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. 2014 May 20;12(5):e1001868. doi: 10.1371/journal.pbio.1001868

Figure 7. Adaptive gene amplification in RAD51 −/− parasites.

Figure 7

(A) Diagram of the RAD51 locus in L. infantum WT with the NEO and HYG disruption cassettes (left panel). Southern blot analysis (right panel) of WT (lane 1); RAD51/RAD51::NEO (lane 2) and RAD51::NEO/RAD51::HYG (lane 3) genomic DNAs digested with PvuII (P) and hybridized with the 5′ UTR RAD51 probe. (B and C) Circular amplicon selection in RAD51 −/− parasites. L. infantum WT pSPαZEOα (1 and 4), RAD51 −/− pSPαZEOα (2 and 5), and RAD51 −/− pSPαZEOαRAD51 (3 and 6) were either cultured in the absence of drug (−) or in the presence of 0.2 µM MTX for five passages. Total DNA was extracted from cells, and semiquantitative PCRs were performed to detect DHFR-TS (B) or PTR1 (C) circular amplicons. Amplification of the chromosomal GAPDH gene was used as reference to normalize the amount of template DNA. The data shown are averages of three independent experiments (*p≤0.05; **p≤0.005; ***p≤0.0005, two-tailed Student's t test).