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. 2004 Dec 15;32(22):6575–6584. doi: 10.1093/nar/gkh991

Figure 3.

Figure 3

Developmental silencing of ESs in ΔtbKU80 trypanosomes. (A) Schematic representation of integrated reporter cassette. VSG 221 gene, GFP-reporter (GFP) and selectable marker (bsr) are indicated as boxes. Endogenous ES promoter (ES) is shown as arrow. Telomeres are represented by a hatched box. The GFP-reporter cassette was introduced into the active ES of two independent tbKU80-deficient cell lines (ΔtbKU80/R3 and ΔtbKU80/R4) and wild-type trypanosomes (Wild Type/R1 and Wild Type/R2). To analyze developmental silencing of ES promoter GFP expression was monitored by FACS analysis in bloodstream forms (red bars) and two weeks post-differentiation into procyclic-form trypanosomes (blue bars). Wild-type cells without GFP reporter cassette served as a negative control to evaluate background fluorescence intensity (control). (B) Schematic representation as in (A). Additional T7 promoter is shown as an open arrow. The T7 promoter driven GFP-reporter was introduced into the ESs of tbKU80-deficient parasites generating cell lines ΔtbKU80/R7 and ΔtbKU80/R8 and wild-type cells generating Wild Type/R5 and Wild Type/R6. To analyze chromatin access of the T7 polymerase as a marker for chromatin remodeling, GFP expression was monitored as described above.