The effect of progressive denaturation of open circular molecules (component II) and supercoiled covalently closed circular molecules (component I) of rat liver mitochondrial DNA has been followed by heating in the presence of formaldehyde and examination in the electron microscope. After heating at 49°C, two, three, or four regions of strand separation were visible in 25% of the component II molecules. Comparisons of the patterns of distribution of these regions in individual molecules indicated that they occurred at at least three specific positions around the molecule. Also, these regions, which were assumed to be rich in adenine and thymine, were within a segment which was less than 50% of the length of the molecule. After heating at 50°C, up to 14 regions of strand separation were observed, but when comparisons were made no clear groupings were found. At 51°C, component II molecules were completely separated into a single-stranded circle and a single-stranded linear piece of similar length. Strand separation was accompanied by shortening of the molecule. At 70°C, single-stranded circles had a mean length of 2.7 µ, compared with 5.0 µ for native molecules. Progressive heating of component I molecules resulted first in conversion to an open circle (I') and then to a second supercoiled form (I''). Visualization of further denaturation products of component I was prevented by crosslinking of the molecule by formaldehyde at high temperatures.

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