Mitochondrial DNA. V. A 25 micron closed circular duplex DNA molecule in wild-type yeast mitochondria. Stucture and genetic complexity.

Abstract

Abstract 1. 1. Electron micrographs of DNA released by osmotic shock from isolated mitochondria of Saccharomyces carlsbergensis NCTC 74 and S. cerevisiae H1 contained closed circular duplex DNA molecules. 2. 2. The mean length of 6 closed circular molecules from S. carlsbergensis mitochondria was 25.6±0.9 μ and the mean length of fourteen molecules from S. cerevisiae mitochondria was 25.0±1.0 μ . The number of cross-overs for both yeast species was 6.5 per μ length. The majority of the linear molecules was longer than 15 μ, with lengths up to 27 μ. The DNA released from the mitochondria of S. cerevisiae H1 also contained three smaller closed circular molecules (3, 4 and 10 μ). 3. 3. All attempts to isolate the 25-μ circles intact were unsuccessful. DNA isolated from yeast mitochondria by a direct lysis method followed by CsCl equilibrium gradient centrifugation consisted exclusively of linear molecules of 2–24 μ with a modal length at 7–8 μ. 4. 4. A closed circular DNA fraction of nuclear density was present in the CsCl equilibrium gradients of yeast lysates in the amount of 1–4% of the nuclear DNA. The majority of these circles had an average contour length of 2 μ, but circles up to 7 μ were present. 5. 5. The renaturation of mitochondrial DNA of S. carlsbergensis and S. cerevisiae followed second-order kinetics with a renaturation constant about 1.1–1.6 times that of T 4 DNA, determined under identical conditions. 6. 6. We conclude that the intact mitochondrial DNA of S. carlsbergensis and S. cerevisiae consists of a circular molecule with a contour length of approx. 25 μ and a genetic complexity equivalent to this size.