Amplification and cloning of T. brucei RNase H1 in pX63-Neo. (A) Ethidium bromide-stained agarose gel showing the PCR amplification product of T. brucei RNase H1 (lane 2); lane 1, 1-kb ladder molecular size marker (GIBCO-BRL). (B) Map of the pXNeo-TbRH1F plasmid. (C) Map of the pXNeo-TbRH1R plasmid. Amp, Neo and TbRH1 indicate the beta-lactamase, neomycin phosphotransferase, and T. brucei ribonuclease H1 genes, respectively. Arrowheads before the genes indicate Leishmania trans-splicing signals. These trans-splicing signals are needed for the expression of the genes cloned behind them. In these plasmids, nucleotide sequences 0–821, 4752–5710, and 6619–7166 are originated from the Leishmania DHFR locus [39]. The TbRH1 gene sequence is 822–1925. The rest of the sequences are from bacterial plasmids [39]. The orientations of the genes with respect to their promoter (Amp gene) or trans-splicing signal (Neo or TbRH1 genes) are indicated as solid arrows.