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The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.

Kinetoplast DNA (kDNA) of the protozoan Crithidia acanthocephali consists mainly of an association of approximately 27,000 covalently closed, 0.8-micron (1.58 X 10(6) daltons) circular molecules apparently held together in a particular structural configuration by topological interlocking. The sensitivities of circular kDNA molecules to the restriction endonucleases EcoRI and HindIII have been studied using agarose gel electrophoresis and electron microscopy. Digestion with EcoRI or HindIII of collections of single circular molecules obtained from sonicated kDNA associations resulted in a single cleavage of 9.3 and 12% of the molecules, respectively. Digestion of intact kDNA associations with EcoRI or HindIII resulted in cleavage of 9.2 and 10.4%, respectively, of the component circular molecules, but not in detectable disruption of the characteristic structure of the associations. Analysis of the products of sequential digestion of kDNA with the two enzymes indicated that approximately 8% of the circular molecules each contain a single site sensitive to EcoRI and a single site sensitive to HindIII; 1.5-3% contain only an EcoRI-sensitive site; 3-4% contain only a HindIII-sensitive site; and the remainder (approximately 86%) are insensitive to either enzyme. Further, data obtained from sequential digestion experiments and from studies of the partial denaturation products of the circular molecules digested with EcoRI or HindIII indicated that when they occur the EcoRI site and the HindIII site are each at a unique position in all molecules, 10-13% of the circular contour length apart. Similar digestion products were found for kDNAs from different cloned organisms, suggesting that the four different kinds of circular molecules, in regard to EcoRI and HindIII sensitivity, are found in similar proportions in the kDNA association of different organisms.