Electron microscopic mapping of deletions on a streptococcal plasmid carrying extraordinarily long inverted repeats

Abstract

Deletions [Delta]101, [Delta]102, and [Delta]103 which occurred within the extraordinarily long inverted repeats of the self-ligated large EcoRI fragment of the Streptococcal MLS (macrolides, lincosamides, streptogramin B)-resistance plasmid pSM19035 led to the formation of plasmids pDB101, pDB102, and pDB103. Their molecular lengths were determined by contour length measurements to be 17.8, 17.4, and 13.9 kb, respectively. Electron microscopic examination of self-annealed molecules revealed stem-loop structures with inverted repeats comprising 41 to 91% of the mass of plasmids. Two unique sequences (US1 and US2) separated the inverted repeats in the case of pDB101 and pDB103, while in pDB102 the repeats were joined at one end and separated at the other by a unique sequence (US2). The size of the unique sequence US2 was identical for all three plasmids, and the location of the resistance determinant was determined by electron microscopic examination of self-annealed molecules of the recombinant plasmid pDB201. Mapping of the deletion termini, accomplished by combining electron microscopic and HindIII restriction data, suggested that deletions may occur at preferential sites.