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1.
Genome Res ; 15(1): 36-43, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15632088

ABSTRACT

Trypanosomatids are an ancient family that diverged from the main eukaryotic lineage early in evolution, which display several unique features of gene organization and expression. Although genome sequencing is now complete, the nature of centromeres in these and other parasitic protozoa has not been resolved. Here, we report the functional mapping of a centromere in the American trypanosome, Trypanosoma cruzi, a parasite with an unusual mechanism of genetic exchange that involves the generation of aneuploidy by nuclear hybridization. Using a telomere-associated chromosome fragmentation approach, we show that the region required for the mitotic stability of chromosome 3 encompasses a transcriptional "strand-switch" domain constituted by a 16-kb GC-rich island. The domain contains several degenerate retrotransposon-like insertions, but atypically, lacks the arrays of satellite repeats normally associated with centromeric regions. This unusual type of organization may represent a paradigm for centromeres in T. cruzi and other primitive eukaryotes.


Subject(s)
Centromere/genetics , Chromosomes/genetics , DNA Fragmentation/genetics , Physical Chromosome Mapping/methods , Trypanosoma cruzi/genetics , Animals , Chromosome Aberrations , Genetic Vectors/genetics , Genome, Protozoan , Mitosis/genetics , Molecular Sequence Data , Monosomy/genetics
2.
Int J Parasitol ; 34(1): 63-71, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14711591

ABSTRACT

WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the beta-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that mitochondrial import is mediated by an alternative targeting signal.


Subject(s)
Antigens, Protozoan/genetics , Mitochondria/metabolism , Trypanosoma cruzi/genetics , Animals , Base Sequence , Gene Deletion , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , Sequence Analysis, DNA , Signal Transduction , Transduction, Genetic/methods
3.
J Biol Chem ; 277(19): 17062-71, 2002 May 10.
Article in English | MEDLINE | ID: mdl-11842085

ABSTRACT

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.


Subject(s)
Glutathione Peroxidase/chemistry , Glutathione Peroxidase/physiology , Glutathione/analogs & derivatives , Glutathione/metabolism , Microbodies/enzymology , Oxygen/metabolism , Spermidine/analogs & derivatives , Spermidine/metabolism , Thioredoxins/metabolism , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Animals , Blotting, Western , Cloning, Molecular , Cytosol/metabolism , DNA/metabolism , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Genetic Vectors , Green Fluorescent Proteins , Hydrogen Peroxide/pharmacology , Immunoblotting , Kinetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Models, Chemical , Molecular Sequence Data , Oxidation-Reduction , RNA/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Subcellular Fractions/metabolism , Time Factors , Transfection , Glutathione Peroxidase GPX1
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