THE MUTATIONAL ANALYSIS OF THE PLUS ORIGIN AND THE IDENTIFICATION OF THE MINUS ORIGIN OF THE PLASMID PKYM WHICH REPLICATES VIA A ROLLING-CIRCLE MECHANISM

The plasmid pKYM isolated from the Gram-negative bacterium Shigella so nnei 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-pair s by cloning the fragment in pUC18 plasmid of Escherichia coli (E. col i). A derivative of pUC18 which contains this fragment multiplies norm ally in an E. coli polA when the Rep protein of pKYM is supplied in tr ans. The 173 base-pairs of pKYM contains the following characteristic structures indispensable for replication in double-stranded form: 1) t he consensus sequence, 5'CTTAaggGATAaaT, of the plus origins of pUB110 plasmid family, 2) three directly repeated sequences of AATGPuC/AG do wnstream of the consensus sequence and 3) forty-five nucleotides upstr eam of the consensus sequence. The minus origin was found on a fragmen t containing nucleotides 281 base-pairs, that was localized upstream o f the plus origin. Both the plus and minus origins are necessary for t he stable multiplication of pKYM in E. coli. Although the nucleotide s equence of plus origin of pKYM has a strong homology to plus origins o f the pUB110 plasmid family (14), the minus origin shares no homology to those of pUB110 plasmid family.