cruzi CL Brener  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 . Epimastigote
forms of T. cruzi strains or clones Colombiana, G, Sylvio X-10, Selleckchem RAD001 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 . 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 . 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) , 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 SCH727965 solubility dmso chromosomes were transferred to nylon filters and 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 selleck chemical fragments corresponding to each T. cruzi amastin sequence as described . 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. 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 . The membranes were exposed to X-ray films (Kodak) or revealed using the STORM840 PhosphoImager (GE HealthCare).