Fig. 1. Targeted replacement of the AP3 gene. (A, from left to right) Schematic representation of the AP3 locus and the constructs used for gene replacement. Genes are shown as arrows, intergenic and flanking DNA sequences as boxes. The restriction sites and the locations of the probes used for the Southern blot analyses, as well as the expected sizes of the labelled fragments, are displayed. HYG, hygromycin-resistance gene; BLE, phleomycin-resistance gene; DHFR, dihydrofolate reductase gene. Southern blot analyses of L. major wild-type (WT), AP3 heterozygote (ap3/AP3) and AP3-null mutant (ap3) with probes specific for HYG and AP3. (B) RT-PCR analysis of specific mRNA expression in promastigotes of the cell lines described in A and the additional AP3 re-expressing cell line (ap3::P_RRNA AP3). Re-expression of AP3 is from the ribosomal DNA locus and contains a different 3'UTR from the wild type gene, so the AP3 mRNA is a different size. +RT and –RT, initial reaction with and without reverse transcriptase, respectively. VPS4-specific primers were used as a positive control.