Abstract The use of the microdiffusion spreading technique, positive staining and dark field electron microscopy, to analyse isolated purified kinetoplast DNA (kDNA) has resulted in a considerable improvement in image quality. Combined with a survey of the literature on the ultrastructure of kDNA which has been published over the last 10 years this image improvement allows some generalizations to be made and a clear picture of how networks of kDNA are constructed of mini-circles, maxi-circles, oligomers and linear DNA has emerged. The kinetoplast in situ within the single mitochondrion, in the species covered by this review, is in the form of a concave disc 1–2μm in diameter with a height, in any one species, which corresponds to less than half the contour length of the mini-circle in that species. The mini-circles may be compressed into bundles of fibres which lie perpendicular to the long axis of the kinetoplast. The gross morphology of kDNA, which is released as a cap structure and expands during isolation and purification into a network of DNA fibres, is probably genera specific in the three genera dealth with primarily in this review. Crithidia networks are 10–20μm and have a ring fishnet structure with a prominent rim of mini-circle rosettes. Trypanosoma networks are 4–6μm and ar e usually flat and circular. Leishmania networks are 3–7μm and are elongated in one dimension as ovals. In networks which have not been overstretched by spreading tensions most mini-circles are linked at two sites at equal distance from each other. The size of the mini-circles and the number of mini-circles which bridge between two interconnection points (particularly apparent in the rims on the edge of networks) is constant within groups of closely related species. Crithidia have mini-circles of 0.78–0.8μm contour length and 16 mini-circles bridge to the sides of a connection point. These mini-circles may break their second linkage site to form rosettes of 32 mini-circles with only one linkage. In the same way in the trypanosomes like T. mega which are parasites of amphibians 8 mini-circles of 0.73μm interconnect on each side of a point. In both the trypanosomes we have studied like T. b. brucei which infect mammals and in the leishmanias only 4 mini-circles of 0.3μm bridge between two interconnection points and can break to form rosettes of 8 mini-circles. The nature of the linkage of mini-circles to each other is still not certain. Catenation may be involved but the weight of evidence now suggests that some form of covalent closure (concatenation) may take place between regions of partial homology in each circle. Overstretching in isolation or in spreading for electron microscopy leads to the separation of rosettes, and tandem repeated chains of mini-circles. It is suggested that these can form fused dimers and oligomers in the form of long loops of DNA both within the inferior and at the periphery of networks. Maxi-circles with a genetic complexity and sequence homology consistent with their postulated role as coding mitochondrial DNA have now been found by most laboratories. The question is still open whether these maxi-circles are bound to the mini-circle networks where they may appear as supercoiled loops in addition to the stretched oligomers of mini-circles. The role of the satellite B of the brucei group of trypanosomes is as yet unclear and further research is also required on those dyskinetoplastic strains which have DNA of the same buoyant density as kDNA but not in mini-circular form. The extra-fast-banding false satellites probably contain histone depleted chromosomes of the same protein scaffold kind as have been found in other systems. The kinetoplast mini-circular network may be involved in mitochondrial division as its relationship to the basal body of the flagellum has similarities to the centriole-kinetochore-centromere complex in chromosome division in primitive eukaryotic organisms.
Read full abstract
Apr 27, 2000
Douglas A Galbi 
Open Access