The unusual properties of mtDNA from a “low-density” petite mutant of yeast

Abstract

1. We have purified the mtDNA from the “low-density”, ethidium-induced petite mutant RD1A by repeated CsCl gradient centrifugation and hydroxylaptite chromatography. 2. The density in neutral CsCl of this DNA is 1.671 g/cm 3 and its T m in 0.2 M Na + is 69 °C. Base composition analysis gives A = T and G = C and G+C = 6 mole percent. 3. Quantitative renaturation studies show: (a) Denatured RD1A mtDNA renatures with second-order kinetics and contains no self-complementary single strands detectable by hydroxylapatite chromatography. (b) RD1A mtDNA must consist of a perfect repetition of a sequence of 300 nucleotides or less. (c) RD1A mtDNA is complementary to roughly 0.5 % of wild-type yeast mtDNA. 4. The behaviour of purified RD1A mtDNA on hydroxylapatite suggests that it largely consists of aggregates that are mechanically trapped on the column. Elution required a temperature near the T m of the DNA. 5. RD1A mtDNA sedimented through 3 M alkaline CsCl as heterogeneous linear DNA with a median molecular weight of about 1.7 · 10 6. 6. In neutral CsCl gradients containing ethidium the bulk of RD1A mtDNA showed a restricted uptake of ethidium; this was abolished by treatment with pancreatic deoxyribonuclease I. 7. Electron micrographs of RD1A mtDNA spread by the protein monolayer technique contained very large networks and an occasional small circle; spreading of the DNA under denaturing conditions confirmed that less than 0.1 weight percent is circular. 8. We propose that most of the isolated RD1A mtDNA is present in large fishnet concatenanes; the unwinding restriction imposed on DNA of which the strands are plaited into a fishnet concatenate explains the restricted ethidium uptake.