Fine mapping of replication origins (oriA and oriB) in Nicotiana tabacum chloroplast DNA

Using a partially purified replication complex from tobacco chloroplasts, replication origins have been localized to minimal sequences of 82 (pKN8, positions 137683-137764) and 243 bp (pKN3, positions 130513-130755) for oriA and oriB respectively, Analysis of in vitro replication products by two-dimensional agarose gel electrophoresis showed simple Y patterns for single ori sequence-containing clones, indicative of rolling circle replication. Double Y patterns were observed when a chloroplast DNA template containing both oris (pKN9) was tested. Dpnl analysis and control assays with Escherichia coli DNA polymerase provide a clear method to distinguish between true replication and DNA repair synthesis, These controls also support the reliability of this in vitro chloroplast DNA replication system, EM analysis of in vitro replicated products showed rolling circle replication intermediates for single ori clones (oriA or oriB), whereas D loops were observed for a clone (pKN9) containing both oris, The minimal ori regions contain sequences which are capable of forming stem-loop structures with relatively high free energy and other sequences which interact with specific protein(s) from the chloroplast replication fraction, Apparently the minimal ori sequences reported here contain all the necessary elements for support of chloroplast DNA replication in vitro.