Isolation and characterization of kinetoplast DNA from 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.
Fairlamb
A H
AH
Section for Medical Enzymology and Molecular Biology, University of Amsterdam, The Netherlands.
Weislogel
P O
PO
Hoeijmakers
J H
JH
Borst
P
P
D000818:Animals; D004262:DNA Restriction Enzymes; D018105:DNA, Kinetoplast / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure*; D008854:Microscopy, Electron; D051381:Rats; D015183:Restriction Mapping; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000302:isolation & purification; D014353:Trypanosomiasis, African / Q000097:blood
D018105:DNA, Kinetoplast; D004262:DNA Restriction Enzymes