Contribution of yeast artificial chromosome-based physical maps to the final assembly of the Trypanosoma cruzi genome.

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.

Telomere and subtelomere of Trypanosoma cruzi chromosomes are enriched in (pseudo)genes of retrotransposon hot spot and trans-sialidase-like gene families: the origins of T. cruzi telomeres.

Here, we sequenced two large telomeric regions obtained from the pathogen protozoan Trypanosoma cruzi. These sequences, together with in silico assembled contigs, allowed us to establish the general features of telomeres and subtelomeres of this parasite. Our findings can be summarized as follows: We confirmed the presence of two types of telomeric ends; subtelomeric regions appeared to be enriched in (pseudo)genes of RHS (retrotransposon hot spot), TS (trans-sialidase)-like proteins, and putative surface protein DGF-1 (dispersed gene family-1). Sequence analysis of the ts-like genes located at the telomeres suggested that T. cruzi chromosomal ends could have been the site for generation of new gp85 variants, an important adhesin molecule involved in the invasion of mammalian cells by T. cruzi. Finally, a mechanism for generation of T. cruzi telomere by chromosome breakage and telomere healing is proposed.

A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi.

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.

A refined molecular karyotype for the reference strain of the Trypanosoma cruzi genome project (clone CL Brener) by assignment of chromosome markers.

We present a useful refinement of the molecular karyotype of clone CL Brener, the reference clone of the Trypanosoma cruzi Genome Project. The assignment of 210 genetic markers (142 expressed sequence tags (ESTs), seven cDNAs, 32 protein-coding genes, eight sequence tagged sites (STSs), 21 repetitive sequences) to the chromosomal bands separated by pulsed field gel electrophoresis (PFGE) identified 61 chromosome-specific markers, two size-polymorphic chromosomes and seven linkage groups. Fourteen new repetitive elements were isolated in this work and mapped to the chromosomal bands. We found that at least ten repetitive elements can be mapped to each chromosomal band, which may render the whole genome sequence assembly a difficult task. To construct the integrated map of chromosomal band XX, we used yeast artificial chromosome (YAC) overlapping clones and a variety of probes (i.e. known gene sequences, ESTs, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 1.3 Mb, covering 37% of the entire chromosome. We found some degree of polymorphism among YACs derived from band XX. These results are in agreement with data from phylogenetic analysis of T. cruzi which suggest that clone CL Brener is a hybrid genotype [Mol. Biochem. Parasitol. 92 (1998) 253; Proc. Natl. Acad. Sci. USA 98 (2001) 7396]. The physical map of the chromosomal bands, together with the isolation of specific chromosomal markers, will contribute in the global effort to sequence the nuclear genome of this parasite.

Actividad de diterpenoides de azorella compacta, llareta, sobre amastigotes de trypanosoma cruzi / Activity of dipernoids isolated from azorella compacta, llareta, on trypanosoma cruzi amastigotes

The trypanocidad activity against amastigote forms of SPA-14, Tulahuen and G strains and CL Brener clone of Trypanosoma cruzi of diterpenoids isolated from Azorella compacta. Phil. (Llareta), a plant with ethnomedicinal prestige from prespanish age, was investigated. Amastigocidal activity was shown in azorellanol (2), diterpene isolated by first time, with an inhitory concentration 50 (IC) that varied between 60 M (CL Brener clone) and 84 M (SPA-14 strain), and in mulin -11,13 -dien-20-oico acid (5) with IC between 41 µM (G strain) and 87 mM (CL Brener clone). The cytotoxicity levels of both compounds against Hela and Vero cells and macrophages J144 are lower than nifurtimox and similar to gentian violet

Niveles de anticuerpos anti-Gal en personas infectadas y no-infectadas por trypanosoma cruzi: probable inducción por bacterias y por el parásito / Anti-Gal antibodies levels in trypanosoma cruzi infected and non-infected persons: probable induction by bacteria and by the parasite

Se estudiaron los niveles de anticuerpos contra epítopes Gal Ó 1,3 Gal en 407 sueros humanos chagásicos (92) y no chagásicos (315), mediante la reacción de hemaglutinación con eritrocitos de conejo; con inmunoelectrotransferencia se investigó la reactividad de sueros con altos títulos de anticuerpos anti-Gal frente a antígenos de escherichia coli y serratia marcescens. Finalmente, utilizando un anticuerpo anti-Gal purificado se identificó epítopes Gal Ó 1,3 Gal en formas metacíclicas de 12 cepas altoandinas chilenas de trypanosoma cruzi. Entre los 92 sueros chagásicos, se demostró que en el 68,5 por ciento (63) de los menos chagásicos se detectó anticuerpos anti-Gal a títulos ò 1:1.600, mientras que entre los sueros no chagásicos, sólo el 15,6 por ciento (49) mostró respuesta anti-Gal a títulos similares. Estos datos sugieren que la determinación de estos anticuerpos podría contribuir a complementar el diagnóstico de la infección, especialmente cuando se establezcan títulos de corte ò 1:3.200. La inmunoelectrotransferencia mostró que sueros de personas infectadas con T. cruzi reconocen varios antígenos presentes en E. coli y S. marcescens, lo que refuerza la idea de que a lo menos en parte estas bacterias serían capaces de estimular estas respuestas. El análisis autorradiográfico utilizando anticuerpo anti-Gal purificado, mostró diferencias en la expresión de los epítopes Gal Ó 1,3 Gal en las diferentes cepas de T. cruzi. Estos resultados sugieren que los anticuerpos anti-Gal podrían tener real significado en los mecanismos de inmunidad natural y protección de la infección en chilenos infectados con T. cruzi