CHARACTERIZATION OF REPLICATION ORIGINS FLANKING THE 23S RIBOSOMAL-RNA GENE IN TOBACCO CHLOROPLAST DNA

Using 5' end-labeled nascent strands of tobacco chloroplast DNA (ctDNA ) as a probe, replication displacement loop (D-loop) regions were iden tified. The strongest hybridization was observed with restriction frag ments containing the rRNA genes from the inverted repeat region. Two-d imensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb BamHI fragment containing part of the rRNA operon. Analysis of in vitro rep lication products indicated that templates from either of the origin r egions supported replication, while the vector alone or ctDNA clones f rom other regions of the genome did not support in vitro replication. Sequences from both sides of the BamHI site in the rRNA spacer region were required for optimal in vitro DNA replication activity. Primer ex tension was used for the first time to identify the start site of DNA synthesis for the D-loop in the rRNA spacer region. The major 5' end o f the D-loop was localized to the base of a stem-loop structure which contains the rRNA spacer BamHI site. Primer extension products were in sensitive to both alkali and RNase treatment, suggesting that RNA prim ers had already been removed from the 5' end of nascent DNA. Location of an origin in the rRNA spacer region of ctDNA from tobacco, pea and Oenothera suggests that ctDNA replication origins may be conserved in higher plants.