Figure 5.

Etoposide-mediated cleavage sites on T. brucei chromosome 4. (a) Procyclics were treated with 500 μM etoposide for 1 h and chromosomal DNA fractionated by CHEFE and Southern blotted. Red arrows identify DIREs and green arrows indicate putative ORFs and the implied direction of transcription. The location of the AT-rich repeat array is highlighted (striped box). The positions of probes and location of the putative centromeric region (yellow oval) are indicated. Lane N, non-treated parasites; lane E, etoposide-treated. (b) Fine mapping of cleavage sites. NotI digested DNA was fractionated by CHEFE as in Figure 4b and Southern blotted. Fragment sizes are shown above the schematic, with their predicted sizes (GeneDB) in parentheses. Black triangles identify these fragments on the autoradiograph and show the major cleavage products. As control, the membrane was hybridized with probe Tb18, from a gene 500 kb downstream of the putative centromere.