Mitochondrial biochemical activities and heteroplasmy evolution in established D. subobscura cell line.

A mutant strain of drosophila (D. subobscura) has two types of mitochondrial genomes: a small population (20%) identical to that of the wild strain (15.9 kb) and a predominant population (80%) which has undergone a 5-kb deletion affecting more than 30% of the coding zone. Two cell lines were established from homogenates of embryos from mutant and wild strains. The activities of the respiratory complexes measured in the different cell lines are much lower than in the flies, indicating a glycolytic metabolism. Various modifications of the medium composition did not change this metabolic pathway. The mutant cell line has two types of populations of mitochondrial genomes and the heteroplasmy is equivalent to that measured in the mutant strain. However, the biochemical characteristics differ from those observed in the flies (i.e., the decrease of complex I and III activities), and the various systems of compensation for the consequences of the deletion that are showed in the mutant strain are no longer observed. Furthermore, in contrast with observations made on mutant flies, the heteroplasmy appears unstable in the mutant cell lines: after 60 or so generations, it progressively decreases until it disappears completely. The limited importance of mitochondrial energy metabolism in cells may explain the low impact of the mutation on the established cell line, in contrast to what is seen in the mutant strain.

The respiratory chain complex thresholds in mitochondria of a Drosophila subobscura mutant strain.

Analysis of a mutant strain of Drosophila subobscura revealed that most (80%) mitochondrial genomes have undergone a large scale deletion (5 kb) in the coding region. Compared with the wild-type strain, complex I and III activities are, respectively, reduced by 50% and 30% in the mutant. However, the ATP synthesis capacities remain unchanged. In order to elucidate how the ATP synthesis is maintained at a normal level, despite a significant decrease in complex I and III activities, we progressively inhibited respiratory chain complex activities, respiration rate and ATP synthesis. Complex I, III and IV activities were inhibited by rotenone, antimycin and KCN, respectively. Threshold curves were thus determined for each complex. Our results demonstrated that in the mutant strain, both mitochondrial respiration and ATP synthesis had decreased when complex I activity was inhibited by more than 20%, whereas 70% inhibition is required to induce similar changes in the wild-type. The complex I inhibition pattern of the wild-type was restored by a backcross (mutant female/wild-type male). The complex III activity threshold is below 20% in both strains, and we observed some difference in antimycin sensitivity, suggesting a modification of the complex enzymatic properties in the mutant. In contrast, threshold values of 70% were measured for complex IV inhibition. Our data suggest that the difference in the complex I threshold curves between the wild-type and mutant strains could partially account for the absence of pathological phenotype in the mutant.

Technical knockout, a Drosophila model of mitochondrial deafness.

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 tko(25t) 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 tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.

Quantitative analysis, by ultrastructural in situ hybridization, of mitochondrial genomes and their expression in mid-gut and ovarian cells of a mutant strain of Drosophila subobscura.

In the mitochondrial deletion mutant strain studied here, two types of DNA coexist (heteroplasmy): intact mtDNA (15.9 kb) and mutant mtDNA (10.9 kb), which represents about 80% of the mitochondrial genomes in somatic tissues. The heteroplasmy level is lower in ovary (63%). Mutation is transmitted unchanged through generations. Quantitative analysis of in situ DNA hybridization demonstrated that for the 12SrDNA probe, of a gene outside the deletion, the mitochondrial DNA cellular content in the studied cells of the mutant strain is 1.5 times higher than in the wild-type strain. For the probe encoding Cyto b, a mitochondrial gene affected by the mutation, the ratios (mutant versus wild-type content) differ according to cell type: close to 0.4 in MGE cells and 0.7 in ovary cells. These values indicate heteroplasmic levels of about 72% in MGE cells and 50% in stage 10 oocytes, which is lower than that previously reported for stage 14 oocytes (60%) and embryos (69%). Analysis of in situ RNA hybridization showed that for the 12SrDNA probe, the transcript concentrations do not differ significantly between MGE cells and cells of germinal origin from the two strains. For the Cyto b probe, the mutant RNA/wild-type RNA ratios are lower in somatic cells than in stage 10 nurse cells and oocytes, but in each case less than expected. These studies indicate that the progressive heteroplasmy increase may be related to intense phases of mitochondria biogenesis and that different compensatory phenomena may exist.

Biochemical and molecular consequences of ethidium bromide treatment on Drosophila cells.

KC167 Drosophila cells were incubated with low concentrations of ethidium bromide (200 ng/ml), causing changes in mitochondrial DNA (mtDNA) content (2-184% of that of controls). SSCP (single strand conformational polymorphism) analysis of mtDNA indicated that the incubation with ethidium bromide also generated mutations. Compared with controls, there were marked reductions in the activities of respiratory complexes III and IV measured in these cells, and in respiration and ATP synthesis capacities measured in isolated mitochondria. These reductions matched that in mtDNA content. In contrast, no link could be demonstrated between mtDNA content and steady-state concentrations of the transcripts of genes COIII and Cyt b.

Developmental changes in heteroplasmy level and mitochondrial gene expression in a Drosophila subobscura mitochondrial deletion mutant.

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.

Biochemical and molecular consequences of massive mitochondrial gene loss in different tissues of a mutant strain of Drosophila subobscura.

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.

Quantitative decrease of human cytochrome c oxidase during development: evidences for a post-transcriptional regulation.

In an earlier study, we showed that cytochrome c oxidase activity, measured in mitochondria isolated from human muscular biopsies, decreased steadily and substantially between the age of four years and adulthood (P < 0.05), whereas complexes I and III activity remained constant. The present study investigates a number of possible causes for this change in activity: although there is a drop in the apparent Vmax, neither the apparent enzyme Km, nor the cellular mtDNA concentration shows any variations over the studied period. Steady-state concentrations of mitochondrial gene transcripts (CO I. CO II, CO III, but also 12S, cytochrome b, or ND4) increase within this age group, indicating an overall increase in mitochondrial genome expression. Concentrations of transcripts of nuclear genes CO IV, CO Vb, and CO VIaH likewise show an increase, albeit less marked. On the other hand, heme aa3 levels and concentrations of mitochondrial (CO II) or nuclear (CO IV, CO VIIaH) subunits, estimated using specific antibodies, correlate closely with enzymatic activity and show a parallel decrease between 4 and 20 years. The observed decrease in complex IV activity is thus quantitative, and subject to post-transcriptional and/or post-translational regulation.

Changes in the respiratory chain complexes activities and in the mitochondrial DNA content during ageing in D. subobscura.

The time course (age 0-8 weeks) of the enzyme activities of respiratory chain complexes I, III and IV and of citrate synthase, and the cell mitochondrial/nuclear DNA content ratio were studied in Drosophila subobscura. The activities of the three respiratory complexes decreased with age, but with different kinetics. The activities of complexes I and III remained nearly stable between weeks 0 and 3 (falling by 6% and 15%, respectively), and then gradually decreased; after 8 weeks residual activities were about 50% of the initial value for complexes I and III. The activity of complex IV fell in the first week, decreasing continually to week 8, where residual activity was 30% of the initial value. No significant age-related change in citrate synthase activity was observed. Mitochondrial DNA (measured by mitDNA/nucDNA) increased linearly up to week 5 (2.6-fold) and then dropped by 40% in week 6 though it remained higher than initial values.

Enzymatic activities of mitochondrial respiratory complexes from children muscular biopsies. Age-related evolutions.

Measurements were performed to determine maximum enzymatic activities of citrate synthetase and respiratory complexes I, III, and IV of mitochondria obtained from muscular biopsies in control children. The significant number of determinations carried out (43 different biopsies in controls aged 3.8 to 19.1 years) permits the formulation of a table of statistically validated reference values for these activities. These values are independent of sex of the controls, and of the studied muscles. Citrate synthetase activity, which remains stable in this age range, thus constitutes a good internal indicator of mitochondrial activity. Complexes I and III manifest activity which does not vary with age. On the other hand, cytochrome oxidase activity shows a highly significant decrease in this age group. This decrease may be correlated with qualitative changes (subunits VIa and VIIa) in composition of this complex.

Tissular distribution of heteroplasmy and ultrastructural studies of mitochondria from a Drosophila subobscura mitochondrial deletion mutant.

A mutant strain of Drosophila subobscura possesses two mitochondrial genome types: a minority population (20%) identical to the wild strain mtDNA (15.9 kb), and a largely predominant population (80%) of shorter genomes (10.9 kb), presenting a deletion of more than 30% of its coding region. Study of tissular distribution of heteroplasmy shows it to be identical--about 80%--in the head (nervous tissue) and thorax (muscles). On the other hand, a lower percentage (64%) is observed in the ovaries. The strain is apparently unaffected despite this massive loss of genes, coding for four tRNA and for complex I and III subunits. Contrary to observations of similar situations in man, the mutant strain shows no accumulation or structurally abnormal mitochondria. Furthermore, cytochemical studies fail to detect mitochondria devoid of cytochrome oxidase activity (COX-). Finally, mitoribosome populations are identical in mitochondria from both strains. These results suggest that, in the mutant strain, there are no mitochondria containing deleted genomes only: heteroplasmy would thus be intramitochondrial.

Biochemical consequences of a large deletion in the mitochondrial genome of a Drosophila subobscura strain.

A mutant strain of D. Subobscura possesses two populations of mitochondrial genomes: a population identical to that of the wild strain (20%) and a dominant population (80%) which has lost more than 30% of its coding zone by deletion. Spectrophotometric determination of respiratory complex activities shows that: complex I (5 genes implicated in deletion) presents maximal activity reduced by 40%, whereas that of complex III (concerned by cytochrome b) is lowered by 30%. Nevertheless, polarographic determinations of substrate oxidation show activity of complex I to be reduced by 30%. In contrast, complex III activity is similar to that measured in the wild strain. The predominant use of one part of the respiratory chain may account for the fact that the mutant strain is apparently unaffected by mutation.

Mitochondrial genome expression in a mutant strain of D. subobscura, an animal model for large scale mtDNA deletion.

A mitochondrial mutant strain of D. subobscura has two mitochondrial genome populations (heteroplasmy): the first (20-30% of the population, 15.9 kb) is the same as could be found in the wild type; the second (70-80% of the population, 11 kb) has lost by deletion several genes coding for complex I and III subunits, and four tRNAs. In human pathology, this kind of mutation has been correlated with severe diseases such as the Kearns-Sayre syndrome, but the mutant strain, does not seem to be affected by the mutation (1). Studies reported here show that: a) Transcripts from genes not concerned by the mutation are present at the same level in both strains. b) In contrast, transcript concentrations from genes involved in the deletion are significantly decreased (30-50%) in the mutant. c) Deleted DNA was expressed as shown by the detection of the fusion transcript. d) The mtDNA/nuc.DNA ratio is 1.5 times higher in the mutant strain than in the wild type. The mutation leads to change in the transcript level equilibrium. The apparent innocuousness of the mutation may suggest some post-transcriptional compensation mechanisms. This drosophila strain is an interesting model to study the consequence of this type of mitochondrial genome deletion.

Translation of polyuridylic acid in lysed mitochondria.

After osmotic shock with 50 mM Tricine buffer (pH 7.9), isolated mitochondria from D. Melanogaster embryos are treated with a low concentration of Triton X-100 (25 micrograms/mg of protein). The lysed mitochondria are still capable of RNA and protein synthesis. While incorporation of labeled precursor is often higher in lysed than in intact mitochondria, neosynthesized proteins exhibit similar electrophoretic patterns. Studies of labeled precursor incorporation in the presence of various effectors indicate a better accessibility to the translation machinery in lysed mitochondria than in intact mitochondria. Such a system has proven capable of translating an exogenous synthetic mRNA, i.e., poly (U).

RNA mapping on Drosophila mitochondrial DNA: precursors and template strands.

Drosophila melanogaster mitochondrial DNA (mtDNA) is closely related to the mammalian and amphibian mtDNA except for gene organization. In Drosophila, genes are distributed in clusters alternatively coded on each strand. Besides the eleven major foreseeable transcripts previously described (MERTEN and PARDUE, 1981, J. Mol. Biol., 153, 1-21), we have characterized two poly A+ transcripts, one major and one minor which could correspond respectively to the ND3 and ND6 reading frames, and 27 poly A+ minor transcripts (0.2 to greater than 3.2 kb) which are distributed along the mtDNA except in the rRNAs, ND 1 and A+ T rich regions. The mapping and length of 25 of these transcripts strongly suggest a precursor role. They would be processed at the level of tRNA or tRNA-like sequences. Most of them are transcribed from the template strand of each gene cluster and their distribution is in agreement with the hypothesis of several transcription origins and terminations located near the extremities of each gene cluster. Quantitatively our results show a large variation in each presumptive mature transcript compared to the other, even in a given gene cluster, suggesting a specific degradation of some of the mature transcripts.

Mitochondrial DNA expression in Drosophila melanogaster: neosynthesized polypeptides in isolated mitochondria.

The expression of mitochondrial genome of D. melanogaster in isolated mitochondria was followed by incorporation of 35S methionine in neosynthesized polypeptides. A high level of protein synthesis was obtained after optimization of all the incubation parameters. Two kinds of energy-generating systems were used: an endogenous system where an oxidizable substrate were added for ATP synthesis; an exogenous system with an energy-rich compound for ATP regeneration, the latter proved to be the most effective. The effect of the oxidative phosphorylation uncoupler (Clccp), and an ATPase inhibitor (oligomycine) allow us to postulate the role of the electrochemical potential in the expression of the mitochondrial genome. Electrophoresis and autoradiography of neosynthesized mitochondrial proteins exhibits 18 to 24 protein bands, ranging from 6.5 to 65 Kd; incubation of KC 0% drosophila cells with 35S methionine and cycloheximide gave similar results. Both our results and those published elsewhere suggest that the expression of mitochondrial genome in higher organisms could be more complex than simple translation of the 13 genes presents on these genomes.

[Isolation of the RNA-poly A+ fraction from mitochondria of Drosophila melanogaster embryos. Incorporation of methionine-35S into newly-synthesized proteins in whole and lysed mitochondria]. / Isolement de la fraction d'ARN-poly A+ des mitochondries d'embryons de Drosophila melanogaster. Incorporation de méthionine-35S dans les protéines néosynthétisées dans les mitochondries entières et lysées.

The poly A+ RNA fraction of embryos mitochondria of Drosophila melanogaster was prepared by fractionation by oligo dT-cellulose chromatography. It accounted for 5% of total insoluble RNA. 15 bands were identified by electrophoresis, from 500 to 2,000 base pairs. This poly A+ RNA fraction hybridizes with mitochondrial DNA clones. It contains mitochondrial transcripts: messenger RNA and the large ribosomal RNA. The sum of the transcripts (29 kb) exceed the coding capacities of the mitochondrial DNA. Some of the transcripts may be precursors, greater in size, of the mRNA corresponding to a gene. Our results are compared to those previously published (9, 11, 12). The relationship between mitochondrial transcripts and proteins was studied in whole and lysed isolated mitochondria. In the former, incubated in the presence of 35S-methionine, 20 to 30 radioactive bands were identified (M. Wt. 100 to 16 kd). This incorporation was totally inhibited by chloramphenicol. When the mitochondria were opened in the presence of detergent (lysis). 35S-methionine incorporation was markedly enhanced. In all cases the number of proteins identified was greater than the number of potential genes (12). This finding is discussed.