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

Abstract

• 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. 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. 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. 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. 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. 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. In alkali, mitochondrial DNA sedimented as a heterogeneous collection of fragments.

• 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 10·10⁶–11·10⁶. 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. The close similarity of the physicochemical properties of mitochondrial DNA and the circular viral DNA molecules is stressed.