Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Kinetoplast DNA (? = 1.703 g/ml.) was isolated by preparative cesium chloride ultracentrifugation in a fixed-angle rotor from total cell DNA of Leishmania tarentolae and examined in terms of sedimentation properties, melting characteristics, and appearance in the electron microscope. It consisted of several molecular types, either free or bound together in associations of variable size: minicircles (molecular weight = 0.56 ± 0.03 × 10⁶), catenated minicircles, “figure 8” molecules, and long molecules. The associations seem to be held together by the long molecules threading through the smaller circles and catenanes. The large associations could be broken down by sonication, DNase II-treatment, or shear forces. Minicircles, catenated dimers, trimers, and small linear fragments were separated on preparative sucrose gradients of sonicated DNA, and S_20,w values were assigned to each molecular type by band sedimentation in the analytical ultracentrifuge.