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. 2010 Dec 14;8(12):e1000560. doi: 10.1371/journal.pbio.1000560

Figure 1. Global variability in transcription.

Figure 1

(A) Variation in BrU incorporation. Hela cells were incubated for 30 min with 5 mM BrU. Br-RNA signal shows a large variation between cells (confocal section). (B). Kinetics of RNA pol II in vivo. FLIP analysis of GFP–RNA pol II. Half of the nucleus was bleached continuously as confocal images were collected approximately every 5 s. (C) Decay in fluorescence intensity of the unbleached area of the nucleus in the FLIP experiment (intensity, arbitrary units) (n = 60). (D) Log plot analysis of FLIP data showing two populations of RNA pol II molecules, as described in Hieda et al [23]. (E) Distribution of t 1/2 for the DNA-associated form of RNA pol II in the cell population (n = 60). (F) BrU incorporation is correlated with the half-life of the DNA-associated RNA pol II population. After FLIP, cells were incubated with BrU, fixed, and immunolabelled for Br-RNA. Individual cells were identified, and the values of t 1/2 for the DNA-associated form of RNA pol II and the corresponding Br-RNA intensity were plotted. (G–I) Histone H2B–GFP fast-exchanging population is a reporter of transcription elongation. (G) Hela cells expressing histone H2B–GFP were used for FLIP analysis (left panel). These cells were subsequently subjected to BrU incorporation. After identification of the same cells (middle panel), we measured Br-RNA production (right panel), which shows a good correlation (I). In (H) we show the variability in t 1/2 for the rapid-exchangeable histone H2B–GFP (transcription-dependent fraction, Figure S4) (n = 100). Again, this shows a high variability between cells in a population. (J and K) RNA pol II molecules track at similar speed inside a given cell. High-power images of Br-RNA foci in two nuclei with different mean intensity of Br-RNA after 15 min of incorporation of BrU. The images have been pseudo-coloured to improve visualisation (purple = low, red = high). All the foci in an individual nucleus show a consistent level of Br-RNA intensity. (L) Analysis of the noise in Br-RNA production in transcription foci in individual cells. The plot shows 40 cells (>200 foci were analysed per cell). Transcription rates of Br-RNA foci appear to be correlated. Bars: (A), 10 µm; (K), 0.200 µm.