Flies.
 Drosophila wild-type strains Dahomey (DAH),
Canton S (CS) and Oregon R (OR) were obtained from stock-centers or
collaborators. The dj-1βGE23381 mutant [31 (link)]: and
AOX-transgenic lines F6 and F24 [30 (link)] were as described previously. Flies were
maintained in a standard medium [30 (link)], collected using CO2 anesthesia
within 24 h of eclosion, and then kept at a density of 20 flies per vial at 25
ºC in a controlled 12 h light:-dark cycle. Vials were changed every 2-3 days.
We have created two new wild type strains of Drosophilamelanogaster backcrossing for eleven generations DAH virgin females with OR males and OR
virgin females with DAH males. The new strains of Drosophila melanogaster are
called DAHOR (flies with nuclear DAH DNA and mitochondrial OR DNA) and ORDAH
(flies with nuclear OR DNA and mitochondrial DAH DNA).


Lifespan studies.
 Between
180 and 400 flies were used for each study. Each independent study was repeated
twice: data were pooled and analysed together. Flies were collected within 24 h
after eclosion using CO2 anaesthesia and kept at a density of 20
flies per vial at 25 ºC in a controlled 12 h light:-dark cycle.
Every 2-3 days vials were changed and the number of dead flies was counted,
from which mean and maximum lifespan (MLS, the last 10% of surviving flies)
were calculated. Prism GraphPad software was utilized to build survival curves
that were further analysed using the Kaplan Meier Log-Rank Test.


Mitochondrial
biochemistry.
 Mitochondria were isolated according to Miwa et al.[31 (link)] with some minor modifications [30 (link)]. Mitochondrial respiration rates
were measured by polarography using a Clark-type oxygen electrode as previously
[30 (link)], in the absence or presence of KCN (100 μM), antimycin
A (10 μM) or rotenone (5 μM). Mitochondrial
ROS production was assayed according to the method described by [32 ] adapted to
flies [30 (link)].


RNA
quantification.
 Total RNA was extracted from 10 days old flies
according to [30 (link)]. For cDNA synthesis, 13
μl reaction mixes containing 2 μg RNA, 1 μl DEPC 10 mM dNTP mix (Fermentas),
0.4μl Random Primers (0.5ug/μl Promega) and DEPC-treated water were
incubated at 90°C for 3 min, then transferred to ice, where 4 μl 5x M-MuLV
reaction buffer (Fermentas) and 1μl 40U/μl RNase inhibitor
(Fermentas) were added. The reactions were mixed and incubated at 25°C for 10
min. On ice, 2μl of 20U/μl M-MuLV reverse transcriptase (Fermentas)
was added, and the reaction was incubated for a further 10 min at 25°C, 1 h at
37°C and 70°C for 10 min. mRNA levels were analyzed by Q-RT-PCR. The transcript
levels of RpL32, Catalase, Superoxide dismutase 1 and 2 and Glutathione
Peroxidase were measured using primers pairs shown in supplementary Table 2. All RNA extractions were performed in triplicate, with each used as a
template for three separate cDNA synthesis reactions which were then pooled.
Each cDNA pool was itself analysed in triplicate. Expression of the target
genes was measured relative to that of RpL32 (rp49), in order to
normalize for sample and run to run variations. A series of 10-fold dilutions
of an external standard was used in each run to produce a standard curve.
Analytical reactions were performed using 20-fold diluted cDNA samples, in 25
μl reaction volume consisting of 2 μl of the cDNA template,
0.4μl of 20 μM forward and reverse primers, and 12.5ul of 2x MAXIMA
SYBR GREEN Master Mix (Fermentas). The PCR program consisted of a 10 min
pre-incubation at 95°C, 40 cycles of 35 secs denaturation at 95°C, 30 secs
annealing at 60°C and 30 secs extension at 72°C. Melting curve analysis,
consisting of a 15 secs denaturation step at 95°c followed by a 1 min
annealing step at 60°C and a 0.3°C/s denaturation ramp to 95 °C, was performed
after the amplification step to verify that only a single, specific extension
product had been amplified. Data were extracted and analysed using Applied
Biosystems StepOne software version 2.0.


Resistance to inhibitors of the ETC.
 To
check the expression and activity of AOX in vivo experiments with a
variety of ETC inhibitors were performed. 20 flies
were kept (males and females separately) in fresh vials. To measure resistance
to KCN, the drug was dissolved in water at varying concentrations and added
directly to the food vial. Resistance to antimycin and rotenone was assayed
essential as described by Fridell et al. [33 (link)]. In brief, 2-3 day old flies were
starved for two hours in empty vials, following this flies were placed in vials
containing Whatman paper (3 mm x 1 mm) impreg-nated with 5% (w/v) sucrose
solution and the appropriate drug (3 mM antimycin or rotenone). Under these
conditions without any drug, flies are able to survive more than 72 h so any
effect before this time should be considered to be provoked by exposure to the
drug. The proportion of flies surviving was recorded over 24 h.


Sequencing
of Mitochondrial gene cytochrome c oxidase subunit I.
 Mitochondrial
DNA was extracted using standard procedures from mitochondria isolated from
around 150 flies according to Miwa et al. [31 (link)] High fidelity PCR using
specific primers CoIF2 and CoIR5 (Supplementary Table 2) were used to amplify a 2.6 kb
fragment containing the cytochrome c oxidase subunit I (CG34067, CoI). PCR
products were purified using a Machary - Nagel PCR purification kit according
to manufacturer's instructions. Products were sequenced using Big dye
Terminator Chemistry 3.1v (Applied Biosystems) and a 3130 AB genetic analyser.
AB sequencing analysis software was used for analysis of electropherograms.


Statistical analysis.
 Data were
analysed using GraphPad Prism 4 and one-way ANOVA was used for statistical
testing. When ANOVA was significant (p < 0.05) Newman-Keuls Multiple
Comparison test was also used. Lifespan data were analysed using the Kaplan
Meier Log-Rank Test. The statistically significant value was established as p < 0.05.


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