522 N. Sienkiewicz, S. Jarosławski, S. Wyllie and A. H. Fairlamb Fig. 2. Genotypic analysis of WT, SKO and DKO cells. A. Structure of the DHFR–TS locus and predicted replacements. The black bars represent the 5′-UTR region upstream of the open reading frames of DHFR–TS, PAC and HYG used as a probe in Southern blot analysis. Only relevant restriction enzyme sites with expected fragment sizes are shown. B. Southern analysis of DNA digested with ClaI and ScaI from WT, SKO (containing PAC and HYG respectively) and DKO cells. DNA size markers are on the left-hand side of blots and the estimated size of detected fragments on the right. 28.0 1.3 h. However, in the absence of drug selection, growth of DKO parasites was similar to that of the other cell lines (13.8 0.1 h). Folate-deﬁcient medium (FDM) was developed to approximate the physiological levels found in mouse (95 nM) (Clarke et al., 2000) and human plasma (16–66 nM) (Hannon-Fletcher et al., 2004; Lin et al., 2004). Similar generation times were obtained for WT and SKO trypanosomes in FDM that are marginally slower compared with HMI9 (18.0 0.4 and 15.8 0.4 h respectively) (Fig. 3B). DKO grew considerably slower (44 2 h) in this medium in the absence of drug selection. Removal of thymidine from either medium did not affect growth of WT or SKO cells, whereas the DKO failed to grow and died by 72 h (Fig. 3). To determine the effects of thymidine concentration on growth, WT, SKO and DKO cell lines were cultured in thymidine-free HMI9 supplemented with varying amounts of thymidine or other pyrimidines (Fig. 4). Thymidine had no effect on growth of WT or SKO cells up to 1 mM, but was toxic at higher concentrations giving similar EC50 values of 7.3 0.6 and 6.7 0.7 mM for WT and SKO respectively (Fig. 4A). In contrast, DKO cells display a narrow window of growth, optimal at 16–64 mM, with essentially no growth with < 2 mM or > 2 mM thymidine (Fig. 4B). Higher concentrations were toxic to DKO cells with an EC50 of 380 45 mM. Other pyrimidines (thymine, uracil or uridine) were unable to replace thymidine (Fig. 4B). To determine at which point ‘thymidine-less death’ was irreversible in the DKO line, cells were incu- bated in thymidine-free HMI9 and then transferred back into thymidine-replete HMI9 medium at intervals. Samples were cultured for up to 14 days and examined daily for the emergence of live, motile parasites. Under these condi- tions, viable parasites were recovered from cultures depleted of thymidine for up to 54 h, but not at 60 h or thereafter (data not shown). DNA content and morphology of WT and DKO cells Prokaryotic and eukaryotic organisms that are aux- otrophic for thymidine undergo cell death by necrosis or apoptosis in response to thymidine starvation. To estab- lish whether the DKO cells undergo a similar response, the DNA content was analysed using a ﬂuorescence- activated cell sorter (FACS) as illustrated in Fig. 5A–C. For the WT cell population, 72% were in G1 phase, with 7% in S (DNA synthesis) phase and 16% in G2/M phase (Fig. 5A). In contrast, DKO cells grown in thymidine- deﬁcient medium were mainly arrested in S phase (58%), with proportionally less in G1 or G2/M (Fig. 5C). In con- trast, DKO cells grown with thymidine showed a similar proﬁle to WT cells (Fig. 5B). Thus, thymidine starvation © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533