DNA in the mitochondria of fixed ECV-304 cells expressing subunit VIII of cytochrome oxidase tagged with YFP. (A,B) Two views taken with a confocal microscope of fixed cells after immunolabelling DNA. Mitochondria marked with YFP (A) contain discrete foci of mtDNA (B); nuclear DNA appears weakly labelled, presumably because most of it is inaccessible to the anti-DNA antibody, an IgM. Pretreatment (60 min; 20°C) with 25 μg/ml DNase removed >96% foci like those in (B). Insets: high-power views (bar: 2 μm). (C,D) Two similar views after growth (24 h) in BrdU, and immunolabelling the resulting Br-DNA; Br-DNA is found in nuclei and mitochondrial foci. Bar: 4 μm. (E) The distribution seen experimentally (exp) of distances between consecutive DNA foci within mitochondria (determined using images like (B); n > 500) differs significantly from a random one (p = 0.0013); foci tend to be closer together than expected. Distances (μm) are binned (that is, 0–0.4, 0.4–0.8, etc). (F) Experimental distribution (determined using 15 images like (D)) of intensities of mtDNA foci. Intensities (arbitrary units, au) are binned (that is, 0–0.5, 0.5–1, etc), the first bin contained no examples, and the arrow marks the average intensity. If the weakest focus (in bin 2) contains one genome, then the average contains ~9.2. (G) Cross-correlation analysis of mtDNA foci and the YFP-tagged subunit (using images like those in the insets in (A,B)); the low Pearson's coefficient (r
) at Δx values close to zero indicates that mtDNA is excluded from sites containing YFP.