We evaluated the role of natural mitochondrial DNA (mtDNA) variation on mtDNA copy number, biochemical features and life history traits in Drosophila cybrid strains. We demonstrate the effects of both coding region and non-coding A+T region variation on mtDNA copy number, and demonstrate that copy number correlates with mitochondrial biochemistry and metabolically important traits such as development time. For example, high mtDNA copy number correlates with longer development times. Our findings support the hypothesis that mtDNA copy number is modulated by mtDNA genome variation and suggest that it affects OXPHOS efficiency through changes in the organization of the respiratory membrane complexes to influence organismal phenotype.

Drosophila melanogaster , like most animal species, displays considerable genetic variation in both nuclear and mitochondrial DNA (mtDNA). Here we tested whether any of four natural mtDNA variants was able to modify the effect of the phenotypically mild, nuclear tko 25t mutation, affecting mitochondrial protein synthesis. When combined with tko 25t , the mtDNA from wild strain KSA2 produced pupal lethality, accompanied by the presence of melanotic nodules in L3 larvae. KSA2 mtDNA, which carries a substitution at a conserved residue of cytochrome b that is predicted to be involved in subunit interactions within respiratory complex III, conferred drastically decreased respiratory capacity and complex III activity in the tko 25t but not a wild-type nuclear background. The complex III inhibitor antimycin A was able to phenocopy effects of the tko 25t mutation in the KSA2 mtDNA background. This is the first report of a lethal, nuclear-mitochondrial interaction within a metazoan species, representing a paradigm for understanding genetic interactions between nuclear and mitochondrial genotype relevant to human health and disease.

The Drosophila tko25t point mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding, and to vary with mitochondrial genetic background. Here we show similarly profound effects conferred by nuclear genetic background. We backcrossed tko25t into each of two standard nuclear backgrounds, Oregon R and w1118, the latter used as recipient line in many transgenic applications requiring selection for the white minigene marker. In the w1118 background, tko25t flies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygous tko25t females in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years, tko25t flies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in the w1118 background. Phenotypic severity did not correlate with the expression level of the tko gene. Analysis of tko25t hybrids between the two backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal and parent-of-origin dependent determinants. Although some of these effects may be tko25t-specific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of non-lethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.

TheDrosophila tko25tpoint mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding, and to vary with mitochondrial genetic background. Here we show similarly profound effects conferred by nuclear genetic background. We backcrossedtko25tinto each of two standard nuclear backgrounds, Oregon R andw1118, the latter used as recipient line in many transgenic applications requiring selection for thewhiteminigene marker. In thew1118background,tko25tflies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygoustko25tfemales in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years,tko25tflies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in thew1118background. Phenotypic severity did not correlate with the expression level of thetkogene. Analysis of tko25t hybrids between the two backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal and parent-of-origin dependent determinants. Although some of these effects may betko25t-specific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of non-lethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.

Although replication of the mitochondrial DNA genome has been studied extensively in mammalian and yeast models, the mechanisms of mitochondrial DNA (mtDNA) replication in the widely‐used model organism Drosophila melanogaster remain largely unknown. We employ a comparative approach to analyze mtDNA replication intermediates using two‐dimensional neutral agarose gel electrophoresis (2DNAGE), and to visualize their architecture by transmission electron microscopy (TEM). Our studies target the nuclear‐encoded replisome proteins (DNA polymerase γ, mtDNA helicase and single‐stranded DNA‐binding protein), as well as transcription factors (mTTF and mTerf5), and their roles in the regulation of mtDNA replication through overexpression of both wild type proteins and biochemical variants in cultured insect cells and fly strains. Whereas 2DNAGE allows the analysis of minimally manipulated DNA preparations, TEM provides molecular details regarding the length of Okazaki fragments and on the presence of double‐stranded DNA and/ or RNA loops at the replication fork. In addition to the evaluation of the modes of replication in normal and perturbed cells, biotin labeling of the replisome proteins enables the determination of their positions on the replicating mtDNA molecule. Together, these methods can be used to identify novel structures and proteins involved in the progression of the replication process. Grant Funding Source: Supported by Academy of Finland and NIH GM45295