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Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 microM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.

As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.

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This chapter describes the methodology used both in performing the electrophoretic karyotype of the protozoan parasite Trypanosoma cruzi and mapping the genetic markers of the chromosomal bands, the construction of chromosome-specific YAC contigs, and their use to assign a chromosomal location to whole genome shotgun sequences.

Resumen Describimos los parámetros hematológicos del roedor subterráneo endémico de Chile Spalacopus cyanus (Rodentia, Octodontidae). Nuestros resultados indican que presenta una condición hematológica que difiere en los altos valores de Hb por eritrocito (HCM) encontrados en roedores de hábitos típicamente subterráneos, donde su HCM representa 49.9 y 47.2% de lo informado para Ctenomys talarum y Ctenomys australis. Sin embargo, S. cyanus presenta algunas características hematológicas que incrementarían su capacidad de transporte de oxígeno en la sangre, tales como un elevado número de glóbulos rojos y un reducido tamaño de éstos. Aunque nuestros resultados contribuyen a la comprensión de los atributos que explican el uso del ecotopo subterráneo por esta especie, se requieren estudios que informen sobre las condiciones atmosféricas de sus galerías, el efecto del tipo de suelo sobre estas últimas, afinidad del O 2 por la hemoglobina (efecto del 2.3 DPG) y los niveles de expresión de los genes EPO y HIF-1α bajo normoxia e hipoxia.