labelled probe specific to the MCA4 5' flank. After detection the Southern blot was stripped and reprobed using second labelled probe, specific to the MCA4 ORF ...
Figure S1. Flow cytometry profiles of propidium-iodide stained cells determined at time points throughout the course of MCA4 RNAi induction. The x axis shows fluorescence intensity in the FL2-A channel. Time points and the ploidies of the peaks are indicated. Figure S2. Generation of MCA4 null mutants. (A) Schematic representations of the MCA4 locus in T. brucei 427 before and after integration of the targeted hygromycin (HYG) and neomycin (NEO) gene replacement constructs (underlined) and MCA4 re-expression from an ectopic locus. Arrows depict open reading frames with flanking DNA sequences used for targeting indicated in boxes. Yellow represents α/β tubulin intergenic regions. Predicted sizes of DNA fragments following PvuI and AvrII digest of genomic DNA with their corresponding Southern blot probes are indicated. (B) Southern blot analysis of MCA4 null mutants. Genomic DNA was digested with PvuI and AvrII, separated on a 0.8% agarose gel and transferred to a Hybond-N nylon membrane before hybridization with a chemiluminescent labelled probe specific to the MCA4 5’ flank. After detection the Southern blot was stripped and reprobed using second labelled probe, specific to the MCA4 ORF. Lane 1, wild type T. brucei BSF 427; Lane 2, NEO-resistant heterozygote; Lane 3 ∆mca4; Lane 4 ∆mca4:MCA4. Figure S3. Phenotype analysis of MCA mutants. (A) Growth analysis of Δmca4 parasites after initial mutant generation. Growth rate of wild type and Δmca4 parasites isolated early after transfection. Data presented as mean of three independent clones, ± standard deviation. (B) DNA configuration of wild type and Δmca4 BSF T. brucei was determined by DAPI staining and fluorescence microscopy. Classification was based on number of nuclei (N) and kinetoplasts (K) per cell, with >200 cells counted per time point. Cells with abnormal DNA content were classified as ‘other’. Data presented as mean of 3 replicates, ± standard deviation (C) Motility analysis. The motility of wild type and ∆mca4 parasites was analysed by assessing their ability to resist sedimentation in culture. Cultures were divided into experimental and control cuvettes and the optical density (600 nm) recorded over a 6 hour period. Before each measurement control cells were resuspended. The density of experimental cultures was subtracted from control cells to produce ∆OD600 value. Data presented as mean of 3 replicates, ± standard deviation. (D) In vitro culture of parasites isolated from mice. In vitro growth of wild type and mutant parasite cell lines isolated from ICR mice at first peak of parasitemia. Figure S4. MCA3 (5μg) was incubated with 1mM CaCl2 for the indicated time and the samples analysed by SDS-PAGE. The major processing product was sequenced by Nterminal Edman degradation. The deduced processing site, Lys22, is indicated (*).
