Genetic control systems in prokaryotes are traditionally thought to fall into two broad classes: transcriptional and translational controls. The former involves an effector molecule that affects transcriptional initiation by interacting with DNA sequences and the transcriptional machinery. Translational controls act through RNA transcripts and are limited by the extent of the transcribed DNA. By contrast, DNA replication controls are more complex. Where they are understood they appear to act via typical transcriptional or translational control systems. Recently, control mechanisms that act in cis but that originate at points some distance from the site of the affected function have been observed. Two examples are 1) Cis specific inhibition of Tn3 transposition by Tn3 and 2) Transcriptional activation by """"""""enhancer sequences"""""""". The goal of the proposed research is to determine the basis of cis specific interactions and to see if this constitutes a different and previously unappreciated genetic control system. Complex replicon systems in which cointegrate plasmids display cis specific inhibition of replication will be studied, with particular attention to pT181/pE194 cointegrates. Inhibitory loci will be mapped by mutation and deletion, further defined by DNA sequencing, and analyzed for transcriptional and translational effects as well as long range effects of DNA structure. Complex replicons composed of a chromosome and a plasmid also will be studied. In the E. coli initiation defective, temperature sensitive dnaA strains, an inserted replicon can """"""""integratively suppress"""""""" the Ts phenotype by serving as the origin of replication. There is evidence that the inserted replicon's origin may be active only at the non-permissive temperature. To study this possible conditional inhibition, origin activity in integratively suppressed strains will be assayed. Two assays will be employed. One method detects """"""""branched structures"""""""" at the inserted replicon's origin and utilizes Southern blotting versus a probe corresponding to that origin. This method will be most definitive if replication is unidirectional or delayed bidirectional. The second method assays gene dosage by quantitative DNA blotting and assumes that genes near the replicative origin are present in greater concentration than genes distal to the origin. The characterization of these specific systems may well define an important genetic control mechanism.
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