Figure 6.
Effects of TFAM overexpression and knockdown on mtDNA topology. One-dimensional agarose gel blots, hybridized with OH probe. (a) mtDNA from uninduced cells and from cells induced to overexpress TFAM-stop for 48 h, fractionated on a 0.4% agarose gel run in TBE. Only the high molecular weight portion of the gel is shown. Samples were either untreated (U) or treated with T7 gp3 endonuclease (gp3), topoisomerase I (tI), topoisomerase IV (tIV) or topoisomerase IV plus T7 gp3 endonuclease. Identity of the main topoisomers was inferred from enzymatic sensitivity and confirmed by other treatments (data not shown). DNA from cells treated with TFAM-specific siRNAs (RNAi) for 24 h was run on a separate gel. (b) MtDNA from TFAM-induced, uninduced and siRNA-treated cells, fractionated on 0.4% agarose gels run in TBE. Only the low molecular weight portion of each gel is shown. First 4 lanes of upper panel are equally exposed, whereas the right-most two lanes are ∼3-fold overloaded, to reveal the presence of 7S DNA in induced cells. Samples were either heated for 2 min at 95°C (+) or left unheated, as indicated.