Materials and Methods Fly Stocks and Culture Flies were raised on standard yeast-glucose- agar medium at 25uC and 65% relative humidity in 12 h light/dark cycles, unless otherwise indicated. To determine the chromosomal location of the transgenes and for the manipulation of transgenic lines, the stock w; CyO/If;TM6B/MKRS was used. Other GAL4 stock lines used were: w;;da-GAL4, w;ey-GAL4 and w;en-GAL4-UAS GFP, obtained from the Bloomington Drosophila Stock Center at Indiana University. For more details of Drosophila lines, see Information S1. Generation of Transgenic UAS-d-mtDNA Helicase Lines The constructs used to overexpress the different versions of the d-mtDNA helicase gene comprised cDNA fragments from d-mtDNA helicase cloned into the pUAST Drosophila transformation vector. The d-mtDNA helicase insert was obtained as previously described [24]. Recombinant pUAST- d-mtDNA helicase constructs were transformed in DH5a competent bacterial cells (Stratagene). Transgenic lines were generated by microinjection of the pUAST- d-mtDNA helicase plasmids in y1 w1118 embryos following standard procedures [24]. Ten independent transgenic lines for each construct were obtained; all of them overexpressed d-mtDNA helicase. Each of them carried a single independent insertion in chromosome II or III, which was brought to homozygosis. For more information see Information S1. UAS-GAL4 Experiments d-mtDNA helicase fragments were overexpressed using the UAS- GAL4 system described by Brand and Perrimon [23]. The daughterless-GAL4 driver (da-GAL4) yields a high and ubiquitous transgenic expression throughout development and was used to induce the overexpression of d-mtDNA helicase. The homozygous da-GAL4 driver was crossed to homozygous UAS lines and the progeny were subjected to study. The same procedure was followed with the w; en-GAL4 (posterior compartment) driver. Crosses were set up in standard food vials and environmental conditions, and the flies were passed every three days. Additional details on Drosophila lines are presented in the Information S1 tables. Viability and Longevity Curves of D. Melanogaster For viability curves, 100 embryos from lines overexpressing the various mutations of d-mtDNA helicase were collected. Embryos were incubated at 25uC with wet yeast, and the number of animals reaching the developmental stages of third instar larvae, pupae and adults were scored. For longevity curves, 200 adult males were collected from the same lines and placed in a vial containing fresh culture medium. Flies were transferred to fresh medium three times a week, and dead flies were counted every day and a Kaplan- Meier survival curve was performed. Real-time RT-PCR Total RNA was extracted using TRIzol reagent (Invitrogen), and 1 mg was converted into cDNA using the QIAGEN quantitect reverse transcription kit following the manufacturer’s instructions. Genomic DNA was isolated from larvae by standard methods. Real-time PCR was performed using the 7900 HT Fast Real-Time PCR System (Applied Biosystems), and PCR products were quantified fluorometrically using the taqman probe (Applied Biosystems); d-mtDNA helicase: Dm01809992_g1; d-COX I (Fw): TGACTACATCCTCCTGCTCTTTCTT, d-COX I (rev): AGCGGATAAAGGTGGATAAACAGTT, FAM probe: TTCCAGCTCCATTTTC; d-ND5 (Fw): AGGATTTT- Drosophila adPEO Mutations TAAAATTAGCTATATTTCATTTATTAACTCATGCTTT, d-ND5 (Rev): ACCCCCCTATTAAACGAATATCTTGAGA, FAM probe: CCCAGCACATATAAAC. At least three indepen- dent amplifications were performed in triplicates for each transcript, and mean values were normalized to the mean value of the reference rRNA 18S: (Fw): AGCCTGCGGCTTAATTT- GACT, (rev): CTTACACACTTATGTTCGACCTGGT and FAM probe: AACACGGGAAAACTT. The PCR amplification program was 95uC for 15 s, 60uC for 1 min, 40 cycles with an initial step of 2 min 50uC and 95uC for 10 min. For mtDNA/ nuclear-DNA relative quantification genomic DNA from Drosoph- ila third instar larvae was purified and quantified by standard methods. 10 ng of DNA were added to Master Mix-PCR Applied Biosystem. The PCR amplification program was 95uC for 15 s, 60uC for 1 min, 40 cycles with an initial step of 2 min 50uC and 95uC for 10 min. Software used was SDS 2.3 (Applied Biosystems). Assays were performed with two different taqman probes: ND5 and 18S. Immunocytochemistry in Imaginal Wing Discs Imaginal discs from third instar larvae were dissected in PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. They were blocked in PBS containing 1% bovine serum albumin and 0.3% Triton X-100 for 1 h, incubated with the primary antibody overnight at 4uC (1:50 dilution), washed four times in blocking buffer, and then incubated with the appropriate secondary antibody (1:200 dilution). for 2 h at room temperature in the dark. Finally, they were washed and mounted in Vectashield (Vector Laboratories). Primary antibodies used were: rabbit anti- phosphohistone 3 (Sigma-Aldrich) and rabbit anti-caspase 3 (Cell Signaling Technology). Secondary antibodies were coupled to the fluorochromes Alexa Fluor 647 (Invitrogen) or Alexa 555 (Invitrogen). In addition, the nucleus was stained with TO-PRO 3 iodide (Invitrogen) and actin with Phalloidin-TRITC (Sigma- Aldrich). Preparations were imaged with a Leica TCS SP2 laser- scanning microscope (Heidelberg, Germany). The number of apoptotic cells was measured using analySIS FIVE software (Olympus Digital Imaging Solutions). Oligonucleotides All oligonucleotides were purchased from Isogen (the Nether- lands) and Sigma-Aldrich. All sequences are given in the 5’ to 3’ direction. Probes used for Southern and Northern hybridization were PCR products obtained from the amplification of Drosophila nuclear and mitochondrial DNAs using the following primers: d- mtDNA helicase (dir): ATGAGACGCGCCGGTTTAA; d-mtDNA helicase (rev): TCAGTTCTCGGATGGCGTCT; d-histone (dir): ACACACGGAACACGAATGCTCG; d-histone (rev): AGC- GAAGCCAAAGCCTGTAGTAGC. Oligonucleotides used for quantitative PCR (qPCR) are as follows: d-mtDNA helicase: TaqMan Gene Expression Assays Dm01809992_g1; d-COX I (dir): TGACTACATCCTCCTGCTCTTTCTT, d-COX I (rev): AGCGGATAAAGGTGGATAAACAGTT, FAM probe: TTCCAGCTCCATTTTC; d-ND5 (dir): AGGATTTT- TAAAATTAGCTATATTTCATTTATTAACTCATGCTTT, d-ND5 (rev): ACCCCCCTATTAAACGAATATCTTGAGA, FAM probe: CCCAGCACATATAAAC. For more details see Information S1. Quantitative RT-PCR Assays ,10 frozen larvae from each genotype were used for RNA extraction with TRIzol reagent (Invitrogen). Total RNA from each sample (2 mg) was reverse transcribed into cDNA using oligo(dT) as a primer and the SuperScript III First-strand PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43954