Abstract:
This thesis describes aspects of genetic regulation within and near a replication origin (ori-1) of the F plasmid. A number of transcriptional promoters were isolated, precisely mapped, and characterized with respect to their strengths and modes of regulation. The principal techniques employed in these investigations were: "shotgun" molecular cloning of restriction fragments into a galactokinase-based promoter selection vector, assays for galactokinase activities, DNA sequencing and S1 nuclease mapping of transcripts. Major findings from this study can be summarized as follows: 1). Promoters for the essential replication genes pifC and E were cloned and shown to be autoregulated at the transcriptional level. 2). An E.coli protein, integration host factor (IHF), was found to modulate the activity of the pif operon promoter. 13). Two promoters which direct transcription in opposite directions from within the minimal ori-1 region were discovered. 4). Transcription from both ori-1 promoters was shown to be repressed by the mini-F encoded D protein. 5). Precise transcriptional startsites of the pifC gene and the two ori-1 promoters were determined. 6). A mini-F protein (D) was shown to resolve dimers of a plasmid which contains a site-specific recombination locus from near ori-1, and a facile assay system for this function was developed.
