Evidence for two distinct origins of replication in the large endogenous plasmid of Anacystis nidulans R2

Summary Shuttle cloning vectors, capable of replication in Escherichia coli and in the cyanobacterium Anacystis nidulans R2, have been used to localize a putative origin of replication of the large endogenous plasmid (pANL) of A. nidulans R2. Utilizing the cloning flexiblity of the polylinker containing E. coli plasmid pDPL 13, we have constructed a series of deletion derivatives of a 11.7 kilobase segment of pANL believed to contain a functional origin of replication. Two distinct segments of pANL that are 5.7 and 4.7 kilobases in size carry sufficient information to support transformation of A. nidulans R2. These DNA fragments, designated by us ORI 1 and ORI 2, do not overlap and show no DNA homology by blot hybridization analysis. Additionally, neither of these fragments are homologous to the replication origin of the other endogenous plasmid (pANS) of A. nidulans R2. Intact hybrid plasmids capable of transforming A. nidulans R2 have been re-isolated from transformed cells indicating that these plasmids exist autonomously in both A. nidulans R2 and E. coli.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal