Heterogeneity in the level of ampicillin resistance conferred by pBR322 derivatives with different DNA supercoiling

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Abstract
Cloning of an EcoRI restriction fragment, containing the 900 bp γ-terminal sequence of transposon Tn1000, into pBR322, resulted in two plasmids, pICV63 and pICV64, which differed in the orientation of the cloned fragment within the replicon and in the level of ampicillin resistance conferred on the host cell. The DNAs of these plasmids differ in superhelicity and we suggest that a change in supercoiling of pICV63 DNA leads to this plasmid conferring resistance to only low levels of ampicillin, probably by reducing the expression of the bla gene. This hypothesis is supported by the fact that topA or supX mutations, which abolish topoisomerase I, reduce still further the level of resistance to ampicillin of pICV63-containing cells, whereas the gyrB226 compensatory mutation renders these cells more ampicillin resistant. Plasmid pICV63, therefore, enables mutant alleles of genes governing DNA topology to be recognized.