Characterization of the pea chloroplast DNA OriA region

One of the two origins of replication in pea chloroplast DNA (oriA) maps in the rRNA spacer region downstream of the 16S rRNA gene, and further characterization of this origin is presented here. End-labeling of nascent DNA strands from in vivo replicating ctDNA was used to generate probes for Southern hybridization. Hybridization data identified the same region that was previously mapped to contain D-loops by electron microscopy. Subclones of the oriA region were tested for their ability to support in vitro DNA replication using a partially purified pea ctDNA replication system. Two-dimensional agarose gel electrophoresis identified replication intermediates for clones from the region just downstream of the 16S rRNA gene, with a 450-bp SacI-EcoRI clone showing the strongest activity. The experiments presented in this paper identify the 940 base pair region in the rRNA spacer between the 3' end of the 16S rRNA gene and the EcoRI site as containing oriA. Previous studies by electron microscopy localized the D-loop in the spacer region just to the right of the BamHI site, but the experiments presented here show that sequences to the left of the BamHI site are required for replication initiation from oriA. DNA sequence analysis of this region of pea ctDNA shows the presence of characteristic elements of DNA replication origins, including several direct and inverted repeat sequences, an A + T rich region, and dnaA-like binding sites, most of which are unique to the pea ctDNA oriA region when compared with published rRNA spacer sequences from other chloroplast genomes.