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Figure 1 Physical maps of the vsg221 expression site and T.brucei promoters. (A) Salient non-coding motifs in the vsg221 ES. The arrow represents the vsg ES promoter. Repeats that occur in tandem arrays are labeled below the map. Other sequences of interest are labeled above the map; 8/16, octamer/hexadecamer conserved sequences; STR, sub-telomeric repeat. (B) The promoters used in this study are shown. The arrows indicate the transcription initiation points, which are designated as position +1. Black boxes upstream of the initiation point indicate regions encompassing elements that are critical for promoter activity in transient transfection assays.
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 | Figure 2 Transcription from promoters inserted into the non-transcribed rRNA gene spacer. (A) Physical map of the ble–promoter–neo cassette and the integration site (vertical arrow) upstream of the endogenous rRNA promoter. The exogenous promoter and the endogenous rRNA promoter are indicated by horizontal arrows. (B) A Northern blot was hybridized with the probes indicated to the left of each panel. Results are shown for three clones with the rRNA promoter cassette (R) and four with the ES promoter cassette (E) in bloodstream forms (BF) and one and two procyclic form (PF) derivatives respectively. The tubulin (tub) probe served as a loading control. Cells with a cassette integrated in an active ES are shown as a positive control for transcription through the entire cassette (active ES). (C) neo mRNA signals were quantified by phosphorimager analysis. These values were corrected for loading variations based on tub mRNA signals and then related to a reference value of 100% for neo mRNA in bloodstream or procyclic clones where neo was driven by an rRNA promoter. The numbers above each bar represent the number of independent clones in each data set and error bars indicate standard deviation. Symbols are as in (B).
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Figure 3 Transcription repression and promoter orientation. (A) Physical map of the ble–promoter–neo cassettes integrated, in either orientation, upstream of vsg221 in the 'silent' vsg221 ES. The exogenous promoters are indicated (arrows). (B) A Northern blot was hybridized with the probes indicated to the left of each panel. Arrowheads indicate the direction of transcription in each clone. BF, bloodstream forms; PF, procyclic forms; R, cassette with rRNA promoter; E, cassette with ES promoter. Cells with a cassette integrated in an active ES are shown as a positive control (active ES). (C) neo mRNA signals were quantified by phosphorimager analysis. Symbols are as in (B).
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 | Figure 4 Transcription repression and telomere proximity. (A) Physical map of the ble–promoter–neo cassette and locations of four integration sites (vertical arrows) in the 'silent' vsg221 ES. The exogenous promoter and the endogenous ES promoter are indicated by horizontal arrows. (B) A Northern blot was hybridized with the probes indicated to the left of each panel. The position of integration (relative to the vsg221 start codon) in each clone, in kbp, is indicated. For the inserted rRNA promoter (R) results are shown for two independent bloodstream-form (BF) clones and one procyclic form (PF) derivative at each integration site. Only one clone was derived for each of the two insertion sites of the ES promoter (E). Cells with a cassette integrated in an active ES are shown as a positive control (active ES). (C) Bloodstream-form neo and vsg221 signals were quantified by phosphorimager analysis. neo signals were normalized against clones with the same cassettes integrated at each position into an active ES (see text). The numbers above each bar represent the number of independent clones in each data set and error bars indicate standard deviation. Symbols are as in (B).
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Figure 5 Transcription repression at the non-telomeric vsg118 locus. (A) Physical map of the ble–promoter–neo cassette and the integration site (vertical arrow). (B) A Northern blot was hybridized with the probes indicated to the left of each panel. BF, bloodstream forms; PF, procyclic forms; R, cassette with rRNA promoter; E, cassette with ES promoter. Cells with a cassette integrated in an active ES are shown as a positive control (active ES). (C) neo signals were quantified by phosphorimager analysis. The numbers above each bar represent the number of independent clones in each data set. Standard deviation was negligible in these samples. Symbols are as in (B).
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 | Figure 6 Neo expression in the vsg221 ES in individual cells. Rabbit anti-Neo primary antibody and rhodamine-conjugated goat anti-rabbit secondary antibody were used to detect Neo (red). DAPI (Sigma) was used to stain nuclear and mitochondrial DNA (blue). In bloodstream forms Neo expression was driven by the endogenous active ES promoter (A) or an ES promoter integrated at a repressed ES (B). In procyclic forms Neo expression was driven by an ES promoter within an ES (C) or an rRNA promoter within an ES (D).
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