ANALYSIS OF SOYBEAN CHLOROPLAST DNA-REPLICATION BY 2-DIMENSIONAL GEL-ELECTROPHORESIS

Chloroplast DNA replication was studied in the green, autotrophic susp ension culture line SB-1 of Glycine max. Three regions (restriction fr agments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were i dentified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragmen ts of the organelle genome, suggesting that these clones contain seque nces that are able to direct initiation of DNA replication in vitro. I n order to confirm that the potential in vitro origin sites are functi onal in vivo as well, replication intermediates were analyzed by two-d imensional gel electrophoresis using cloned restriction fragments as p robes. The two Pvu II fragments that supported deoxynucleotide incorpo ration in vitro apparently do not contain a functional in vivo replica tion origin since replication intermediates from these areas of the pl astome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a re plication bubble originating within its borders, which is indicative o f an active in vivo origin. Closer examination of cloned Sac I 14.5 su b-fragments confirmed high template activity in vitro for two, S/B 5 a nd S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replic ation intermediate patterns obtained for these sub-fragments are consi stent with the double D-loop model for chloroplast DNA replication wit h both origins being located in the large unique region of the plastom e [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo func tion.