The mitochondrial DNA (mtDNA) molecules of some rats obtained from the SASCO colony contain six EcoRI-sensitive sites while the mtDNA molecules of other rats obtained from the same source contain only four EcoRI sites. By mapping the positions of the EcoRI sites on the molecules relative to the D-loop it was determined that mtDNA molecules from all rats have four EcoRI sites in common, but all the mtDNA molecules of some rats have two extra EcoRI sites. EcoRI sensitivity of mtDNA molecules was shown to be maternally inherited. Construction of heteroduplex molecules failed to reveal evidence of gross nucleotide sequence changes associated with differences in EcoRI sensitivity. Mitochondria were isolated from rat livers as described (7), except that mitochondria were pelleted by centrifugation at 15,000 X g, and DNA was prepared from mitochondria and separated into covalently closed circular molecules and open (nicked) circular molecules as described in Wolstenholme and Fauron (4). Fragments resulting fr i either EcoRI or HindIII digestion (4, 9) were circularized as follows. The restriction enzyme was destroyed by heating to 630 for 5 min and the DNA fragment-containing solution was dialyzed againsts 33 mM Tris-HCI (pH 7.6). MgCl2 was added to 6.6 mM, ATP to 0.06 mM, and dithiothreitol to 10 mM (10). One unit of DNA ligase isolated from T4-infected E. coil cells (Miles Laboratories) was added to 100 ,1 of solution containing DNA at a concentration of less than 10 ,gg/ml, and the mixture was incubated for 48 hr at 40 and prepared for electron microscopy at room temperature.Heteroduplexes were constructed as follows. A 1:4 mixture of nicked circular mtDNA molecules containing six EcoRI sites and fragments resulting from complete EcoRI digestion of mtDNA molecules containing four EcoRI sites were dialyzed against 95% formamide/10 mM EDTA (pH 8.2) for 1 hr to denature the DNA, then against 50% formamide/100 mM Tris. HCI (pH 8.2)/10 mM EDTA for 1 hr to achieve approximately 50% renaturation, and prepared directly for electron microscopy with a hypophase containing 20% formamide/10 mM Tris-HCI (pH 8.2)/i mM EDTA (11). The lengths (as percentage circular genome) of double and single-stranded segments of heteroduplex molecules were determined by comparison with the mean lengths of totally double-stranded and totally single-stranded circular molecules contained in the same preparation.All of the confidence limits given (+) are standard deviations, and the number of observations in each case is given by n.
RESULTSA preparation of mtDNA obtained from the pooled livers of six rats was shown by electron microscopy to contain approximately 95% circular molecules. The molecular weights of these circular molecules had a unimodal distribution with a mean of 10.34 + 0.26 X 106 (n = 30). Analysis of this mtDNA (or of separated covalently closed and nicked circular molecules) by agarose gel (1.6%) electrophoresis after EcoRI digestion revealed eight bands at positions expected for fragments with mean lengths equivalent to a...