Asci restriction enzyme

Molecular karyotype was obtained from L. infantum WT, mrpA^−/-, mrpA^−/- Sb-resistant mutants, and revertants by separation of chromosomes through pulse field electrophoresis. 10⁸ mid-log phase parasites were embedded in low melting point agarose blocks, digested with proteinase K, and electrophoresed in a contour-clamped homogenous electric field apparatus (CHEF Mapper, Bio-Rad, Hercules, CA, USA). The blocks were mounted in 1% agarose gel and electrophoresed in 0.5x Tris-Borate-EDTA running buffer at 5 V cm⁻¹ with a 120° separation angle at 14 °C for 30 h. A range of 150–1500 kb was applied for a wide chromosomal separation, resolving most Leishmania chromosomes in a single molecular karyotype gel. Saccharomyces cerevisiae chromosomes were used as a DNA size marker (Bio-Rad, Hercules, CA, USA). For Southern blots, genomic DNA was isolated using DNAzol reagent (Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions, then digested with the AscI restriction enzyme (New England Biolabs Inc, Ipswich, MA, USA). Digested genomic DNA or PFGE-derived molecular karyotype was transferred by capillarity onto nylon membranes (Hybond-N⁺, Amersham Pharmacia Biotehc, Sunnyvale, CA, USA) and cross-linked with a crosslinker. Blots were hybridized with [α-³²P] dCTP-labeled DNA probes according to standard protocol.