The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels.

Abstract

Recombinant DNA techniques were used to study various aspects of rep gene function in Escherichia coli. In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the lambda pL promoter. By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction. Cells carrying such plasmids showed no ill effects from the high concentration of the protein. To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique. The viability of E. coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions. We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.