Further analyses based on sequencing data generated from large inserts previously mapped on specific T. cruzi chromosomes are warranted to solve this question. Figure 2 Genomic localization of amastin genes in different T. cruzi strains. Chromosomal bands from different T. cruzi strains, separated by Pulsed Field Gel Electrophoresis (PFGE) and transferred to membranes, were hybridized with 32P-labelled Selleck Ilomastat probes corresponding to β2-amastin (A), δ-Ama40 (B), δ-amastin (C) and tuzin genes (D). T. cruzi strains or clones are SylvioX-10 (Sylvio), Colombiana (Col.), G and Dm28c, Y and CL Brener (CLBr). Sizes of yeast chromosomal bands (Sc) are buy Ferrostatin-1 indicated on the left. Distinct patterns of amastin gene expression Because

analyses of amastin gene expression have been limited to members of the δ sub-family and these studies have not been conducted with different strains BAY 11-7082 datasheet of the parasite, we decided to evaluate by northern blotting the expression profiles of members of the δ- and β-amastin sub-families. We also decided to compare the expression levels of different amastin genes in parasite strains representative of T. cruzi I (Sylvio X-10 and G), T. cruzi II (Y) and in CL Brener (a T. cruzi VI strain). As shown in Figure 3, the levels of amastin transcripts derived from δ- and β- sub-families are differentially modulated throughout the T. cruzi life cycle. Most importantly, clear

differences in expression levels were found when different T. cruzi strains are compared: whereas in CL Brener , Y and Sylvio X-10 strains, transcripts of δ-amastins are up-regulated in amastigotes, as previously described in the initial

characterization of amastins performed with the Tulahuen Sclareol strain (also a T. cruzi VI strains) [6], the same was not observed with the G strain. Even though it presents a more divergent sequence and is transcribed from a different locus in the genome, the expression of δ-Ama40, similar to other δ-amastins, is also up-regulated in amastigotes in all strains analysed except in the G strain. In contrast, in all parasite strains, the expression of β1- and β2-amastin transcripts is up-regulated in epimastigotes. Similar to β2-amastin from CL Brener, two distinct δ-Ama40 transcripts with different sizes were detected in Y and G strains. It can be speculated that transcripts showing different sizes derived from δ-Ama40 and β2-amastin genes may result from alternative mRNA processing events. Recent reports on RNA-seq analyses indicated that alternative trans-splicing and poly-adenylation as a means of regulating gene expression and creating protein diversity frequently occur in T. brucei[17]. Current analyses of RNA-seq data will help elucidating mechanism responsible for the size variations observed for this sub-set of β- and δ-amastins. Moreover, the striking difference in the expression of δ-amastins observed in the G strain is also currently being investigated.