misincorporation into RNA and DNA in other cells (Van Triest et al., 2000). Interestingly, the sensitivity of T. brucei to raltitrexed and 5-ﬂuoro-orotate is the inverse of what is observed for malaria parasites, where raltitrexed (ICI D1694) is virtually inactive (EC50 >20 mM) (Rathod and Reshmi, 1994), yet 5-ﬂuoro-orotate is highly active (EC50 6 nM) (Rathod et al., 1989). In conclusion, we have evaluated DHFR–TS as a drug target in African trypanosomes and obtained deﬁnitive genetic evidence and compelling chemical evidence sug- gesting that these enzymes are sufficiently validated to warrant entry into a drug discovery pipeline. Enzymatic and structural studies of this enzyme are underway to facilitate this process. Experimental procedures Trypanosome culture Trypanosoma brucei bloodstream-form ‘single marker’ S427 (T7RPOL TETR NEO) and knockouts were cultured in HMI9 medium (Hirumi and Hirumi, 1989) supplemented with 2.5 mg ml-1 G418 to maintain expression of T7 RNA poly- merase and the tetracycline repressor protein (Wirtz et al., 1999). HMI9 medium contains high concentrations of folate principally from the Iscove’s modiﬁed Dulbecco’s medium (IMDM) and the 10% (v/v) Serum Plus components (SAFC Biosciences). Serum Plus is a proprietary medium supple- ment containing 20% fetal bovine serum (FBS), growth- promoting factors, hormones, lipids and 2.72 mg l-1 folic acid (6.2 mM folate, excluding FBS; personal communication from SAFC Biosciences). HMI9 therefore contains 7.9 mM folate, not including folate from FBS (12% v/v, ﬁnal). Folate free IMDM is not commercially available, so a FDM equivalent to HMI9 was prepared using folate-free RPMI 1640 supple- mented with 50¥ MEM amino acid solution (Invitrogen) to provide amino acid and other nutrient concentrations equiva- lent to those found in HMI9 (see Table S1). Other compo- nents were the same as HMI9 (14 mM glucose, 25 mM HEPES, 2 mM pyruvate, 0.5 mM bathocuproine disulphonic acid, 1.5 mM cysteine, 0.2 mM 2-mercaptoethanol, 1 mM hypoxanthine, 0.16 mM thymidine and 10% FBS) except Serum Plus was reduced to 0.5% so that the total FBS was 10.1% (see Table S1). Excluding the contribution from the serum components, FDM contains 31 nM folate. Cultures were initiated with 1 ¥ 105 cells ml-1 and subcul- tured when cell densities approached 1–2 (¥ 106) ml-1. The single marker WT, SKO and DKO cell lines were also grown continuously in the presence of the appropriate drug selec- tion (see below for further details). Once established, DKO cell lines were also grown in the absence of additional drug selection. Generation times (g) were determined by non- linear regression using GraFit with proportional weighting ﬁtted to the following equation: N = N0 2t g where N0 is the starting cell density at zero time and N is cell density at time, t. Validation of DHFR–TS as a drug target 529 Generation of knockout and recovery constructs Primers used in this study (Table S2) are based on the T. brucei DHFR–TS GeneDB sequence (Tb927.7.5480, 927 strain) and E. coli ThyA (Accession No. J01710, K-12, MG1655 strain). All constructs were sequenced to conﬁrm correct assembly. To generate the T. brucei gene replace- ment cassettes, the region containing the 303 bp of the 5′-UTR to the 286 bp 3′-UTR ﬂanking the open reading frame (ORF) of T. brucei DHFR–TS was ampliﬁed from genomic DNA, using Pfu polymerase. This was used as a template for the ampliﬁcation of the individual regions for the assembly of the replacement cassettes containing the selectable drug resistance genes puromycin N-acetyl transferase (PAC) and hygromycin phosphotransferase (HYG), as previously described (Martin and Smith, 2006b). To generate a recovery construct pLew100_TbDHFR–TS, the DHFR–TS ORF was ampliﬁed using TbDHFR–TS-XhoI fwd and TbDHFR–TS- BamHI rev primers, and cloned into pCR-Blunt II TOPO (Invitrogen). This was used to introduce silent mutations to remove two HindIII sites within the gene at Leucine 236 (L236) and Alanine 452 (A452) sequentially with the following sets of primers HindIII-L236 fwd, HindIII-L236 rev and HindIII- A452 fwd HindIII-A452 rev respectively (Table S2). This TOPO clone was used as a template to generate a PCR product containing 5′-HindIII and 3′-PacI restriction sites which was subcloned into a modiﬁed pLew100 vector con- taining blasticidin S transferase (BSD) replacing NEO as the selectable gene marker (kindly supplied by Dr Kirstee Martin). The recovery construct, pLew82_EcTS containing the E. coli gene for thymidylate synthase (ThyA), was engineered as follows. ThyA (Accession No. J01710) was ampliﬁed from E. coli (K-12, MG1655 strain) gDNA using EcThyA-HindIII fwd and EcThyA-BamHI rev primers (Table S2) and the product cloned into pCR-Blunt II TOPO (TOPO_EcTS). The ORF was subcloned into pLew82 containing the resistance marker gene bleomycin. Generation of an E. coli thyA - cell line and functional complementation of TS recovery constructs An E. coli thymidylate synthase deﬁcient (thyA -) cell line derived from E. coli BL21 (DE3) Star cells (Invitrogen) was engineered using the TargeTron™ Gene Knockout System (Sigma-Aldrich) as described in the manufacturer’s instructions. Brieﬂy, the TOPO_EcTS clone was used as the template for the intron retargeting PCR using primers IBS- EcThyA and EBS2-EcThyA (Table S2) that were chosen at Sigma’s TargeTron Design Site (http://www.sigma-genosys. com/targetron/). The disrupted thyA - cells were selected using kanamycin and ThyA-speciﬁc disruption was veriﬁed by colony-PCR screening using EcThyA-HindIII fwd and EBS2- EcThyA primers. The resulting thyA - E. coli was transformed with the pLew82_EcTS or pLew100_TbDHFR–TS recovery constructs and plated out onto LB containing 50 mg ml-1 car- benicillin and 1 mg ml-1 tetracycline to conﬁrm that these plas- mids contained a functional thymidylate synthase gene. Negative controls of the empty vector and cells alone were included. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533