FIGURE 1.

Generation of the ATG4.1 null mutant. A, schematic representation of the ATG4.1 locus and the plasmid constructs used for gene replacement. The ATG4.1 and antibiotic resistance genes are shown as arrows, flanking DNA sequences are shown as boxes. Restriction enzymes used for the different constructs and the expected sizes of the XhoI fragments are shown. 5′-DHFR and 3′-DHFR, dihydrofolate reductase flanking regions; BSD, blasticidin resistance gene; HYG, hygromycin resistance gene. B, Southern blot analysis of genomic DNA digested with XhoI, separated on a 1% agarose gel, blotted onto a nylon membrane, and hybridized with a ³²P-labeled DNA probe corresponding to the 5′-flanking region of ATG4.2. C, Western blot analysis using the α-His antibodies of extracts from early stationary phase promastigotes. EF1α served as a loading control.