Cloning and characterization of Paramecium mitochondrial DNA replication initiation regions

Fragments containing the replication-initiation region of mitochondrial (mt) DNA from four species of Paramecium aurelia were ligated to the pBR322 plasmid and used to transform Escherichia coli. The criteria for identifying the desired mitochondrial DNA restriction fragments were based on the palindromic dimer structure of the replicative intermediate of this DNA. The nature of the cloned sequences was verified by hybridization to mitochondrial DNA fragments containing the initiation region, by determining the size of snapback renaturation products, and by determining the symmetry of restriction-endonuclease sites within the clones. Heterologous hybridization experiments showed that one species' initiation region is not homologous to the other three. Cleavage patterns of each of the clones by 18 different restriction enzymes were determined. There are no sites within approx. 250 bp of the center of the insert of any clone although there are numerous cleavages in more distal regions. These sites are generally symmetric about the center of the insert as expected for a palindromic structure. The characteristics of the cloned sequences support a proposed replication model for all four species studied.