Isolation and characterization of kinetoplast DNA from the bloodstream form of Trypanosoma brucei.
We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease 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 Trypanosoma 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 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), 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 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) 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. We conclude 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. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.
|Keywords||0 (DNA, Kinetoplast), Animals, DNA Restriction Enzymes, DNA, Kinetoplast/*chemistry/isolation & purification/*ultrastructure, EC 3.1.21 (DNA Restriction Enzymes), Microscopy, Electron, Rats, Restriction Mapping, Support, Non-U.S. Gov't, Support, U.S. Gov't, P.H.S., Trypanosoma brucei brucei/*genetics/isolation & purification, Trypanosomiasis, African/blood|
Fairlamb, A.H., Weislogel, P.O., Hoeijmakers, J.H.J., & Borst, P.. (1978). Isolation and characterization of kinetoplast DNA from the bloodstream form of Trypanosoma brucei.. The Journal of Cell Biology, 76, 293–309. Retrieved from http://hdl.handle.net/1765/2945