Figure 5.

DNA transcription assay of a recombinant mitochondrial genome by in organello RNA synthesis using isolated mouse ρ⁰ mitochondria. The full mouse mtDNA clone, pMusMtTN-COXII, was electroporated into isolated ρ⁰ mitochondria and in organello RNA syntheses were performed for 2 h at 37°C. RT–PCR was carried out using 16S rRNA-specific (A) or ND6-specific (B) primers. 16S rRNA-R and ND6-R primers were used as gene-specific primers for the first strand synthesis of 16S rRNA and ND6 transcription, respectively, and the 16S rRNA-F and MusMt-MluIA primer set or ND6-F and ND6-R primer set was used for subsequent PCR amplification, respectively (see Materials and Methods). Expected band sizes of the RT–PCR products were 232 bp for 16S rRNA (see panel A) and 123 bp for ND6 (see panel B), respectively. Lane M, 100 bp DNA ladder; lanes 1 and 2, 12 kV/cm electroporation; lanes 3 and 4, 16 kV/cm electroporation; lanes 5 and 6, no electroporation control with plasmid. Control lanes in which reverse transcriptase (RT) was omitted are indicated below each panel by minus signs.