Drosophila adPEO Mutations Figure 2. Phenotypes and expression levels of Drosophila lines overexpressing mutants of d-mtDNA helicase. (A–D); Phenotypes of the lines that overexpress the d-mtDNA helicase mutants with a constitutive and ubiquitous driver; (A) w1118;;, control line; (B) w;;UAS helicaseWT/da-GAL4, the phenotype is similar to control line; (C) w;;UAS helicaseK388A/da-GAL4, 100% of lethality at third larval instar (D) w;;UAS helicaseW441C/da-GAL4, the phenotype is similar to that of the control line; (E) w;;UAS helicaseA442P/da-GAL4, 100% of lethality at pupal stage. White arrowheads show necrotic tissue within the unformed pupae. (F) Quantitation of the viability of the overexpressed lines. (G) Expression levels of d-mtDNA helicase. 30 mg of mitochondrial protein from the overexpressing and control lines was used for immunoblot analysis with a polyclonal antibody versus d-mtDNA helicase. The d-mtDNA helicase protein levels are similar in all of the overexpressing lines whereas the endogenous level in the control line was undetectable. A monoclonal porin antibody was used as an internal loading control. doi:10.1371/journal.pone.0043954.g002 Discussion A previous study of d-mtDNA helicase knockdown in Drosophila Schneider cells using RNAi [24] showed cellular phenotypes of slow growth, reduced viability and a 5-fold reduction in mtDNA copy number. Whereas overexpression of the wild type helicase modestly increased mtDNA copy number, overexpression of an active site mutant K338A resulted in a dose-dependent mtDNA depletion, more severe than that observed upon d-mtDNA helicase knockdown. Furthermore, whereas overexpression of the A442P mutant significantly reduced mtDNA copy number, overexpres- sion of the W441C mutant did not. To extend these findings and in consideration of the need to develop tractable animal models to study mitochondrial diseases, PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43954