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T. brucei ribonuclease H1 is a non-essential nuclear protein.

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posted on 13.12.2018, 18:25 by Emma Briggs, Kathryn Crouch, Leandro Lemgruber, Craig Lapsley, Richard McCulloch

A. Representative immunofluorescence images of T. brucei cells expressing Ribonuclease H1 (TbRH1) as a fusion with 12 copies of the myc epitope (TbRH112myc); wildtype (WT) cells are shown for comparison. Anti-myc signal is shown in red and DNA is stained with DAPI (blue); merged DAPI and anti-myc signal is also shown, as is the cell outline (by differential interference contrast microscopy; DIC). Scale bars, 5 μm. B. Super-resolution structure-illumination imaging of TbRH1 and nuclear DNA colocalisation; TbRH112myc expressing cells are shown stained with anti-myc antiserum and with DAPI; representative images are shown of different cell cycles stages, and only in the merge of anti-myc (magenta) and DAPI (cyan) images is colour provided. Graphs plot length across the nucleus (x, pixels) versus mean pixel intensity at each position (y, arbitrary units) for DAPI (aqua) and TbRH1 (pink). Scale bars, 5 μm. C. Growth of WT cells and T. brucei TbRH1 heterozygous (TbRH1+/-) or homozygous Tbrh1-/- mutants in culture, with mean population density shown at 24 hr intervals; error bars denote SD from three experiments. D. Percentage of the population of WT and Tbrh1-/- cells in discernible cell cycle stages, determined by DAPI staining and fluorescent imaging followed by counting the number and shape of nuclear (N) and kinetoplast (K) structures in individual cells: 1N1K, 1N2K and 2N2K and ‘other’ cells that do not conform to these patterns (>200 cells were counted for each cell type).