CONJUGATIVE MOBILIZATION OF PLASMID R1162

广宿主质粒R1162的共轭动员

Conjugative mobilization of broad host-range plasmid R1162 requires both oriT, a cis-active, 38 base-pair site, and the products of at least four plasmid mob genes. Mutagenized oriT DNA will be tested to identify the base-pairs important for mobilization, conjugation-dependent recombination, and nicking by relaxation complex. In addition, the sites of recombination, and relaxation complex-induced nicking within oriT will be mapped. The mob gene products will be identified by examining the polypeptides made in minicells containing cloned fragments R1162 DNA; the corresponding open reading frames will then be located by DNA base sequencing. The direction and strandedness of plasmid DNA transfer during mobilization will be determined by hybridizing newly-transferred DNA, isolated from minicells, with specific DNA probes. Mutations in mob genes whose products interact with oriT will be obtained as pseudorevertants of nonmobilizable, oriT-plasmids. The altered mob genes will be mapped by complementation and recombination. Finally, the polypeptide covalently joined at the relaxation complex nick site will be isolated, and the amino acid sequence partially determined. This sequence will then be compared to the base sequence of the mob genes, in order to identify the gene encoding the polypeptide.