Maxi-circles and mini-circles in kinetoplast DNA from trypanosoma cruzi

Abstract

Maxi-circles are a minor component of kinetoplast DNAs from all trypanosomatids studied, but they have not previously been found in Trypanosoma cruzi; We have spread intact kinetoplast DNA from the epimastigotes of strain Y in protein monolayers and analysed the mini-circle networks by electron microscopy. Long loops up to 10 micrometer were present, extending from the network rim; these are considered typical of maxi-circles. The presence of maxi-circles was proven by digestion of kinetoplast DNA with restriction endonucleases and S1 nuclease. This released a minor DNA component, detectable by agarose gel electrophoresis, which hybridized to maxi-circle DNA from Trypanosoma brucei. The molecular weight of the linearized maxi-circle of Trypanosoma cruzi is 26 . 10(6), as judged from its electrophoretic mobility in 0.6% agarose. Our restriction enzyme analysis of the mini-circles of Trypanosoma cruzi has confirmed their sequence heterogeneity and internally-repeated structure. We have found that more than 90% of the mini-circles are cut into 1/4 length molecules by endonuclease TaqI. Denaturation and renaturation of mini-circles, cut once with endonuclease MboI, mainly yields linear and circular molecules with single-stranded eyes and tails in electron micrographs. This shows that 1/4 repeats contain sub-segments in which sequence divergence is extensive. Our EcoRI and HapII digests differ in fragment size distribution from those previously reported. This suggests that this distribution may not be a stable characteristic of the Y strain.