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. 2017 May 2;292(25):10696–10708. doi: 10.1074/jbc.M117.778480

Figure 2.

Figure 2.

Generation of L. major Δafkp80− and Δfkp40− null mutants singly. A, map of the A/FKP locus. The ORFs for AFKP80 (LmjF16.0480) and FKP40 (LmjF16.0440) are shown in red and blue, respectively. The ORFs for the three intervening genes LmjF16.0470 (hypothetical protein), LmjF16.0460 (60S ribosomal protein L21), and LmjF16.0450 (hypothetical protein) are shown in gray (labeled 70, 60, and 50). Narrow bars flanking A/FKP ORFs depict conserved flanking regions shared by the two genes (gray, 5′; white, 3′) or the boundaries of the targeting fragments used for gene replacement (black). The locations of PCR primers and predicted fragments described in B–D are shown above or below the central map. For clarity, the labels for AFKP80 or FKP40-specific products have been colored red or blue, respectively; similarly, l or r designate PCR products probing the left (5′) or right (3′) flanking regions, respectively. Primer sequences can be found in supplemental Table S1. The analysis of the marker replacement alleles is shown in supplemental Fig. S1. B, PCR analysis of left (5′) flanking regions of FKP40 and AFKP80 in WT, Δfkp40−, and Δafkp80−. These PCRs establish the presence or loss of either or both of the A/FKP genes. Primer e (SMB2453) is common for both A/FKP genes, primer d (SMB 2830) is specific for the AFKP80, and primer f (SMB2783) is specific for FKP40. C, PCR analysis of right (3′) flanking regions of FKP40 and AFKP80 in WT, Δfkp40−, and Δafkp80−. These PCRs establish the presence or loss of A/FKP genes as expected. Primer a (SMB2451) is common for A/FKP, primer b (SMB2793) is specific for AFKP80, and primer c (SMB2784) is specific for FKP40. D, PCR analysis of intervening genes in WT, Δfkp40−, and Δafkp80−. These PCRs establish that the intervening genes are maintained intact as expected. Primers 2 + 3 (SMB3522, 3) amplify regions including the LmF16.0450 ORF, primers 4 + 5 (SMB3524, 5) amplify the region including LmjF16.0460 ORF, and primers 6 + 7 (SMB3526, 7) amplify the region encompassing the LmjF16.0470 ORF.