Mitochondrial transcription. Cells with YFP-tagged mitochondria were grown in 2.5 mM BrU for different times, fixed, Br-RNA immunolabelled with Cy3, and confocal images collected. In some cases, cells were grown for 30 min, washed, and regrown in 5 mM U for up to 2 h (I,J); in others, DNA was immunolabelled with Cy5 (E-G,J) or stained with Hoechst 33342 (K). (A,B) Two views of one field; after growth in BrU for 20 min, Br-RNA is seen mainly in mitochondria. (C,D) Two views of one field; after growth in BrU for 60 min, Br-RNA is seen mainly in nuclei. Bar: 10 μm. (E-G) Three views of mitochondria of one cell, after growth in BrU for 30 min and immunolabelling DNA with Cy5; the merge reveals Br-RNA (red) in some, but not all, mtDNA foci (green). Bar: 2 μm. (H) On growth in BrU, the intensity of Br-RNA labelling (arbitrary units/μm2) in mitochondria increases progressively. (I) After growth for 30 min in BrU and regrowth in U, the intensity of mitochondrial Br-RNA labelling falls exponentially (red line; t1/2 ~ 45 min). (J) The effects of incubation time on distance between a Br-RNA focus and the nearest mtDNA focus. Distances (μm) are binned (that is, 0–0.2, 0.2–0.4, etc). After 20 or 30 min in BrU, most Br-RNA foci lie between 0–0.2 μm of the nearest mtDNA foci (labelled with Cy5); however, after 1 h (or a 30 min pulse followed by a 2 h chase), Br-RNA foci appear to be spread randomly. The decline in Br-RNA colocalizing with mtDNA (that is, lying within 0.2 μm) between 20 and 60 min is consistent with a residence half-life of 43 min. (K) After growth in BrU for 30 min, the intensity of labelling in a Br-RNA focus (au, arbitrary units) does not correlate with the DNA concentration seen in the associated mtDNA focus. The line (slope = 0.0042) is that of best fit. In an analogous experiment in which mtDNA was marked with the antibody, the line of best fit had a similar slope of 0.0068.
Iborra et al. BMC Biology 2004 2:9 doi:10.1186/1741-7007-2-9