Physico-chemical characterization of mitochondrial DNA from potato tubers

Abstract

1. A procedure was described for the mass isolation of mitochondria from potato tubers. The deoxyribonuclease-treated crude mitochondrial pellet was fractionated into several layers by sucrose gradient centrifugation. The light layer ( d = 1.18) contained highly purified mitochondria, as seen by electron microscopy. 2. The DNA extracted from this fraction gave only a sharp and symmetrical band at 1.706 g/ml in analytical CsCl gradient ultracentrifugation. Analysis of the very heavy and very light fractions of the preparative CsCl DNA band, revealed only molecules banding at 1.706 g/ml, which is evidence of the intermolecular homogeneity of this plant mtDNA. The buoyant density was shifted by 0.016 and 0.058 g/ml when mtDNA was respectively thermally denatured and run in an alkaline CsCl gradient. No band splitting was observed in an alkaline CsCl gradient. The melting curve was characterized by a sharp transition at T m = 88.8°C in 1 × SSC . 3. The intramolecular homogeneity has been demonstrated by CsCl gradients and thermal denaturations achieved with mtDNA of different molecular weights. No heterogeneity was detectable on the CsCl banding pattern and on derivatives of the melting curves. 4. The mtDNA renatured rapidly as seen by the buoyant density shift and hypochromicity, like other organelle DNAs. Except for the initial minutes the optical reassociation curves appeared homogeneous and the kinetic complexity was close to 100 · 10 6 daltons. 5. The size of the mtDNA molecules was studied by sedimentation velocity and by length measurement on the electron microscope. The molecular weight calculated from the s 2 0, w and from the molecular length was close to 60 · 10 6. Electron microscopy revealed that mtDNA molecules are linear and heterogeneous in size.