Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function.
Bottom Line: In the present study, we found that the decrease in cytochrome c oxidase (COX) activity was ascribable to a temperature-dependent destabilization of cytochrome a heme.Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt:CoI(T300I) homoplasmy in the eye caused severe neurodegeneration at 29°C.Our results demonstrate a novel approach for Drosophila mtDNA genetics and its application in modeling mtDNA diseases.
Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.Show MeSH
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Mentions: Wild-type mtDNA has an XhoI site, whereas the mt:CoIT300I mitochondrial genome does not and is therefore resistant to XhoI digestion. We reasoned that expression of a mitochondrially targeted form of XhoI, MitoXhoI (Xu et al., 2008), in a heteroplasmic background would destroy wt mtDNA specifically and shift mt:CoIT300I heteroplasmy toward homoplasmy (Supplemental Figure S4A). As a test of principle, we ubiquitously expressed MitoXhoI under control of a tubulin-Gal4 driver (tub-Gal4) in heteroplasmic larvae and quantified mtDNAs based on their sensitivity to XhoI digestion (Supplemental Figure S4B). Expression of MitoXhoI efficiently eliminated wt mtDNA, resulting in nearly 100% mt:CoIT300I. The absolute amount of mtDNA in the heteroplasmic flies expressing MitoXhoI was similar to that in wt flies (Supplemental Figure S4C), suggesting that the copy number of mtDNA remains constant after removal of wt mtDNA. We further tested the efficacy of this approach by generating homoplasmy in specific tissues—larval fat body and eye disk—because of the ease of dissecting homogeneous tissues. Expression of MitoXhoI in fat body driven by Cg-Gal4 or in eye disk by eyeless-Gal4 (ey-Gal4) in the heteroplasmic background completely removed wt mtDNA in the fat body or eye disk, respectively, whereas other tissues remained heteroplasmic (Figure 4A).
Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.