ALBERT

Description: Culture of Drosophila expressing the steroid-dependent GeneSwitch transcriptional activator under the control of the ubiquitous α -tubulin promoter was found to produce extensive pupal lethality, as well as a range of dysmorphic adult phenotypes, in the presence of high concentrations of the inducing drug RU486. Prominent among these was cleft thorax, seen previously in flies bearing mutant alleles of the nuclear receptor Ultraspiracle and many other mutants, as well as notched wings, leg malformations, and bristle abnormalities. Neither the α -tubulin -GeneSwitch driver nor the inducing drug on their own produced any of these effects. A second GeneSwitch driver, under the control of the daughterless promoter, which gave much lower and more tissue-restricted transgene expression, exhibited only mild bristle abnormalities in the presence of high levels of RU486. Coexpression of the alternative oxidase (AOX) from Ciona intestinalis produced a substantial shift in the developmental outcome toward a wild-type phenotype, which was dependent on the AOX expression level. Neither an enzymatically inactivated variant of AOX, nor GFP, or the alternative NADH dehydrogenase Ndi1 from yeast gave any such rescue. Users of the GeneSwitch system should be aware of the potential confounding effects of its application in developmental studies.

Description: A point mutation [ technical knockout 25t ( tko 25t )] in the Drosophila gene coding for mitoribosomal protein S12 generates a phenotype of developmental delay and bang sensitivity. tko 25t has been intensively studied as an animal model for human mitochondrial diseases associated with deficiency of mitochondrial protein synthesis and consequent multiple respiratory chain defects. Transgenic expression in Drosophila of the alternative oxidase (AOX) derived from Ciona intestinalis has previously been shown to mitigate the toxicity of respiratory chain inhibitors and to rescue mutant and knockdown phenotypes associated with cytochrome oxidase deficiency. We therefore tested whether AOX expression could compensate the mutant phenotype of tko 25t using the GeneSwitch system to activate expression at different times in development. The developmental delay of tko 25t was not mitigated by expression of AOX throughout development. AOX expression for 1 d after eclosion, or continuously throughout development, had no effect on the bang sensitivity of tko 25t adults, and continued expression in adults older than 30 d also produced no amelioration of the phenotype. In contrast, transgenic expression of the yeast alternative NADH dehydrogenase Ndi1 was synthetically semi-lethal with tko 25t and was lethal when combined with both AOX and tko 25t . We conclude that AOX does not rescue tko 25t and that the mutant phenotype is not solely due to limitations on electron flow in the respiratory chain, but rather to a more complex metabolic defect. The future therapeutic use of AOX in disorders of mitochondrial translation may thus be of limited value.