Size and structural variations of mitochondrial DNA.

Mitochondrial DNA's from many vertebrates (mammals,'-7 birds,", I and amphibians') and at least one invertebrate (sea urchin9) heretofore examined exhibit a remarkable structural similarity. All of these DNA's were found in the form of closed circular duplex molecules of approximately 5 u in length, with an estimated molecular weight of 107 daltons. However, the estimates of the DNA content per mitochondrion vary in different types of mitochondria from one or two9 up to 14 molecules' 7 of 107 mol wt DNA. In fungi, Luck and Reich'? isolated a linear DNA filament of 13 million daltons from Neurospora mitochondria, and the presence in mitochondria of at least two distinct density species of DNA has been indicated." In yeast mitochondria the presence of rather heterogeneous-size linear filaments at least 5 A in length was observed by Sinclair et al.,12 but circular DNA species of varying length also present in the mitochondrial fraction were found to possess buoyant density identical to the corresponding nuclear DNA. This implies that the circular DNA might represent nuclear DNA contaminating the mitochondrial fraction. However, in separate experiments, Avers13 also observed in yeast mitochondria the presence of circular DNA molecules of up to 10.1 g in length and estimated that an equivalent of 10 A DNA molecule should be present per mitochondrion. In previous studies'4, 1 with protozoa (Tetrahymena and Paramecium) and several plants, we found DNA contents of 300 to 500 million daltons per mitochondrion. Purified Tetrahymena mitochondrial DNA had a relatively uniform sedimentation coefficient of 41S16 and was believed to represent an intact form existing in vivo. However, we were unsure whether the 41S DNA assumed a circular or linear structure. Studies were therefore made to examine Tetrahymena mitochondrial DNA by the Kleinschmidt's protein monolayer spreading technique."7 For comparison, DNA's of plants and monkey liver mitochondria were also included in these studies. Materials and Methods.-Tetrahymena mitochondria and mitochondrial DNA used in the present studies were isolated from ST strain of Tetrahymena pyriformis by the method described previously.'6 The isolated mitochondrial DNA showed a density of 1.686 gm cm-3 and less than 3% nuclear DNA contamination. Monkey (Macaca mulata) mitochondria were isolated from the liver (a courtesy of Dr. L. Mastroianni, Pennsylvania University Hospital) which was homogenized under aseptic conditions with a mortar and pestle in 0.25 M sucrose. The homogenate was centrifuged at 180 g for 6 min, and the resulting supernatant fluid was centrifuged at 5000 g for 6 min. The mitochondrial pellet was resuspended in the same sucrose medium and washed three times by repeated centrifugation at 10,000 g for 15 min. The final mitochondrial pellet was treated with DNase (16 ,g/ml at 0.05 M MgSO4), and DNA was isolated as described.4' 16 Purified DNA was prepared for electron-microscopic examinations by the method of Kleinschmidt and Zahn.17 To examine purified DNA, a DNA-cytochrome c (0.01%) mixture in 1 M ammonium acetate was layered over a water-hypophase surface. To examine DNA released by osmotic disruption, isolated mitochondria suspended in cold