Haemophilus influenzae is a useful system for studying DNA recombination. Under appropriate conditions, all cells in a culture can be induced to take up Haemophilus-specific DNA from the medium and recombine it with the resident chromosome (genetic transformation). A major aim of this study is to combine electron microscopy with recombinant DNA techniques to visualize the recombination associated with transformation. Cells will be transformed with a cloned fragment of the H. influenzae chromosome to see the interaction of a defined DNA with a unique region of the recipient chromosome. Digestion of the recipient-transforming DNA complex with appropriate restriction endonucleases will reduce the branched intermediates to a predictable size so that a uniform population of intermediates can be analyzed. If multiple classes of intermediates are seen, they will be arranged in a logical sequence to reconstruct the recombination pathway. Experiments using modified transforming DNAs containing partial homology to the resident chromosome may give some insight into the initial intractions between transforming and recipient DNAs as well as the role of branch migration in the formation of the final recombinant product. My earlier work has shown that recombination between resident plasmids represents a different pathway from that of genetic transformation. A novel intermediate composed of two plasmids linked by a single strand of DNA may represent an early intermediate formed by asymmetric transfer of a single strand from one plasmid to its recombining partner, or may represent an artifact that arises when two fused plasmids partially dissociate. Plasmid recombination can be measured using mutant strains and modified plasmids to test various models for the initiation of plasmid recombination. Knowledge about the in vivo mechanisms of recombination will provide a basis for determining the fidelity of future in vitro systems of recombination. A complete understanding of recombination may give insight into its biological role and how it might contribute to the maintenance and pathogenesis of H. influenzae.
| Trieu, V N; McCarthy, D (1989) Identification of Escherichia coli DNA helicase IV with the use of a DNA helicase activity gel. J Bacteriol 171:2128-35 |
| McCarthy, D (1989) Cloning of the rec-2 locus of Haemophilus influenzae. Gene 75:135-43 |
| McCarthy, D; Kupfer, D M (1987) Electron microscopy of single-stranded structures in the DNA of competent Haemophilus influenzae cells. J Bacteriol 169:565-71 |
| McCarthy, D; Cox, S S (1986) rpe, a cis-acting element from the strA region of the Haemophilus influenzae chromosome that makes plasmid establishment independent of recombination. J Bacteriol 168:186-91 |
