FINE MAPPING OF REPLICATION ORIGINS (ORIA AND ORIB) IN NICOTIANA-TABACUM CHLOROPLAST DNA

Using a partially purified replication complex from tobacco chloroplas ts, replication origins have been localized to minimal sequences of 82 (pKN8, positions 137683-137764) and 243 bp (pKN3, positions 130513-13 0755) 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 r olling circle replication. Double Y patterns were observed when a chlo roplast DNA template containing both oris (pKN9) was tested. Dpnl anal ysis and control assays with Escherichia coil DNA polymerase provide a clear method to distinguish between true replication and DNA repair s ynthesis, These controls also support the reliability of this in vitro chloroplast DNA replication system, EM analysis of in vitro replicate d 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 o ri sequences reported here contain all the necessary elements for supp ort of chloroplast DNA replication in vitro.