In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): A remnant of bacterial rolling circle and conjugativeplasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from t he chromosome of an alpha-proteobacterium-like ancestor and developed durin g evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid m p1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmi ds. Several mp1 subclones were tested for their ability to support DNA repl ication using a newly developed in vitro system. Neutral/neutral two-dimens ional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replic ation. Replication activity was very high for a BamHI-restricted total plas mid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragmen t. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the stro ngest in vitro DNA replication activity, while a 1161 bp-template outside o f this region resulted in a substantial loss of activity. Electron microsco pic studies of in vitro DNA replication products from the highly active clo nes also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between position s 628 and 776 on the plasmid map. This sequence shares homology with double -stranded rolling circle origin (dso) or transfer origin (oriT) nicking mot ifs from bacterial plasmids. mp1 is the first described rolling circle plas mid in eukaryotes. (C) 1998 Academic Press.