Synthesis System for RT-PCR (Invitrogen), according to the manufacturer’s instructions. The gene encoding the 18S rRNA was used as a normalizing internal control [25]. Exon-specific oligonucleotide primers for the d-mtDNA helicase were designed with Primer3 software. Real time PCRs were performed in a Rotor Gene thermocycler (Corbett Research) using Taqman probes (Applied Biosystems) as double-stranded DNA binding dye and under the following conditions: 50uC for 2 min, 95uC for 10 min; 40 cycles (95uC for 15 sec and 60uC for 1 min annealing/ extension. Each sample contained 1X Taqman probe, 0.2 mM dNTPs, 1X enzyme buffer, 4 mM MgCl2, 0.5 units of AmpliTaq Gold DNA polymerase UP, 0.3 mM of each oligonucleotide, and 4 ml of a 1/20 dilution of the reverse transcribed products in a final volume of 20 ml. Three separate samples were collected from each genotype and triplicate measurements were performed. Immunoblotting Protein extracts (30 mg) obtained from mitochondria purified by differential centrifugation were fractionated by 10.5% SDSPAGE and transferred to nitrocellulose filters. Filters were pre-incubated for 1 h with 5% skim milk in TBS, followed by incubation for 1 h with the corresponding primary antibody. Polyclonal antibody against d-mtDNA helicase was used as previously described [24]. As a loading control, an antibody against Drosophila VDAC-porin was used at a 1:2000 dilution. DNA Extraction and Southern Blotting Aliquots of 20 frozen third instar larvae were ground in 800 ml of homogenization solution that contained 10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 60 mM NaCl, 0.5% SDS. Precipitated material was removed by centrifugation and the supernatant fractions were extracted once with phenol/ chloroform (1:1), treated with 10 mg/ml RNase A at 37uC for 15 min, re-extracted with phenol/ chloroform (1:1), and precipitated with 2 volumes of ethanol. DNA precipitates were washed with 80% ethanol and resuspended in 50 ml of H2O. Southern analysis was performed as described previously [25]. Mitochondrial Preparations For enzymatic measurements, aliquots of fresh third instar larvae were ground in buffer containing 250 mM sucrose, 2 mM EDTA, 100 IU/liter heparin, 10 mM Tris-HCl, pH 7.4 and mitochondria were isolated by differential centrifugation; purified mitochondria were sonicated for 6 s at 4uC, frozen and thawed. Protein determination was performed using the DC Protein Assay kit (Bio-Rad). References 1. Kaguni LS (2004) DNA polymerase gamma, the mitochondrial replicase. Annu Rev Biochem 73: 293–320. 2. Garesse R, Vallejo CG (2001) Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes. Gene 263: 1–16. 3. Spinazzola A, Zeviani M (2005) Disorders of nuclear-mitochondrial inter- genomic signaling. Gene 354: 162–168. 4. Zeviani M, Servidei S, Gellera C, Bertini E, DiMauro S, et al. (1989) An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 339: 309–311. 5. Van Goethem G, Dermaut B, Lofgren A, Martin JJ, Van Broeckhoven C (2001) Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet 28: 211–212. 6. Spelbrink JN, Li FY, Tiranti V, Nikali K, Yuan QP, et al. (2001) Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 28: 223–231. 7. Kaukonen J, Juselius JK, Tiranti V, Kyttala A, Zeviani M, et al. (2000) Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 289: 782– 785. Drosophila adPEO Mutations OXPHOS Activities Mitochondrial OXPHOS complex and citrate synthase activ- ities were measured as described previously [23]. Incubation was at 30uC except for complex IV, which was measured at 38uC. Enzyme activities are expressed in nanomoles of substrate catalyzed per minute and per citrate synthase activity. Reactive Oxygen Species (ROS) Production Mitochondrial superoxide was assessed using the fluorogenic dye MitoSOX (Molecular Probes) and dihydroethidium (Invitro- gen). 20 wing imaginal discs from third instar larvae were dissected in 1X PBS. Cell dissociation of wing discs was performed as described previously [32] and incubated with 5 mM MitoSox in this medium for 10 min at 37uC in the dark. The cell suspension was analyzed by flow cytometry (BD Biosciences FACS Vantage SE) and data were processed with the BD FACS Diva and Cell Quest Pro software (Becton Dickinson). Alternatively, 20 wing imaginal discs from third instar larvae were dissected and incubated in vivo with the dye as previously described [26]. Preparations were imaged with a Leica TCS SP2 laser-scanning microscope (Heidelberg, Germany). Statistical Analysis Statistical analysis was performed with the Prism 4.03 software (GraphPad Software). The one-way ANOVA test and the Bonferroni post-test were used to determine the statistical significance of the results. Supporting Information Information S1 (DOCX) Acknowledgments Supporting tables. We thank Ernesto Sa´nchez-Herrero (CBMSO) for his advice and support during this work. We are also indebted to Vero´nica Domingo for excellent technical assistance. Author Contributions Conceived and designed the experiments: ASM RG. Performed the experiments: ASM MC SP RHS. Analyzed the data: ASM RG. Contributed reagents/materials/analysis tools: ASM MC YM AJW LSK. Wrote the paper: ASM RG. Critical reading and suggestions: LSK YM AJW. 8. Clayton DA (1982) Replication of animal mitochondrial DNA. Cell 28: 693– 705. 9. Clayton DA (2000) Transcription and replication of mitochondrial DNA. Hum Reprod 15 Suppl 2: 11–17. 10. Holt IJ, Lorimer HE, Jacobs HT (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100: 515–524. 11. Tyynismaa H, Sembongi H, Bokori-Brown M, Granycome C, Ashley N, et al. (2004) Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number. Hum Mol Genet 13: 3219–3227. 12. Tyynismaa H, Mjosund KP, Wanrooij S, Lappalainen I, Ylikallio E, et al. (2005) Mutant mitochondrial helicase Twinkle causes multiple mtDNA deletions and a late-onset mitochondrial disease in mice. Proc Natl Acad Sci U S A 102: 17687–17692. 13. Goffart S, Cooper HM, Tyynismaa H, Wanrooij S, Suomalainen A, et al. (2009) Twinkle mutations associated with autosomal dominant progressive external ophthalmoplegia lead to impaired helicase function and in vivo mtDNA replication stalling. Hum Mol Genet 18: 328–340. 14. Hudson G, Deschauer M, Busse K, Zierz S, Chinnery PF (2005) Sensory ataxic neuropathy due to a novel C10Orf2 mutation with probable germline mosaicism. Neurology 64: 371–373. PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43954