Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko25t exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko25t mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.

Eighty percent of DNA molecules are deleted in the mitochondrial population of an adult mutant strain of D. subobscura. Both intact and deleted genomes are autonomous monomers. The heteroplasmy level, which is lower in germ tissue, increases from the oocytes (60%) to the third larval instar (83%), and is then maintained throughout the life of the fly. The mtDNA/nuclear DNA ratio is on average two-times greater in the heteroplasmic strain than in the wild-type strain, irrespective of the stage, but the cellular content of mitochondria is elevated only in the embryos and pupae of the mutant strain. The steady state concentrations (SSCs) of the transcripts affected by the deletion are greatly reduced at the larval and adult stages, and less so at the pupal stage of the mutant strain compared with the wild-type. The SSCs of these transcripts are identical in the two strains at the embryonic stage. The fusion transcript, indicating that the deleted genome is expressed, was detected at all stages. The mechanisms involved in the changes in the heteroplasmy level during the course of development and in its maintenance from the third larval instar onwards are discussed.

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In the studied mutant strain of Drosophila subobscura, 78% of the mitochondrial genomes lost >30% of the coding region by deletion. The mutations was genetically stable. Despite this massive loss of mitochondrial genes, the mutant did not seem to be affected. Distribution of the two genome types, cell levels of mitochondrial DNA, steady-state concentrations of the mitochondrial gene transcripts, mitochondrial enzymatic activities, and ATP synthesis capacities were measured in the head, thorax, and abdomen fractions of the mutant strain in comparison with a wild type strain. Results indicate that the deleted genomes are detected in all fractions but to a lesser extent in the male and female abdomen. In all fractions, there is a 50% increase in cellular mitochondrial DNA content. Although there is a decrease in steady-state concentrations of mitochondrial transcripts of genes affected by deletion, this is smaller than expected. The variations in mitochondrial biochemical activities in the different fractions of the wild strain are upheld in the mutant strain. Activity of complex I (involved in mutation) nevertheless shows a decrease in all fractions; activity of complex III (likewise involved) shows little or no change; finally, mitochondrial ATP synthesis capacity is identical to that observed in the wild strain. This latter finding possibly accounts for the lack of phenotype. This mutant is a good model for studying mitochondrial genome alterations and the role of the nuclear genome in these phenomena.Various alterations in the mitochondrial genome have been correlated with severe human pathology (1-3). Among the most substantial of these alterations, deletions, which account for a small fraction of the described cases, have always been detected at the heteroplasmic state (4 -6). Once the proportion of deleted genomes reaches a critical threshold, these deletions have often very pejorative effects on mitochondrial function (7,8). Such mutations are generally sporadic, although dominant autosomal factors have been observed (9 -11). A locus has been localized on chromosome 10, but no gene has yet been identified.In the heteroplasmic state, the mutant Drosophila subobscura strain studied in our laboratory presents a substantial mtDNA 1 deletion (30% of the coding region) (12). Contrary to clinical findings (1), this mutation remains stable from one generation to the next. It does not seem to affect the mutant (apparent absence of phenotype) in terms of reproduction (number of embryos, emergence of larvae), life span, or flight capacities. This strain thus offers a good model for studying possible biochemical and molecular consequences of severe mitochondrial genome alteration and the responses that enable the mutant to survive.The first investigations, performed in the whole adult fly (13), showed a strong heteroplasmic tendency in the deleted molecules; this affected 78% of the mitochondrial genomes. mtDNA cell content (per nuclear genome) showed a 50% increase in the mutant strain compared with the D. subobscura ...

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