Electron microscopic analysis of two nonconjugative derivatives of plasmid R1drd-19Km- from Escherichia coli

The plasmids pON5300 and pON5304, nonconjugative variants of the plasmid R1drd-19Km, were analyzed by electron microscopy. It was found by heteroduplex mapping that a 1.4 kb DNA segment was inserted into EcoRI E fragment of both plasmids, where some tra-genes and oriT are localized. Although this DNA segment was mapped to the same region its orientation was different in each of the two plasmids. The inserted DNA segment was identified as an IS10R sequence on the basis of analysis of self-annealed molecules of pON5304 and their cleavage with EcoRV restriction enzyme. These methods enable us not only to map IS10R sequences on 87 kb pON5300 and 65 kb pON5304 molecules, respectively, but also to define their orientation.