Principal features of a model for the control of DNA replication of the broad host-range plasmid R1162 will be tested: (1) Does the directly-repeated sequence of R1162 DNA bind plasmid-encoded protein? This sequence will be chemically synthesized and tested for specific binding of purified plasmid proteins, expression of incompatibility and inhibition of R1162 DNA replication in vitro. Significant base-pairs will be identified from the locations of mutations affecting these properties, and by studies of base protection in the presence of DNaseI and methylating agents. (2) Does DNA synthesis begin within the inverted repeat? The importance of the inverted repeat DNA for replication, and the positions where DNA synthesis is initiated, will be determined by characterizing oriV-mutations and pseudorevertants, and by mapping nascent DNA fragments isolated from replicating molecules. (3) Does a plasmid protein track along the DNA? Protein-DNA complexes will be trapped by UV irradiation and labelled by colloidal gold-antibody. The distribution of protein along the plasmid DNA will be analyzed following the examination of these complexes by electron microscopy. (4) How is expression of the RepI region controlled? The starting point of RepI mRNA in two species will be determined by S1 mapping. Key base-pairs within the promoter will be identified from the isolation and mapping of """"""""up-promoter"""""""" mutations. A radioactive hybridizing probe will be used to detect a putative countertranscript, and its role in regulation determined by the measurement of levels of this RNA in cells containing cop plasmids. The level at which control is exerted will be concluded from the behavior of gene fusions, and by quantitative estimates of the amounts of RepI mRNA made in the presence and absence of a negative regulator.
