Figure 3.

(A) Gene disruption for COX VI in culture adapted, monomorphic forms of T .b. rhodesiense EATRO 795. A nested insertion strategy was used to disrupt each allele of COX VI, to generate the cell line, ΔCOXVI::NEO/∧COXVI::HYG. The first allele was deleted by homologous integration of a neomycin resistance construct; the second allele of COX VI is disrupted by insertional inactivation. (B) Northern blot of RNAs isolated from the COX VI null mutant during differentiation to the procyclic form or in wild-type procyclic forms. A weakly hybridizing band for COX VI is retained, reflecting hybridization to a readthrough RNA after integration of the second drug-resistance cassette into the COX VI gene. COX I, II, and III expression are not affected by COX VI gene ablation. (C) EP procyclin expression of the cell line in which the COX VI gene is disrupted, or wild-type cells. At each time point, cells were assayed for EP procyclin by immunofluorescence. Although differentiation was efficient for each population, being monomorphic the differentiation of the wild-type and the COX VI null mutant was asynchronous with respect to that for stumpy populations.