Heterogeneity in sensitivity to cleavage by the restriction endonucleases ECORI and HindIII of circular kinetoplast DNA molecules of Crithidia acanthocephali.

Abstract

Kinetoplast DNA (kDNA) of the protozoan C. acanthocephali consists mainly of an association of .apprx. 27,000 covalently closed, 0.8-.mu.m (1.58 .times. 106 daltons) circular molecules apparently held together in a particular structural configuration of topological interlocking. The sensitivities of circular kDNA molecules to the restriction endonucleases EcoRI and HindIII were studied using agarose gel electrophoresis and EM. 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 2 enzymes indicated that .apprx. 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 (.apprx. 86%) are insensitive to either enzyme. 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 4 different kinds of circular molecules, in regard to EcoRI and HindIII sensitivity, are found in similar proportions in the kDNA association of different organisms.