Skip to main content
. 2016 May 26;5:e12765. doi: 10.7554/eLife.12765

Figure 3. Mutation of TbRECQ2 impairs survival of T. brucei after induction of a DNA double strand break, either in the active telomeric VSG expression site or in the core of a chromosome.

(A) I-SceI target sequences in HR1 and HRES cells. HR1 cells contain an I-SceI recognition site embedded within an RFP:PUR fusion gene (black), flanked by tubulin sequences (white), located between genes Tb.11.02.2110 and Tb.11.02.2020 on one copy of chromosome 11; HRES cells contain an I-SceI recognition site upstream of a PUR gene, flanked by tubulin sequences, located downstream of the 70 bp repeats of the active VSG221 expression site on chromosome 6. B and C show clonal survival following I-SceI induction in HR1 and HRES cells, respectively. In both cases, wild type and two recq2-/- clones were distributed in three 96 well plates at a concentration of 0.26 cells per well either in the absence (I-SceI uninduced) or presence (I-SceI induced) of 2 μg.mL-1 tetracycline. The number of wells with surviving cells after 7–10 days growth is depicted as percentage of survivors following I-SceI induction relative to survivors without I-SceI induction; error bars represent standard error of the mean between three experimental repeats. Puromycin sensitivity of surviving I-SceI induced and uninduced clones was then tested, and is represented as the percentage of tested clones that grew in the presence (+) or absence (-) of 1 μg.mL-1 puromycin (N: number of clones analysed). (D) Clones from (C), excluding those that were puromycin resistant, were assayed for ESAG1 and VSG221 presence by PCR; data are shown as the percentage that were PCR positive (N: number of clones analysed).

DOI: http://dx.doi.org/10.7554/eLife.12765.006

Figure 3.

Figure 3—figure supplement 1. Generation of TbRECQ2 mutants in T. brucei HR1 and HRES cells.

Figure 3—figure supplement 1.

(A) Agarose gels of PCR products generated from genomic DNA from HRES or HR1 wild type (WT) cells and RECQ2 heterozygote (+/-) and knockout (-/-) clones using primers described in the Figure S1B. Gaps indicate that lanes have been aligned in this figure after excision from multiple gels or from disparate parts of the same gel; size markers are shown (bp). (B) Confirmation by PCR of RECQ2 knockout in HR1 and HRES cell lines. Agarose gels showing products generated when PCR was performed on a region of the PIF6 ORF or the RECQ2 ORF using cDNA (+RT) synthesised from RNA extracted from recq2-/- mutants; control reactions in which no substrate was added (-), or where cDNA from HR1 WT cells was used, are shown. Gaps indicate that lanes have been aligned in this figure after excision from multiple gels or from disparate parts of the same gel; size markers are shown (bp).
DOI: http://dx.doi.org/10.7554/eLife.12765.007