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W2073455917 abstract "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. 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 (1DiMauro S. Moraes C.T. Arch. Neurol. 1993; 50: 1197-1208Crossref PubMed Scopus (356) Google Scholar, 2Wallace D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8739-8746Crossref PubMed Scopus (438) Google Scholar, 3Wallace D.C. Shoffner J.M. Trounce I. Brown M.D. Ballinger S.W. Corraldebrinski M. Horton T. Jun A.S. Lott M.T. Biochim. Biophys. Acta. 1995; 1271: 141-151Crossref PubMed Scopus (207) Google Scholar). 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 (4Holt I.J. Harding A.E. Morgan-Hugues J.A. Nature. 1988; 331: 717-719Crossref PubMed Scopus (1543) Google Scholar, 5Holt I.J. Harding A.E. Petty R.K.H. Morgan-Hugues J.A. Am. J. Hum. Genet. 1990; 46: 428-433PubMed Google Scholar, 6Lestienne P. Ponsot G. Lancet. 1988; i: 885Abstract Scopus (256) Google Scholar). Once the proportion of deleted genomes reaches a critical threshold, these deletions have often very pejorative effects on mitochondrial function (7Hayashi J.-H. Ohta S. Kikuch A.M.T. Goto Y.-I. Nonaka I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10614-10618Crossref PubMed Scopus (513) Google Scholar, 8Attardi G. Yoneda M. Chomyn A. Biochim. Biophys. Acta. 1995; 1271: 241-248Crossref PubMed Scopus (104) Google Scholar). Such mutations are generally sporadic, although dominant autosomal factors have been observed (9Zeviani M. Servidei S. Gellera C. Bertini E. DiMauro S. DiDonato S. Nature. 1989; 339: 309-311Crossref PubMed Scopus (550) Google Scholar, 10Suomalainen A. Kaukonen J. Amati P. Timonen R. Haltia M. Weissenbach J. Zeviani M. Somer H. Peltonen L. Nat. Genet. 1995; 9: 146-151Crossref PubMed Scopus (217) Google Scholar, 11Kaukonen J.A. Amati P. Suomalainen A. Rotig A. Piscaglia M.G. Salvi F. Weissenbach J. Fratta G. Comi G. Peltonen L. Zeviani M. Am. J. Hum. Genet. 1996; 58: 763-769PubMed Google Scholar). A locus has been localized on chromosome 10, but no gene has yet been identified. In the heteroplasmic state, the mutant Drosophila subobscurastrain studied in our laboratory presents a substantial mtDNA 1The abbreviations used are: mtDNA, mitochondrial DNA; W, wild strain; H, mutant strain; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; α-GP, α-glycerophosphate; CO, cytochrome oxydase; ND, NADH dehydrogenase. deletion (30% of the coding region) (12Volz-Lingenhöhl A. Solignac M. Sperlich D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11528-11532Crossref PubMed Scopus (44) Google Scholar). Contrary to clinical findings (1DiMauro S. Moraes C.T. Arch. Neurol. 1993; 50: 1197-1208Crossref PubMed Scopus (356) Google Scholar), 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 (13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar), 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 theD. subobscura wild strain. Determinations of steady-state concentrations of mitochondrial transcripts showed these to be identical to values measured for transcripts of genes unaffected by deletion (12 S, COIII). On the other hand, the transcript concentrations of the concerned genes showed a decrease, although this was less than expected and differed according to the genes (from 55% for ND1 to 35% for cytochrome b). Furthermore, detection of the fusion transcript (13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar) showed that both types of genomes were expressed. Maximum respiratory complex activities were identical in the wild type and mutant strains for complex IV. They were, respectively, 30% and 40% lower for complexes III and I (14Debise R. Touraille S. Durand R. Alziari S. Biochem. Biophys. Res. Commun. 1993; 196: 355-362Crossref PubMed Scopus (20) Google Scholar). Nevertheless, these differences were not seen for complex III in our experimental conditions for respiratory chain observation (oxygraphy), whereas complex I consistently presented a 30% decrease in activity. The respiratory chain would thus seem to be operative as of complex III. No ultra structural mitochondrial alteration was observed irrespective of the studied tissues. Moreover, cytochrome oxidase activity was detectable in all mitochondria. Heteroplasmy was probably intramitochondrial (15Lecher P. Béziat F. Alziari S. Biol. Cell. 1994; 80: 25-33Crossref PubMed Scopus (16) Google Scholar, 16Lecher P. Petit N. Béziat F. Alziari S. Eur. J. Cell. Biol. 1996; 71: 423-427PubMed Google Scholar), since both types of genomes coexisted in each mitochondria. Overall, these results tend to indicate compensation for the consequences of deletion at the level of the whole fly. One possible explanation for this surprising apparent innocuousness of the mutation is that compensation may exist between the various tissues or certain (vital) tissues may be less affected than others. The results presented in this article were obtained from measurements performed in mitochondrially enriched fractions of various tissues with high energy requirements. Whereas studies described in man show that distribution of deleted molecules and effects of deletion may differ substantially from one tissue to another (17Ponzetto C. Bresolin M. Bordini A. Moggio M. Meola G. Bet L. Prelle A. Scarlato G. J. Neurol. Sci. 1990; 96: 207-210Abstract Full Text PDF PubMed Scopus (27) Google Scholar), this is not so for the mutant D. subobscura strain we studied, in which biochemical specificities of the various tissues are upheld. The studied flies belong to the D. subobscura strain and were raised on medium standard cornmeal as described (12Volz-Lingenhöhl A. Solignac M. Sperlich D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11528-11532Crossref PubMed Scopus (44) Google Scholar) at 19 °C. The wild strain (W) acts as a control to study the effects of deletion on the mutant strain (H). The various studied fractions were enriched with a given mitochondrial type: the mitochondria of the nerve tissue for the head, those of the muscle tissue for the thorax, and those of male or female genital organs for the male or female abdomens. The flies were frozen for molecular biology or anaesthetized in ice for biochemistry before dissection of the three parts (head, thorax, abdomen). The obtained fractions were used as quickly as possible for extraction of nucleic acids or mitochondria. Total DNA was obtained from 50–60 heads, thoraces, or abdomens according to the method described by F. Béziat et al.(13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar). 50–60 heads, thoraces, or abdomens were ground in 1 ml of RNA-ZOLTM (Bioprobe system) at 4 °C. 0.1 volume of chloroform, isoamyl alcohol (24:1, v/v) was added. After stirring and incubation (5 min at 4 °C), the aqueous and organic layers were separated by centrifugation at 12,000 × g, 15 min, 4 °C. The RNA was precipitated with a volume of isopropyl alcohol. Heteroplasmy was determined by Southern blotting of DNA fragments obtained after digesting DNA with MspI and hybridization with the COIII probe labeled as described (13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar). The signals were analyzed by densitometry. Mitochondrial DNA content was measured by comparison with nuclear DNA by slot blot of the various DNA fractions followed by hybridization with a mitochondrial probe (12 S RNA) and a nuclear probe (18 S). The signals were analyzed by densitometry, as was heteroplasmy. RNA content was estimated by the Northern technique using 18 S RNA as the control RNA. The studied RNAs are 12 S RNA and COIII (genes not involved in deletion), cytochrome b, and ND1, ND5, ND4-4L (genes involved in deletion), using the probes described in Ref.13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar. The mitochondria were isolated by differential centrifugations of ground tissue samples in buffer containing 0.22 msucrose, 0.12 m mannitol, 1 mm EDTA, 10 mm Tricine, pH 7.6 (18Alziari S. Stepien G. Durand R. Biochem. Biophys. Res. Commun. 1981; 99: 1-8Crossref PubMed Scopus (25) Google Scholar). Protein determination was performed with Bio-Rad reagent using the method of Bradford method (19Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216334) Google Scholar). For enzymatic assays, mitochondria (about 1 mg protein/ml) were sonicated for 6 s at 4 °C then frozen and unfrozen except for α-glycerophosphate dehydrogenase and ATP synthesis determinations. All activities were measured at 28 °C and expressed in nmol·min−1· mg−1. NADH oxidation was monitored at 340 nm in a pH 7.2 buffer containing 35 mmNaH2PO4, 5 mm MgCl2, and 2.5 mg/ml bovine serum albumin in the presence of 2 μg/ml antimycin, 2 mm KCN, 97.5 μm ubiquinone, 0.13 mm NADH, and 50 μg of mitochondrial proteins. Only the rotenone-sensitive activity is noted. Ubiquinol was obtained by reduction of ubiquinone with sodium dithionite followed by extraction with cyclohexane. After evaporation of the cyclohexane, ubiquinol was dissolved in ethyl alcohol and stabilized with 10 mm HCl. To measure the activity of complex III, cytochrome c reduction was monitored at 550 nm (ε = 18,500m−1·cm−1) in 1 ml pH 7.2 buffer containing 35 mm NaH2PO4, 5 mm MgCl2, and 2.5 mg/ml bovine serum albumin in the presence of 2 mm KCN, 2 μg/ml rotenone, 15 μm ubiquinol, 15 μm cytochromec, and 5 μg of mitochondrial proteins. The nonenzymatic reduction of cytochrome c with ubiquinol (in the absence of mitochondria) was deduced from this measurement. Oxidation of partially reduced cytochrome c (reduced OD − oxidized OD ∼ 0.65) was monitored at 550 nm (ε = 18,500m−1·cm−1) in 1 ml of pH 7.4 buffer containing 30 mm KH2PO4, 1 mm EDTA, 56 μm cytochrome c, 5 μg of mitochondrial proteins. The appearance of thionitrobenzoic acid (yellow) derived from the reduction of dithiobis(nitrobenzoic acid) (colorless) with coenzyme A, was monitored at 412 nm (ε = 13,600 m−1·cm−1) in 1 ml of 100 mm Tris-HCl pH 8 buffer, 2.5 mm EDTA, 37 μm acetyl-CoA, 75 μm dithiobis(nitrobenzoic acid), 300 μm oxaloacetate, 5 μg of mitochondrial proteins. 20 μg of mitochondrial proteins were incubated for 30 min in 100 μl of 40 mm Pi buffer, pH 8, containing 1 mmKCN, 0.2% p-iodonitrotetrozolium violet, 10 mmα-glycerophosphate (α-GP). The reaction was stopped with 400 μl of 1 m acetic acid, and the iodoformazan thus formed was extracted with 1 ml of pure ethyl acetate. The organic layer was recovered by centrifugation and the OD was read at 500 nm (ε = 18,500m−1·cm−1). This value allows deduction of the OD of a test not containing α-GP. The reduction of NAD by α-GP was monitored at 340 nm in 100 mm glycine buffer, pH 9.8, containing 0.5 mmNAD, 10 mm hydrazine (to displace the reaction toward NADH formation), 5 mm α-GP, and 50 μg of post-mitochondrial supernatant proteins. Mitochondrial ATP synthesis was measured using a technique derived from that described by Wibomet al. (26Wibom R. Söderlund K. Lundin A. Hultman E. J. Biolumin. Chemilumin. 1991; 6: 123-129Crossref PubMed Scopus (35) Google Scholar) based on the luminescence of luciferin in the presence of luciferase, which is proportional to the ATP concentration in the test medium (27Deluca M. Adv. Enzymol. 1969; 44: 37-68Google Scholar). In the presence of ATP, the photon flux emitted by the reagent (BioOrbit) remains constant for several minutes for a determined ATP concentration. Synthesis kinetics can thus be directly monitored with a BioOrbit 1251 luminometer. Kinetics were recorded at 25 °C, pH 7.5, with 50 μl of the incubation medium containing 0.15 mm ATP and 1 μg of mitochondrial proteins. After 2 min of incubation, 5 μl of substrate and 100 μl of reagent were added simultaneously, and fluorescence was monitored for 1 min. The final concentrations of substrates were 5 mmglutamate + 2 mm malate, 15 mm proline + 2 mm malate, 1 mm palmityl carnitine + 2 mm malate, or 10 mm α-glycerophosphate. Calibration was performed at the end of each measurement by the addition of 200 pmol of ATP. A control run in the presence of oligomycin allows ATP synthesis imputable to intermembranar adenylate kinase activity to be subtracted from the result. Results are compared for the various tissues of a single strain, then for tissues of the two strains (W and H). The values thus determined are considered significantly different where the statistical study (Student's t test) yields a value ofp < 0.05. Three fractions are readily accessible in the Drosophila. One tissue is particularly well represented in each of these three fractions. Hence, mitochondria from this tissue constitute the dominant population: the head, where the mitochondrial pellet is enriched with nerve tissue mitochondria; the thorax, with muscle mitochondria, particularly from flight muscles; and the abdomen, with mitochondria from the germ line, particularly in the abdomens of pubescent females. These samples thus allow evaluation of the characteristics of mitochondria from different tissues, all of which have high energy demands but different biochemical characteristics. The possible influence of mutation is studied on these fractions; thus, on mitochondria from different tissues. Relative proportions of the two types of mitochondrial genomes are determined after incubation of total DNA with the MspI enzyme followed by hybridization with the COIII probe as described above (13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar). Heteroplasmy measured in the fractions from heads and thoraces (containing somatic tissues), 74 ± 5% and 79 ± 6%, respectively, is not significantly different (p < 0.05) from that measured in the whole fly (78 ± 5%). On the other hand, there is a significantly lower percentage of heteroplasmy in the abdomens (containing the germ cells): 71 ± 5% in males, 63 ± 5% in females. Moreover, there is a significant difference in values for male and female abdomens. Cellular mtDNA content extracted from the various fractions of the wild and mutant strains is compared with nuclear DNA content as described previously (13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar) using two types of probes: a mitochondrial probe (12 S) and a nuclear probe (18 S). These cellular contents are compared for different tissues from a given strain and for each tissue, comparing the two strains. To facilitate comparisons between the different fractions of the wild type strain, relative quantities of cellular mtDNA in the thorax and abdomen (Fig. 2) are evaluated by comparison with the content measured in the head (base 100 for the head). The cellular mtDNA content in the thorax is identical to that measured in the male abdomen and exceeds the content measured in the head by 50%. The mtDNA content is higher in the female abdomen than in the other fractions: × 3 compared with the head. Cellular mitochondrial DNA content is thus lower in the head fraction. The difference observed between the head and thorax corroborates electron microscopy findings, 2P. Lécher, unpublished results. where mitochondria are seen to be much more abundant in the thorax, or measurement of citrate synthetase activity, which is 2.5 times higher in the thorax than in the head. Very high levels are observed in female abdomens where maturing ova are prevalent. The same distribution of cellular mtDNA levels in the mutant strain for the various fractions is encountered (Fig. 2). mtDNA contents for each fraction are compared for the two strains. Compared with the wild strain, the mtDNA/cell content in the mutant strain shows a 43–75% increase (respectively, 75 ± 3, 72 ± 3, 43 ± 4, and 62 ± 4 for head, thorax, male and female abdomens). However, these differences do not significantly differ from results obtained for the whole fly (50% increase; Ref. 13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar). For each fraction from the mutant strain, the relative percentage of intact and deleted molecules compared with the wild strain was calculated taking two parameters into account: increased cell content and heteroplasmy (the mtDNA content in each tissue of the wild strain is 100%, Fig. 3). The percentages of intact molecules are identical in the head and thorax fractions (36%) and comparable to those measured in the whole fly (30%). The percentage is significantly higher in the male abdomen (44.5%). The female abdomen contains 54% of intact molecules. Hence, the female abdomen apparently constitutes the fraction least affected by mutation. Total RNA extracted from the different fractions of the mutant and wild strains (see “Experimental Procedures”) is analyzed by Northern blot and hybridization with different probes. The results presented are obtained from six different extractions and Northern blot analyses. The fact that different probes with different specific activities were used makes it very difficult to exactly estimate steady-state concentrations for each transcript in the various fractions of the two strains. Steady-state concentrations are thus determined by comparing hybridization signals obtained with a single probe for transcripts extracted from head and thorax fractions and from male and female abdomens in the two strains. A control of RNA extraction and hybridization is obtained with the 18 S probe. The Rf ratios express hybridization signal ratios (corrected with 18 S) for each transcript in the various fractions of a given strain. The same membranes were used to compare the different transcript concentrations in each fraction between mutant and wild type. The Rs ratios express hybridization signal ratios (corrected with 18 S) for each transcript in the various fractions for the two strains. Head and thorax concentrations of the 12 S transcripts are identical (Rf = 1.1 ± 0.5). The COIII transcript shows a lower concentration (p < 0.05) in the head (Rf = 0.7 ± 0.2). Transcripts of complex I genes constitute identical quantities in both tissues (Rf ND1 = 0.9 ± 0.3, ND4-4L = 0.9 ± 0.2, ND5 = 1 ± 0.5). On the other hand, the concentration for cytochrome btranscript is much higher (p < 0.05) in the head (r = 3.3 ± 1.2). Male and female abdomen comparison gives results similar to those described for head and thorax except for cytochrome btranscript, which is in identical concentration in both abdomens (Rf = 0.7 ± 0.4, p > 0.05). Results indicate that the Rf obtained for transcripts 12 S, ND1, and cytochrome b (head/thorax or male/female abdomens) are identical to those obtained for the wild type. In the head and thorax, the COIII transcripts are in identical concentration (Rf = 1.4 ± 0.4; the difference is not significant). ND4-4L and ND5 transcripts (Rf = 1.3 ± 0.3 and Rf = 1.5 ± 0.1) show levels in male abdomens to be significantly higher than those observed in female abdomens (p < 0.05). The most remarkable finding is the fact that the head/thorax Rf for the cytochrome b transcript is very high (Rf = 2.6 ± 1.3), as in the wild strain. This tissular specificity was thus unaffected by the mutation involving this gene. Results indicate that the concentrations of gene transcripts (12 S, COIII) unaffected by deletion are lower in the head (20–30%); COIII transcript concentration also decrease in thorax. In contrast, they are higher (20–30%) in the abdomens of male or female. Concentrations of gene transcripts affected by mutation are lower in each fraction. Those of complex I decreased by 70% in the head and thorax. The decrease is less important in the abdomens (50% in male, 10–50% in female). The cytochrome b transcript concentration shows a 50% decrease in the different fractions. In each fraction, the mutation has affected the transcript concentrations of the genes involved by the deletion, but as observed in the whole fly, the observed decreases are very often not directly proportional to the quantities of intact genomes. The ND1–ND5 fusion transcript (1300 base pairs) was detected in RNA extractions from the whole fly, where its relative concentration was very similar to that of ND1 (13Béziat F. Morel F. Volz-Lingenhöhl A. Saint-Paul N. Alziari S. Nucleic Acids Res. 1993; 21: 387-392Crossref PubMed Scopus (29) Google Scholar). This transcript is detected in all fractions (results not show), indicating an expression of the deleted genomes in all tissue. The concentration in all fractions is not significantly different from that of the ND1 transcript. Mutation affects genes implicated in respiratory chain function. Biochemical activities of respiratory complexes I, III, and IV, certain subunits of which are coded by the mitochondrial genome, were tested in various fractions of the wild and mutant strains. The activity of citrate synthetase, an enzyme of the Krebs cycle of nuclear origin, was also measured. Enzymatic systems known to be active in insect mitochondria (28Sacktor B. Rockstein M. The Physiology of Insects. 4. Academic Press, New York1974: 271-354Google Scholar) and which possibly play a role in electron transfers toward respiratory chain complexes such as the α-glycerophosphate dehydrogenase and α-glycerophosphate oxidase couple, were likewise measured. Finally, ATP synthesis capacity by isolated mitochondria was evaluated. Activity of NADH dehydrogenase (complex I) by comparison with the head is lower in the abdomen and higher in the thorax. Activity of complex III is higher in the head than in the other fractions: three times the activity measured in the abdomen fractions (the difference between the male and female abdomen is not significant) and five times the measured activity in the thorax. In the head fraction, this activity is superior to that of complex I (× 8) and complex IV (× 2). Such activity in mitochondria of the head fraction correlated with the high concentration of Cytob transcript theoretically corresponds to higher demand on this complex and a different respiratory metabolism ensuring direct electron supply to ubiquinone and to complex III. Complex IV activity shows no significant differences for the studied fractions. This is the highest activity of the respiratory chain: 7–9 times the activity of complex I in the various fractions, except in the head where complex III is dominant. The activity of citrate synthetase is identical in the head, the male abdomen, and the female abdomen. On the other hand, it is very high (× 2.5) in the thorax, showing a much greater Krebs cycle potentiality in the muscles, which are highly solicited in flight (28Sacktor B. Rockstein M. The Physiology of Insects. 4. Academic Press, New York1974: 271-354Google Scholar). Two enzymes, the α-glycerophosphate dehydrogenase cytosolic enzyme and α-glycerophosphate oxidase, located on the outer surface of the internal membrane, allow direct reduction of ubiquinone through cytosolic redox potential (α-GP shunt). Activities of these enzymes are clearly higher in the thorax: thoracic α-glycerophosphate oxidase is 3 times more active than α-glycerophosphate oxidase in the head or male abdomen (p < 0.001), and thoracic α-glycerophosphate dehydrogenase is 4–5 times higher than α-glycerophosphate dehydrogenase in these fractions. Activity of these enzymes is low in the female abdomen. Male and female activities are significantly different (p < 0.05). This metabolic shunt, which plays a major role in insects (28Sacktor B. Rockstein M. The Physiology of Insects. 4. Academic Press, New York1974: 271-354Google Scholar), should be more effective in the muscles, since α-GP is a substrate rapidly used for flight. Hence, mitochondrial metabolism in the fly shows tissular specificity: enzymatic capacities vary and logically reflect different metabolic flux for the different tissues. Comparison of activities in the various studied fractions leads to the same conclusions as above. Mutation did not modify tissular specificities of the studied metabolisms; hence, normal metabolic flux was theoretically upheld. We compared activity for each fraction of the mutant strain with that of the corresponding wild strain: Ra (activity H/activity W) is shown in Fig. 7. Activity of complex I clearly decreases in all fractions: Ra = 0.5 in the head, 0.6 in the thorax, and 0.7 in the abdomen. In the whole fly (Ra = 0.6), this activity decreases as observed in the thorax. The smallest decrease in activity, in the abdomen, is probably attributable to the fact that heteroplasmy is only 62% in females and 71% in males (but the differences between the male and female abdomen is not significant). Activity of the complex III is not affected in the head and male abdomen but drops in the thorax (Ra = 0.8) and in the female abdomen (Ra = 0.7). This decrease is identical to that observed in the whole fly (Ra = 0.8). Hence, mutation does not affect the very important activity of this complex in the head. There is no significant difference in complex IV activity for the two strains irrespective of the studied fraction. Results thus bear out those observed for the whole fly (14Debise R. Touraille S. Durand R. Alziari S. Biochem. Biophys. Res. Commun. 1993; 196: 355-362Crossref PubMed Scopus (20) Google Scholar): mutations affecting complex I and complex III activities do not affect complex IV activity. This indicates that in the mutant strain, the protein synthesis in all mitochondria is normal, as observed previously (15Lecher P. Béziat F. Alziari S. Biol. Cell. 1994; 80: 25-33Crossref PubMed Scopus (16) Google Scholar). Activity of citrate synthetase is identical in the two strains for the head and thorax but is higher in the mutant abdomens (p< 0.05). This possibly indicates increased Krebs cycle activity. The metabolic" @default.
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W2073455917 title "Biochemical and Molecular Consequences of Massive Mitochondrial Gene Loss in Different Tissues of a Mutant Strain of Drosophila subobscura" @default.
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