Figure 4

Etoposide-mediated cleavage sites on T. brucei chromosome 1. (a) Procyclics were treated with 500 μM etoposide for 1 h and chromosomal DNA fractionated by CHEFE. Hybridization was carried out with probes Tb1-Tb4. Their positions and the location of the strand-switch domain (yellow oval) are shown. Lane N, non-treated parasites; lane E, etoposide-treated. (b) Fine-mapping of cleavage sites. Chromosomal DNA from treated/non-treated parasites was immobilized in agar blocks, restriction digested and fractionated by CHEFE. Fragment sizes are shown above the schematic, with their predicted sizes (GeneDB) in parentheses. Black triangles identify the fragments and cleavage products on the relevant autoradiographs. As control, blots were re-hybridized with probe Tb4, from a gene 150 kb upstream of the putative centromere. (c) Comparison of the T. cruzi chromosome 3 centromeric domain with the syntenic region of T. brucei chromosome 1. In the T. cruzi chromosome, the degenerate VIPER/SIRE element and L1Tc retroelements are indicated, together with a truncated cruzipain pseudogene (ψCZP) and an U2snRNA gene. The corresponding region in T. brucei chromosome 1 contains 2 INGI retrotransposons and 1 DIRE. The locations of a leucine rich repeat protein gene (LRRP), a rRNA gene array and a 5.5 kb array of approximately 30 bp repeats are shown. Green arrows indicate putative ORFs and the implied direction of transcription. The dashed lines between the T. cruzi and T. brucei maps identify the equivalent positions of the first ORFs of the conserved directional gene clusters.