The American trypanosome Trypanosoma cruzi is exposed to toxic oxygen metabolites that are generated by drug metabolism and immune responses in addition to those produced by endogenous processes. However, much remains to be resolved about the parasite oxidative defense system, including the mechanism(s) of peroxide reduction. Here we show that reduction of peroxides in T. cruzi is catalyzed by two distinct trypanothione-dependent enzymes. These were localized to the cytosol and mitochondrion. Both are members of the peroxiredoxin family of antioxidant proteins and are characterized by the presence of two conserved domains containing redox active cysteines. The role of these proteins in protecting T. cruzi from peroxide-mediated damage was demonstrated following overexpression of enzyme activity. The parasite-specific features of T. cruzi cytoplasmic peroxiredoxin and T. cruzi mitochondrial peroxiredoxin may be exploitable in terms of drug development.

A BSTR ACTThe unusual DNA base ␤-D-glucosylhydroxymethyluracil, called ''J,'' replaces Ϸ0.5-1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In Trypanosoma brucei, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens Leishmania donovani and Trypanosoma cruzi, J was found to be abundantly present in the (GGGTTA) n telomere repeats. Outside Kinetoplastida, J was found only in Diplonema, a small phagotrophic marine f lagellate, in which we also identified 5-MeCyt. Fractionation of Diplonema DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinof lagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both. T. brucei may have recruited J for the control of genes involved in antigenic variation.In the nuclear DNA of Trypanosoma brucei, Ϸ0.5-1% of Thy is replaced by the modified base ␤-D-glucosyl-hydroxymethyluracil (␤-gluc-HOMeUra) (1). This base that we call ''J'' was detected initially by 32 P-nucleotide postlabeling combined with twodimensional TLC (2D-TLC) (2), and we used this technique to show that approximately one-half of the cellular J is present in both strands of the telomeric (GGGTTA) n repeats (3). To map the location of J more precisely, we have generated antisera that immunoprecipitate J-containing duplex DNA and that detect this DNA with high sensitivity and specificity on dot blots (4). We have used these antisera to demonstrate that J is present in other repetitive DNA sequences but not in housekeeping genes or transcribed repeats (4). Moreover, we have shown that J is responsible for the blocked restriction sites that are present in silent telomeric variant surface glycoprotein (VSG) genes but not in actively transcribed VSG genes (4-6). This result has linked J to the transcriptional control of VSG genes.Thus far, J has been detected only in African trypanosome species that undergo antigenic variation (2). The availability of antibodies acting against J has prompted us to reinvestigate whether J is also present in other organisms. With anti-J-DNA immunoblots, approximately one J per 10 7 bases can be detected, which is Ϸ1,000-fold more sensitive than MATERIALS AND METHODSCells and DNA Analysis. DNA was derived from: T. brucei brucei (427); Trypanosoma congolense (WG81 and TSW13 bloodstream forms and WG81 procyclics); Trypanosoma vivax (Y58); Crithidia fasciculata (ϭ C. luciliae); Leishmania donovani (HU3); Leishmania tarentol...

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Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0.65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28 degrees C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0.6% agarose overlay containing G418 as the selective drug, on top of a 0.8% agar base. Transformation efficiencies were as high as 10(-5) cells per microgram of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of 'shotgun transformation' to generate libraries of T. cruzi recombinants.