Figure 3.

Deletions within the A+T region of mtDNA in Schneider S2 cells overexpressing mtDNA helicase. (A) Agarose gel electrophoresis of HindIII-treated mtNA (4 μg) obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. The uncut material and resulting HindIII A, B and C fragments are indicated on the right. The bracket shows the distribution of RNase A-sensitive material (see Supplementary Figure S2). Fragment size is indicated in kb on the left. (B) Agarose gel electrophoresis of HindIII-SacI-treated mtNA (4 μg) obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. The resulting bands are labeled relative to the HindIII fragment nomenclature on the right: uncut material and control HindIII B fragment (lane 1) remain undigested; deletion-bearing HindIII B fragments of mtDNA from helicase-overexpressing cells (lane 2) are labeled as B1, B2, B3; SacI digestion of the HindIII A fragment generates derivative fragments A, A’, A’’; SacI digestion of the HindIII C fragment generates derivative fragments C, C’. Fragment size is indicated in kb on the left. (C) Southern-blotting analysis of HindIII-treated mtNA samples obtained from control (lane 1) and mtDNA helicase-overexpressing (lane 2) S2 cells. Fragments encompassing the origin of replication were visualized by hybridization with the Ori probe (see Figure 4A, and Materials and Methods). The HindIII B fragment and its deletion-bearing derivatives B1, B2 and B3, as well as uncut material are labeled on the right. (D) Southern-blotting analysis of HindIII-treated mtNA samples obtained from control (lanes 1) and mtDNA helicase-overexpressing (lanes 2) S2 cells. Fragments were visualized by ethidium bromide staining in the agarose gel (EtBr), or by hybridization with probes specific to the replication origin site (Ori), and coding region (6, see Figure 1). M indicates a molecular weight standard with fragment sizes indicated in kb at left.