ANALYSIS OF THE TOBACCO CHLOROPLAST DNA-REPLICATION ORIGIN (ORIB) DOWNSTREAM OF THE 23S RIBOSOMAL-RNA GENE

We have mapped the origin of DNA replication (oriB) downstream of the 23 S rRNA gene in each copy of the inverted repeat (IR) of tobacco chl oroplast DNA between positions 130,502 and 131,924 (IRA) by a combinat ion of approaches. In vivo chloroplast DNA replication intermediates w ere examined by two-dimensional agarose gel electrophoresis. Extended are patterns suggestive of replication intermediates containing extend ed single-stranded regions were observed with the 4.29 kb SspI fragmen t and an overlapping EcoRI fragment from one end of the inverted repea t, while only simple Y patterns were observed with a 3.92 kb BamHI-Kpn I fragment internal to the SspI fragment. Other restriction fragments of tobacco chloroplast DNA besides those at the oriA region also gener ated only simple Y patterns in two-dimensional agarose gels. Several c hloroplast DNA clones from this region were tested for their ability t o support in vitro DNA replication using a partially purified chloropl ast protein fraction. Templates with a deletion of 154 bp from the Ssp I to the BamHI sites near the end of the inverted repeat resulted in a considerable loss of in vitro DNA replication activity. These results support the presence of a replication origin at the end of the invert ed repeat. The 5' end of nascent DNA from the replication displacement loop was identified at position 130,697 for IRA (111,832 for IRB) by primer extension. A single major product insensitive to alkali and RNa se treatment was observed and mapped to the base of a stem-loop struct ure which contains one of two neighboring BamHI sites near the end of each inverted repeat. This provides the first precise determination of the start site of DNA synthesis from oriB. Adjacent DNA fragments con taining the stem-loop structure and the 5' region exhibit sequence-spe cific gel mobility shift activity when incubated with the replication protein fraction, suggesting the presence of multiple binding sites. ( C) 1997 Academic Press Limited.