MITOCHONDRIAL DNA .2. STRUCTURE AND PHYSICOCHEMICAL PROPERTIES OF ISOLATED DNA

The molecular topology and proportions of the DNA forms isolated from mitochondria of mouse fibroblasts (L-cells) were studied after removal of contaminating nuclear DNA by DNase. The DNA consisted of 40 to 60% covalently closed circular DNA monomers (4.74 ± 0.02μ; mol.wt 9.1 × 106), including 5% double-length molecules; in addition, up to 10% of the DNA consisted of multiple-length forms of two to four interlocked monomers; the remaining fraction contained mostly open (nicked) circles and some linear fragments; the latter could not be annealed to form circles. The total yield agreed with the chemically determined DNA content of L-cell mitochondria. A 3 to 5% difference in guanine + cytosine content was found for mitochondrial and nuclear DNA by three methods. A comparison of the contour lengths of circular mitochondrial DNA of ascites tumor cells, human liver, chicken liver and rat liver gave values of 4.76 ± 0.04μ, 5.06 ± 0.06μ, 5.20 ± 0.11μ and 4.94 ± 0.09μ, respectively, with slightly higher values for DNA bound to ethidium bromide. The small differences appear to reflect true differences in length, since co-spreading of mixtures of two types of DNA gave bimodal distributions with peak categories representative of the size of each DNA spread individually. The closed circular DNA component of L-cell mitochondria (DNA I) was isolated and studied with respect to the number of tertiary turns and its resistance to denaturation by alkali and heat. By electron microscopy, 33 ± 3 tertiary turns per molecule or 3.6 turns per 1 × 106 molecular weight were found. DNA I sedimented in alkaline CsCl 3.6 to 3.8 times faster than linear DNA. Electron microscopy showed highly twisted monomeric and some dimeric forms in the fast band, monomeric open duplex and single-stranded circles with linear strands peeling off or lying free in the slower bands. The Tm of DNA I in 0.1 SSC (standard saline citrate is 0.15 m-NaCl-0.015 m-sodium citrate) was about 22 °C higher than that of nicked mitochondrial DNA. © 1969.