In vitro replication of plasmid pKYM

: The crude extract (fraction II) prepared from E. coli could replicate plasmid pKYM, only when the extract contained the rep protein which was produced by the plasmid and essential for its multiplication in vivo. The intermediate of replication was accumulated when a dideoxynucleotide triphosphate was added to the reaction mixture. By analyzing the intermediate, the initiation site of the deoxyribonucleic acid replication and the direction of replication could be determined. The replication initiated inside the ori region of pKYM and the direction was unidirectional. The analysis also suggested that the synthesis of the lagging strand stopped at almost the same site as the initiation site.