THE FIDELITY OF REPLICATION OF MOUSE MITOCHONDRIAL DNA-pSC101 RECOMBINANT PLASMIDS GROWN IN E. COLI K12

ABSTRACT Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoR I endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the mtDNA replicated as part of a plasmid in E. coli lacked the alkali lability characteristic of animal mtDNAs. Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single base replication errors occur at a frequency of less than one nucleotide in three hundred. Electron microscopic analyses of plasmid—LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSClOl and mtDNA fragments.