Figure 5.

Uridine can be exchanged for thymidine in large size DNA imported into isolated plant mitochondria. (A) A double-stranded DNA fragment of 920 bp was PCR-amplified in the presence of increasing concentrations of dUTP. To assess uracil incorporation, the resulting PCR products (U1–U5) were 5′-end-labeled, treated with a commercial uracil excision mix, denatured and analyzed by electrophoresis on agarose gels. The migration of the original full-length DNA fragment is indicated by an arrow. (B) The 920 bp PCR products U1–U5 containing increasing levels of uracil were imported for 45 min into isolated A. thaliana mitochondria preincubated for 20 min in the presence of [α³²P]dTTP (lanes 6–10) or [α³²P]dCTP (lanes 11–15). After import and DNase treatment, mitochondrial nucleic acids were extracted, denatured and analyzed by electrophoresis on agarose gels. (C) PCR products U1–U5 were imported for 1 h into isolated A. thaliana mitochondria preincubated for 20 min in the presence of [α³²P]dTTP (lanes 16–20) or [α³²P]dCTP (lanes 21–25). After import and DNase treatment, an aliquote of the mitochondria was kept for direct analysis (not shown, in this experiment label incorporation into the probes was not detectable yet at that stage), whereas the remaining organelles were post-incubated for 1 h in DNA synthesis conditions. Finally, mitochondrial nucleic acids were extracted from all samples, denatured and analyzed by electrophoresis on agarose gels. The migration of the original full-length DNA fragment and of the mtDNA are indicated by arrows.