Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei

Restriction endonucleases PstI, EcoRI, HapII, Hhal, and S₁ nuclease were used to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in Kinetoplast DNA (kDNA) networks of T. brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S₁ nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 X 10⁶, determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 X 10⁶, determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 X 10⁶ daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 μm) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 μm) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. It is concluded that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. It is concluded that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.