cruzi CL Brener [13] were aligned by reciprocal BLAST against each amastin coding sequences. Unique reads showing at least 99.7% of identity were mapped on the CDS and the coverage for each nucleotide was determined. Coverage values were normalized through z-score and the copy numbers were determined after determining the ratios between z-score and the whole genome coverage. Parasite culture T. cruzi strains or clones, obtained from different sources, were classified according to the nomenclature and genotyping protocols described by [32]. Epimastigote

forms of T. cruzi strains or clones Colombiana, G, Sylvio X-10, Protein Tyrosine Kinase inhibitor Dm28c, Y and CL Brener were maintained at 28°C in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum (FCS) as previously described [3]. Tissue culture derived trypomastigotes and amastigotes were obtained after infection of LLC-MK2 or L6 cells with metacyclic trypomastigotes generated in LIT medium as previously described [3]. Pulse-field gel

electrophoresis and Southern blot analyses Genomic DNA, extracted from 107epimastigotes and included in agarose blocks were separated as chromosomal bands by pulse-field gel electrophoresis (PFGE) using the Gene Navigator System (Pharmacia) as described by Cano et al. (1995) [33], with the following modifications: separation was done in 0.8% agarose gels using a program with 5 phases of homogeneous pulses (north/south, east/west) with interpolation for 135 h at 83 V. Phase 1 had pulse time of 90 s (run time 30 h); phase 2 120 s (30 h); phase 3200 s (24 h); phase 4 350 s (25 h); phase 5 800 s (26 h). Chromosomes from Saccharomyces cerevisiae (Bio-Rad) were used as molecular mass standards. Separated BYL719 cost chromosomes were transferred to nylon filters and MM-102 cell line hybridized with 32P labelled probes prepared as described in the following section. RNA purification and

Northern blot assays Total RNA was isolated from approximately 5 × 108 epimastigote, trypomastigote and amastigote forms using the RNeasy® kit (Qiagen) following manufacturer’s recommendations. RNA samples (15 μg/lane) were separated by denaturing agarose gel electrophoresis, transferred to Hybond-N+ membranes and hybridized with the 32P labeled Thiamet G fragments corresponding to each T. cruzi amastin sequence as described [3]. The probes used were PCR amplified fragments from total genomic DNA extracted from the CL Brener strain using primers described in Table 1, in addition to a PCR fragment generated by amplification of the insert cloned in plasmid TcA21 (corresponding to δ-amastin) and the 24Sα ribosomal RNA[6]. DNA fragments were labeled using the Megaprime DNA-labeling kit (GE HealthCare) according to the manufacturer’s protocol. All membranes were hybridized in a 50% formamide buffer for 18 h at 42°C and washed twice with 2X SSC/0.1% SDS at 42°C for 30 min each, as previously described [3]. The membranes were exposed to X-ray films (Kodak) or revealed using the STORM840 PhosphoImager (GE HealthCare).