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. 2021 Apr 27;15(4):e0009377. doi: 10.1371/journal.pntd.0009377

Fig 5. Disruption of PTR1 in chromosomal DHFR-TS null mutants.

Fig 5

(A) Schematic representation of the PTR1 locus and of the integration of the puromycin N-acetyl transferase (PURO) and zeocin resistance gene (ZEO) deletion cassettes. The expected sizes after digestion with AfeI when hybridized to probe 2 (small black boxes) are shown. Also shown are the expected size of PCR fragments generated with primers FF75 and FF76 amplifying the coding sequence of PTR1, or with primers AB1 and AB2 located in its 5’ and 3’UTRs, respectively. The pSP72αBlastα-PTR1 episome is shown at the bottom. All cells from this Fig were grown in the presence of 50 μg/ml thymidine. (B and C) Southern blot analysis of genomic DNAs digested with AfeI hybridized with probe 2. The genotype of the strains for each lane is indicated on top of the blots. In panel B, the hybridized band with the ZEO integration is more intense than expected. This is likely due to tandem integration of the marker during selection. WT and +, wildtype; N, NEO; H, HYG; P, PURO; Z, ZEO; pBLAST, pSP72αBlastα. (D) Southern blot analysis of total DNAs (genomic and episomal) digested with XbaI and HindIII and hybridized to an intragenic DHFR-TS probe (see also S2 Fig). The genotype of the strains for each lane is indicated on top of the blot. WT and +, wildtype; N, NEO; H, HYG; P, PURO; Z, ZEO; pBLAST, pSP72αBlastα. P25, P50, P75 and P100 mean that parasites harboring the pSP72αBlastα-DHFR-TS episome were grown for 25, 50, 75 and 100 passages in absence of blasticidin in the presence of excess thymidine. (E) Left panel. Amplification of the PTR1 locus with primers AB1 and AB2 shows the replacement of PTR1 alleles by PURO and ZEO markers in the chromosome of DHFR-TSNEO/HYG PTR1PURO/ZEO cells harboring episomes coding for DHFR-TS or PTR1. Right panel. Amplification of the PTR1 coding sequence with primers FF75 and FF76 confirms that the psp72αblastα-PTR1 episome is not lost from DHFR-TSNEO/HYG PTR1PURO/ZEO cells in the absence of blasticidine. The genotype of the strains for each lane is indicated on top of the gels. WT and +, wildtype; N, NEO; H, HYG; P, PURO; Z, ZEO; pBLAST. P50, parasites harboring the pSP72αBlastα-PTR1 episome grown for 50 passages in the absence of blasticidin in the presence of excess thymidine.