Structural organization of the maxicircle variable region ofTrypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites

Abstract

The maxicircle of the parasitic protozoan Trypanosoma brucei, one component of the mitochondrial genome, has size differences among isolates that localize to the variable region (VR) between the ND5 and 12S rRNA genes. We present here the nucleotide sequence of this entire region, thus completing the sequence of the maxicircle genome. We also find heterogeneously sized transcripts from throughout most of the VR. The VR has three distinct sections, each with characteristic repeated sequences. The repeated sequences in two sections are short and highly reiterated; the intraspecies size variation occurs within this region. The third section contains non-repetitive sequences and a large duplication immediately upstream of the 12S rRNA gene. Two repeat units within section I contain a sequence that has homology to the DNA replication origin of minicircies. This region also contains sequences with homology to topoisomerase 11 binding and cleavage sites. These findings suggest a role for the VR in DNA replication of the maxicircle.

Stepwise Transfer of tRNA through the Double Membrane of Leishmania Mitochondria

“…Light microscopy of this fraction revealed the presence of large, presumably fused, membrane lamellae, with no evidence of intact mitoplasts. 2 As expected, malate dehydrogenase was predominantly located in the soluble fraction and succinate dehydrogenase in the particulate fraction (Table I). (Some malate dehydrogenase activity was also detected in the digitonin-soluble fraction; a similar result has been obtained with mammalian mitochondrial preparations (23) and probably represents contamination with the cytosolic form of the enzyme.)…”

“…2 Treatment of mitochondria with digitonin resulted in enhanced sensitivity of inner membrane proteins, such as succinate dehydrogenase, to protease treatment, whereas the matrix marker malate dehydrogenase remained unaffected (Fig. 2B), indicating the intactness of the inner membrane.…”

“…Essentially identical results were obtained when mitoplasts were prepared by hypotonic shock instead of digitonin. 2 The intramitochondrial distribution of RNA changed with time ( Fig. 3B).…”

“…Under these conditions we sometimes observed partial degradation of imported RNA, possibly by residual RNase during the postimport washing and mitochondrial lysis steps. 2 In order to make the assay more reliable in terms of RNA intactness and yield, the RNase concentration was lowered, whereas the incubation temperature was raised to 37°C to increase the rate of degradation. A RNase titration experiment (Fig.…”

“…The mitochondrial genomes of many protozoal species, including leishmania, trypanosomes, and plasmodium, are unusual for their apparently total lack of tRNA genes (1)(2)(3). To sustain the translation of organellar mRNAs, a large number of nuclear-encoded tRNAs are imported from the cytoplasm (4 -10).…”