Figure 3.

POLGβ siRNA results in a 90% decrease in total cellular 7S DNA and the loss of mtDNA multimers held together by protein. Whole cell nucleic acid was isolated from cells treated twice with dsRNA-840 for 120 or 144 h. Aliquots of 3 μg of DNA were digested with PvuII, which cuts human mtDNA once to produce a linear fragment of 16.6 kb, 7S DNA was released from mitochondrial D-loops by incubation at 70°C for 10 min and separated by 1D-AGE. Southern blots were hybridized with a radiolabelled fragment of mtDNA corresponding to part of the D-loop region (∼nt 16 111–191). The amount of 7S DNA was expressed as a fraction of linear mtDNA (7S DNA/16.6 kb) (A). AccI digested DNA extracted from HOS cells, without the use of proteinase K, after transfection without (B) or with dsRNA targeting POLGβ (C) separated by 2D-AGE and hybridized with probe detecting the 2.8-kb fragment of human mtDNA nt 15 255–1504. W—mtDNA trapped in the well of the first dimension gel, D—mtDNA separated by 1D-AGE but unable to penetrate the second dimension gel (drag). X—molecules resolving near the apex of a standard X arc (2). (D) The abundance of the various species relative to the unit length fragment (1n) based on phosphorimager analysis.