Cloning the origin of transfer region of the resistance plasmid R1

Abstract

The insertion of a 7.7-kb EcoRI fragment of the resistance plasmid R1 into pBR325 yielded a plasmid which is mobilizable by pDB12, a multicopy derivative of R1drd-19 lacking most of the resistance determinants. The vector alone was not mobilizable in this system. From this observation we conclude that we have cloned the origin of transfer (oriT) of R1. After inserting a 5.3-kb PvuII-EcoRI fragment of the 7.7-kb region into pUC9 the DNA was cleaved randomly with DNaseI and BamHI linkers were attached to the ends. A subsequent BamHI digestion and electrophoretic separation of the resulting DNA molecules by their size allowed us to generate an ordered series of stepwise shortened plasmids. Plasmids with a deletion of approximately 3400 bp could no longer be mobilized. Since the next larger plasmid with 284 additional base pairs could be mobilized, we are able to confine the oriT location within this extra nucleotide stretch. The DNA sequence of this region was determined. Dominant features within the DNA region are a high AT content and five inverted repeats, which might function as recognition or substrate sites for proteins of the conjugational transfer system.