CHARACTERIZATION AND MODE OF IN-VITRO REPLICATION OF PEA CHLOROPLAST ORIA SEQUENCES

A partially purified replicative system of pea chloroplast that replic ates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and ins ensitive to chain terminators like dideoxynucleotides, arabinosylcytos ine 5'-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of repl ication. When sequences of either of the two chloroplast origins of re plication (OriA or OriB) are used as templates, the replicative interm ediates are found to have sigma structures. Electron microscopic analy sis of the sigma structures restricted with various enzymes reveals th at the initiation site of in vitro replication maps near the displacem ent-loop regions where replication initiates also in vivo. Although th e observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from th at observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication pr oceeds in the theta mode, the mode of replication usually observed in vivo.