Mitochondrial DNA II. Sedimentation analysis and electron microscopy of mitochondrial DNA from chick liver

Abstract

1. 1. The physiochemical properties of pure mitochondrial DNA from chick liver were studied by band sedimentation in the analytical ultracentrifuge and by electron microscopy. 2. 2. Up to 80 % of native chick-liver mitochondrial DNA sedimented in a homogeneous band with an s 20, w = 39 S, the remainder of the high-molecular-weight DNA sedimenting in a homogeneous band with an s 20, w = 27 S. In some preparations a minor third component with an s 20, w = 24 S was present. The 39-S component was not affected by the peptide hydrolase pronase, but it was converted into the 27-S component by treatment with pancreatic deoxyribonuclease (EC 3.1.4.5) or hydroquinone, or by “ageing”. 3. 3. Electron micrographs of all preparations of chick-liver mitochondrial DNA, spread according to the Kleinschmidt protein-monolayer technique, showed predominantly molecules in which no free ends could be distinguished. No branched molecules were seen in any preparation. 4. 4. Micrographs of 39-S DNA, prepared by preparative sucrose-gradient centrifugation, contained 84 % highly twisted circles, 14 % open or half-open circles and 1 % linear molecules. Micrographs of pure 27-S DNA contained only 4 % twisted circles, 77 % half-open or open circular molecules and 19 % linear molecules. 5. 5. The mean circumference of 63 more or less open molecules was 5.35 μ with 90 % of all values falling between 4.85 and 5.85 μ. 6. 6. In mitochondrial DNA denatured in 12 % formaldehyde, 3 major and 3 minor components were found in analytical band-sedimentation studies using 3 M CsCl containing 2 % formaldehyde as bulk solution. The major components were tentatively identified as the formaldehyde double-stranded cyclic coil ( s 20, w = 83 S), the single-stranded ring ( s 20, w = 32 S) and the single-stranded broken ring ( s 20, w = 28 S). 7. 7. In alkali, mitochondrial DNA sedimented as a heterogeneous collection of fragments. 8. 8. We conclude that chick-liver mitochondrial DNA in situ is a double-stranded circular molecule with a molecular weight of the sodium salt of 10·10 6–11·10 6. Both strands are covalently continuous and, on extraction, the DNA is obtained in a twisted circular form ( s 20, w = 39 S) which is converted into an open circular form ( s 20, w = 27 S) after cleavage of at least one phospho-diester bond. The 24-S component is tentatively identified as the linear form of mitochondrial DNA. 9. 9. The close similarity of the physicochemical properties of mitochondrial DNA and the circular viral DNA molecules is stressed.