526 N. Sienkiewicz, S. Jarosławski, S. Wyllie and A. H. Fairlamb Fig. 7. Complementation studies with EcTS and TbDHFR–TS. (A)–(C) Complementation with E. coli TS and (D)–(F) complementation with T. brucei DHFR–TS. A. Growth of E. coli thyA - transformed with pLew82_EcTS or empty vector on thymidine-free medium. B. Southern analysis of T. brucei DKO transfected with pLew82_EcTS (cDKOTS). DNA was digested with SacI and SphI and probed with EcTS ORF. The size of the expected fragment is indicated on the right with standard markers on the left. C. Growth of cDKOTS in HMI9 medium with or without thymidine. Circles, plus thymidine; squares, no thymidine present. Open symbols depict growth without tetracycline induction and closed symbols with tetracycline. Triangles, growth of DKO in the absence of thymidine. The lines represent the best ﬁts to the equation describing exponential growth as described in Experimental procedures. D. Growth of E. coli thyA - transformed with pLew100_TbDHFR–TS or empty vector on thymidine-free medium. E. Southern analysis of T. brucei DKO transfected with pLew100_TbDHFR–TS (cDKODHFR–TS). DNA was digested with HindIII and StuI and probed with TbDHFR–TS. The size of the expected fragment is indicated on the right with standard markers on the left. F. Growth of cDKODHFR–TS in HMI9 medium with and without thymidine (open and closed circles respectively). Open squares, growth of DKO in the absence of thymidine. The lines represent the best ﬁts to the equation describing exponential growth as described in Experimental procedures. Symbols on the x-axis indicate that cell densities are below the limits of detection (i.e. < 103 ml-1). thymidylate cycle in the absence of DHFR (Fig. 7C). The greater enzymatic activity than WT cells (2838 1438 TS add-back grew normally in HMI9 containing thymidine cpm) conﬁrming that functional TS is present in this trans- (generation times 13 and 13.4 h, respectively, with or genic line. The DKO line showed activity that was not without tetracycline), but, in its absence, no viable trypa- signiﬁcantly different from zero (862 1283 cpm), con- nosomes were visible beyond day 4. Nonetheless, when ﬁrming the absence of TS activity as demonstrated by compared with the DKO grown in the absence of thymi- thymidine auxotrophy (Figs 3 and 4). dine, survival was prolonged by 2 and 3 days, respec- To demonstrate that deletion of DHFR–TS had not tively, in the absence and presence of tetracycline. resulted in other genomic mutations, a copy of To demonstrate that the E. coli TS was also functional TbDHFR–TS (with two internal HindIII restriction sites in T. brucei, extracts were prepared from cell lines cul- tured in the presence of thymidine and assayed radio- ablated) was cloned into pLew100 (Wirtz et al., 1999). The resulting construct was able to rescue thyA - E. coli metrically for enzyme activity (Fig. 8). Irrespective of (Fig. 7D) indicating that the modiﬁed TbDHFR–TS was tetracycline induction, cDKOTS cells showed ~10-fold functional. pLew100_TbDHFR–TS was then transfected © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533