Validation of DHFR–TS as a drug target 523 Fig. 3. Growth characteristics of the T. brucei WT, SKO and DKO cells under various conditions. A. Growth in HMI9 medium in the presence/absence of drug selection. WT, open circles; SKO plus puromycin, closed circles; DKO plus puromycin and hygromycin, open squares; and DKO without drugs, closed squares. Open triangles show the growth of DKO without drugs in HMI9 medium lacking the thymidine component. B. Growth in folate-deﬁcient media (FDM) containing 160 mM thymidine. WT, open circles; SKO plus puromycin, closed circles; DKO plus puromycin and hygromycin, open squares; and DKO without thymidine, closed squares. Data are the means of triplicate cultures, where the standard deviations are 5% or less of the value. Symbols on the x-axis indicate that cell densities are below the limits of detection (i.e. < 103 ml-1). The lines represent the best ﬁts to the equation describing exponential growth as described in Experimental procedures. results in cells failing to complete DNA synthesis from 2N (G1) to 4N (G2/M) with arrest within S phase. The morphology of DKO cells grown in the absence of thymidine for 24 h was examined by scanning electron, light and ﬂuorescence microscopy. In contrast to the long slender morphology of WT or DKO trypanosomes grown in the presence of thymidine (Fig. 5A and B), most thymidine-depleted DKO trypanosomes appeared shorter and fatter with loss of their free ﬂagellum. In some respects these are morphologically similar to the non- dividing stumpy forms seen in relapsing infections which are metabolically pre-adapted for growth in the tsetse ﬂy vector midgut (Fairlamb and Opperdoes, 1986). However, unlike stumpy forms, some had lost their ﬂagellum or possessed two ﬂagella (Fig. 5C). A large vacuole at the posterior end of the parasite was also evident, which, by transmission electron microscopy, appeared to be a grossly enlarged ﬂagellar pocket (Fig. 5F) compared with Fig. 4. Effect of pyrimidines on growth. Cells were grown in HMI9 medium supplemented with varying amounts of pyrimidines for 72 h and cell density was determined using Alamar Blue as described in Experimental procedures. A. WT plus thymidine, open circles; SKO plus thymidine, closed circles. B. DKO plus thymidine, closed squares; DKO plus thymine, open squares (a complete lack of growth stimulation was also observed with uracil or uridine). The dashed line is the best ﬁt for growth stimulation and the dotted line is the best ﬁt for growth inhibition. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533