Abstract
The plasmid pKYM isolated from the Gram-negative bacterium Shigella sonnei has been shown to replicate via a rolling-circle mechanism (14). We have confirmed the plus origin of pKYM in a region of 173 base-pairs by cloning the fragment in pUC18 plasmid of Escherichia coli (E. coli). A derivative of pUC18 which contains this fragment multiplies normally in an E. coil polA when the Rep protein of pKYM is supplied in trans. The 173 base-pairs of pKYM contains the following characteristic structures indispensable for replication in double-stranded form: 1) the consensus sequence, 5′CTTAaggGATAaaT, of the plus origins of pUB110 plasmid family, 2) three directly repeated sequences of AATGPuC/AG downstream of the consensus sequence and 3) forty-five nucleotides upstream of the consensus sequence.
The minus origin was found on a fragment containing nucleotides 281 base-pairs, that was localized upstream of the plus origin. Both the plus and minus origins are necessary for the stable multiplication of pKYM in E. coli. Although the nucleotide sequence of plus origin of pKYM has a strong homology to plus origins of the pUB110 plasmid family (14), the minus origin shares no homology to those of pUB110 plasmid family.
