(A) Map of the Tcmpx locus (top line) and integration of the pTEX-CF-mpx construct. The direction of polycistronic transcription, toward the center of the chromosome, is shown. The 1.5-kb fragment used for targeting is indicated by the crossed lines, and Δcyp identifies the disrupted cyclophilin gene formed as a result of the integration event. Expression of the neor gene is under control of the rDNA promoter. Telomeric sequences supplied by the vector are shown as horizontal arrowheads in the truncated chromosome (bottom line). The position of the plasmid sequences used as a probe in Figure 2, B and C is indicated. (B) Autoradiographs of Southern blots of T. cruzi chromosomal DNA separated by CHEFE and hybridized with a β-propeller protein gene (Tcbpp1) (Bromley et al. 2004) or plasmid DNA probes as indicated. (WT) Wild type; (T) transfected cells. (C) Southern blot of restriction-digested genomic DNA from transfected cells (see Fig. 2A for the location of the restriction sites) probed with plasmid DNA. The telomeric sequences are identifiable as a smear ranging from 1.5 to 4 kb. The top band in both lanes allows the position of the corresponding upstream restriction sites in the chromosomal DNA to be established.
