze the properties (in vivo and in vitro) of the proteins, and/or RNA's coded for by the element (one IS1 protein has been identified in minicells, and a model-building study initiated). Another, recently-observed effect by which IS1 can activate the expression of a gene by providing the -35 region of a "hybrid" promoter will be investigated. This effect may have important implications for the understanding of the formation and stability of compound transposons.