Figure 4. Adaptive gene amplification upon drug selection.

(A) Schematic representation of the DHFR-TS locus and the circular amplicon generated by HR between DRs (black arrows) on chromosome 6 in L. major. Genomic DNA was extracted from WT and MTX stressed promastigotes. Amplicon detection in the unstressed WT population (right panel, lane 1), after two passages with MTX (at EC₅₀, 0.2 µM) (right panel, lane 2), and after removing MTX pressure (lanes 3–7). C^a and C^b are the primers used for detecting the rearrangements. (B) Schematic representation of the PTR1 and MRPA locus and the linear amplicon with inverted duplications generated after annealing of IRs (black arrows) on chromosome 23 in L. infantum (left panel). Detection of the PTR1 amplicons by PCR using primers L^a and L^b in the unstressed WT 263 population (right panel, lane 1), after four passages with MTX (at EC₅₀, 0.2 µM) (lanes 2–4), and after removing MTX pressure (lanes 5–8). Lane 9 is a no DNA template control. (C) Kinetics of the selection and loss of the DHFR circular amplicon containing cells in LV39 (filled circle) and PTR1 linear amplicon containing cells in L. infantum (filled square) under MTX pressure (continuous line) and after drug removal (dashed lines). Average of three biological independent experiments is shown. (D) Adaptive MRPA gene amplification upon SbIII selection. Semiquantitative PCR was performed with primers L^a and L^b. Genomic DNA was extracted from L. infantum WT (lane 2) cells stressed with SbIII at 160 µM (4×EC₅₀) (lanes 3–5) and from cultures after drug removal (lanes 6–8). Lane 1 is a no DNA template control. Densitometric ratios of PCR band intensities are indicated at the bottom. One representative experiment out of four is shown. Amplification of the chromosomal GAPDH gene was used as a reference to normalize the amount of template DNA loaded.