Specialized Recombination in the E coli Chromosome

Deonier, Richard

Abstract

This project will investigate specialized recombination processes associated with sites on the bacterial chromosome, in order to explore the role of such sites in the sequence organization of the bacterial genome. Bacterial loci capable of undergoing a variety of specialized recombination processes are revealed by the production of F-prime plasmids containing specific lengths of bacterial DNa from Hfr strains. One category of sites is revealed by the repeated production of Type II F lac ion proC ion purE ion plasmids, which result from recombination between a single site between proA and argF and one of three sites located 250-400 kb clockwise of this first site. Another class of sites is revealed by the production of the transfer-defective tra delta F purE ion, tra delta F purE ion proC ion), or Tra delta F proA ion plasmids, whose bacterial segments terminate at specific loci within one of three general regions. The third category of sites to be examined is associated with the deletion end-points generated by the transposon gamma delta (equals Tn1000), which is also a natural constituent of the F plasmid, and which is capable of producing Type I F-prime plasmids from an Hfr or from a preformed Type II F-prime. Mapping of all of these sites by restriction enzyme digestion, Southern hybridization, and electron microscope heteroduplex methods will be undertaken to determine any relationships among these three categories of sites and to determine whether they are active in other specialized recombination processes. Some of the sites will be sequenced to determine whether they are similar to other known genetic elements (e.g., oriT of f). By cloning appropriate bacterial fragments containing these sites into specially designed small plasmids, improved and efficient in vivo recombination assay systems will be developed which will allow these processes to be examined in a variety of mutant backgrounds (e.g., himA- or hip-). This will permit rapid genetic analysis, and will provide recombination products that are small enough that physical analysis will be facilitated.