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Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function.

Chen Z, Qi Y, French S, Zhang G, Covian Garcia R, Balaban R, Xu H - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.

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Related in: MedlinePlus

Proportion of mt:CoIT300I mtDNA in different tissues of heteroplasmic flies. The heads, thoraxes, guts, and testes were dissected from 10 individual males eclosed and aged at 18°C for 2 d (A) or 4 wk (B) or shifted to 25°C (C) and 29°C (D) for 4 wk after eclosion at 18°C, and degrees of heteroplasmy were determined by restriction digest analysis. Tissues from a single fly are connected by a gray line. (E) Average mt:CoIT300I levels of each organ from 10 individual flies of each group (means ± SD, n = 10). Y, young, 2-d-old flies; O, old, 4-wk-old flies. Note that at 25 and 29°C, mt:CoIT300I levels are significantly decreased in testes compared with other tissues in the same group (*p < 0.05, **p < 0.005).
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Figure 3: Proportion of mt:CoIT300I mtDNA in different tissues of heteroplasmic flies. The heads, thoraxes, guts, and testes were dissected from 10 individual males eclosed and aged at 18°C for 2 d (A) or 4 wk (B) or shifted to 25°C (C) and 29°C (D) for 4 wk after eclosion at 18°C, and degrees of heteroplasmy were determined by restriction digest analysis. Tissues from a single fly are connected by a gray line. (E) Average mt:CoIT300I levels of each organ from 10 individual flies of each group (means ± SD, n = 10). Y, young, 2-d-old flies; O, old, 4-wk-old flies. Note that at 25 and 29°C, mt:CoIT300I levels are significantly decreased in testes compared with other tissues in the same group (*p < 0.05, **p < 0.005).

Mentions: For flies eclosed and aged at 18°C, the proportion of mutant mtDNA among single individual flies varied from 60 to 90% (Figure 3, A and B), suggesting random segregation of mtDNAs during oogenesis. However, there was no significant difference in the average mutation loads of the various organs (Figure 3E). These results suggest a lack of selection against the mutant genome during development and through the aging process at 18°C for all organs examined. Among the flies cultured at 25°C, the average mutant load in heads, thoraces, and guts was ∼80% and demonstrated little variation (Figure 3E). However, the testes had a much lower mutant load (69 ± 7.4%; Figure 3, C and E). When cultured at 29°C, heteroplasmic flies containing >85% mutant genome were not recovered after 4 wk. Analysis of the survivors revealed that the mutation load in three somatic tissues was similar at ∼70% (Figure 3E) but was significantly reduced in testes (52 ± 11.5%; Figure 3, D and E). Although the spatial resolution is difficult to determine in these experiments, our results suggest that mutant mtDNA is subject to negative selection in testis and only at higher temperatures.


Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function.

Chen Z, Qi Y, French S, Zhang G, Covian Garcia R, Balaban R, Xu H - Mol. Biol. Cell (2014)

Proportion of mt:CoIT300I mtDNA in different tissues of heteroplasmic flies. The heads, thoraxes, guts, and testes were dissected from 10 individual males eclosed and aged at 18°C for 2 d (A) or 4 wk (B) or shifted to 25°C (C) and 29°C (D) for 4 wk after eclosion at 18°C, and degrees of heteroplasmy were determined by restriction digest analysis. Tissues from a single fly are connected by a gray line. (E) Average mt:CoIT300I levels of each organ from 10 individual flies of each group (means ± SD, n = 10). Y, young, 2-d-old flies; O, old, 4-wk-old flies. Note that at 25 and 29°C, mt:CoIT300I levels are significantly decreased in testes compared with other tissues in the same group (*p < 0.05, **p < 0.005).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Proportion of mt:CoIT300I mtDNA in different tissues of heteroplasmic flies. The heads, thoraxes, guts, and testes were dissected from 10 individual males eclosed and aged at 18°C for 2 d (A) or 4 wk (B) or shifted to 25°C (C) and 29°C (D) for 4 wk after eclosion at 18°C, and degrees of heteroplasmy were determined by restriction digest analysis. Tissues from a single fly are connected by a gray line. (E) Average mt:CoIT300I levels of each organ from 10 individual flies of each group (means ± SD, n = 10). Y, young, 2-d-old flies; O, old, 4-wk-old flies. Note that at 25 and 29°C, mt:CoIT300I levels are significantly decreased in testes compared with other tissues in the same group (*p < 0.05, **p < 0.005).
Mentions: For flies eclosed and aged at 18°C, the proportion of mutant mtDNA among single individual flies varied from 60 to 90% (Figure 3, A and B), suggesting random segregation of mtDNAs during oogenesis. However, there was no significant difference in the average mutation loads of the various organs (Figure 3E). These results suggest a lack of selection against the mutant genome during development and through the aging process at 18°C for all organs examined. Among the flies cultured at 25°C, the average mutant load in heads, thoraces, and guts was ∼80% and demonstrated little variation (Figure 3E). However, the testes had a much lower mutant load (69 ± 7.4%; Figure 3, C and E). When cultured at 29°C, heteroplasmic flies containing >85% mutant genome were not recovered after 4 wk. Analysis of the survivors revealed that the mutation load in three somatic tissues was similar at ∼70% (Figure 3E) but was significantly reduced in testes (52 ± 11.5%; Figure 3, D and E). Although the spatial resolution is difficult to determine in these experiments, our results suggest that mutant mtDNA is subject to negative selection in testis and only at higher temperatures.

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20892.

Show MeSH
Related in: MedlinePlus