DNA circles with cruciforms from Isospora (Toxoplasma) gondii

Abstract

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-μm circles containing a cruciform with 0.5-μm tails. We also found 23-μm circles with the properties expected of head-to-tail dimers of the 12-μm circles. Some of these dimers have two cruciforms with 0.4-μm tails, some have one cruciform with 0.8-μm tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 μg/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-μm tails are part of a 1.7-μm palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-μm circles may represent the mitochondrial DNA of Toxoplasma.