Cloning and delimiting one chloroplast DNA replicative origin of Chlamydomonas

The EcoRl restriction fragments containing D-loops which marked the replication origin of chloroplast DNA were identified in two different species of Chlamydomonas. Each fragment was cloned in the E. coli plasmid pBK325. The cloned fragments were compared by restriction endonuclease analyses and by heteroduplex analyses in the electron microscope. The relative position of the D-loop regions and the homologous regions between the 2 fragments was determined. The D-loops were located within one short homologous region of 0.42kb in length between the 2 cloned restriction fragments. The homologous region was subcloned in pBR322. Closed circular plasroid DNAs containing the short homologous region showed preferred denaturation in the D-loop region under physiological salt concentration which suggested that D-loop region was AT rich. Sequence divergence was detected at both ends of the D-loop region. Southern blot analyses indicated the presence of species-specific repetitive sequences within the divergent regions.