doi: 10.1016/j.molbiopara.2007.01.013.
Epub 2007 Jan 21.
RNA polymerase I transcription stimulates homologous recombination in Trypanosoma brucei
Affiliations
- PMID: 17316839
- PMCID: PMC3827967
- DOI: 10.1016/j.molbiopara.2007.01.013
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RNA polymerase I transcription stimulates homologous recombination in Trypanosoma brucei
Mol Biochem Parasitol.
2007 May.
No abstract available
Figures
Transcription stimulates homologous recombination. A. The assay strains were established by integrating a cassette containing a Tet-on RRNA promoter (PRRNA) driving transcription of the HR-target (GeneDB ID: Tb927.8.3140) fused to a GFP reporter. TetO, Tet operator. We used 2T1 cells that express TetR:BLE and have a tagged RRNA spacer [3]. The schematic illustrates the HR-assay construct (top) and all three HR-targets in the assay strain, the ectopic Tet-on target (light-grey, transcribed by RNAP-I) and both native targets (black, transcribed by RNAP-II, bottom). The donor construct contains a procyclin promoter (PPRO) to drive expression of the blasticidin S deaminase (BSD) selectable marker which is flanked by target-homologous segments 189 (5′) and 216 bp (3′) in length. The expected double-cross-over is indicated and deletes an 83 bp (NcoI / NdeI) segment from the target. T. brucei strains were grown and manipulated in vitro as previously described [3]. Electroporation was with a BioRad Gene Pulser set at 1.4 kV and 25μF without a pulse controller. B. The assay strains (1 and 2) express GFP in the presence of Tet (1 μg ml−1 for 24 h) as shown by western blotting (top panel, arrowhead) and immunofluorescence analysis (bottom panel, strain 1 is shown) carried out using rabbit anti-GFP as previously described [14]. A lower molecular weight, cross-reactive band on the western blot indicates equal loading and an equivalent coomassie-stained control is shown below the blot. The immunofluorescence images are merged with the phase images. Scale bar: 5μm. C. Summary of key data from Table 1. Two independent strains were analysed in duplicate experiments and transformation efficiency is expressed as the proportion of cells that survive electroporation (see Table 1). The black and light-grey bars represent HR at the native and ectopic targets respectively. Tet was added 24 h prior to electroporation and maintained during manipulation. Survivorship was determined (see Table 1), blasticidin selection (10 μg ml−1) was applied and cultures were distributed in 24-well plates 6 h following electroporation. Wells containing transformed cultures were counted on day six (see Table 1) at which point it is easy to discriminate between positive and negative wells. None of the twenty plates analysed had more than 25% positive wells indicating that the vast majority represented clones. GFP expression was assessed by immunofluorescence in 83 of 120 cultures (see B and Table 1) and were scored as ectopic or native target recombination when GFP negative or positive respectively (see A and Table 1). Error bars, one standard deviation.
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