Human Molecular Genetics, 2000, Vol. 9, No. 18 2733-2742
© 2000 Oxford University Press
A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome
Istituto Nazionale Neurologico ‘C. Besta’, Via Celoria 11, 20133 Milano, Italy, 1Department of Clinical Neurosciences, Royal Free and University College Medical School, University College London, London NW3 2PF, UK and 2Department of Molecular and Cellular Biology, Section of Molecular Biology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands
Received 31 July 2000; Revised and Accepted 5 September 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We report on a novel frameshift mutation in the mtDNA gene encoding cytochrome c oxidase (COX) subunit III. The proband is an 11-year-old girl with a negative family history and an apparently healthy younger brother. Since 4 years of age, she has developed a progressive spastic paraparesis associated with ophthalmoparesis and moderate mental retardation. The presence of severe lactic acidosis and Leigh-like lesions of putamina prompted us to perform muscle and skin biopsies. In both, a profound, isolated defect of COX was found by histochemical and biochemical assays. Sequence analysis of muscle mtDNA resulted in the identification of a virtually homoplasmic frameshift mutation in the COIII gene, due to the insertion of an extra C at nucleotide position 9537 of mtDNA. Although the 9537Cins does not impair transcription of COIII, no full-length COX III protein was detected in mtDNA translation assays in vivo. Western blot analysis of two-dimensional blue-native electrophoresis showed a reduction of specific crossreacting material and the accumulation of early-assembly intermediates of COX, whereas the fully assembled complex was absent. One of these intermediates had an electrophoretic mobility different from those seen in controls, suggesting the presence of a qualitative abnormality of COX assembly. Immunostaining with specific antibodies failed to detect the presence of several smaller subunits in the complex lacking COX III, in spite of the demonstration that these subunits were present in the crude mitochondrial fraction of patient’s cultured fibroblasts. Taken together, the data indicate a role for COX III in the incorporation and maintenance of smaller COX subunits within the complex.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Human cytochrome c oxidase, COX, the terminal component of the mitochondrial respiratory chain, is composed of 13 protein subunits: the three largest are encoded by mtDNA genes, whereas the remaining subunits are encoded by nuclear genes. The sequences of all the COX subunit genes have been completely determined in humans. Combined or isolated defects of COX account for a number of mitochondrial disorders including severe infantile myopathies, progressive encephalomyopathies, cardiomyopathies and Leigh syndrome (LS) (1). Nuclear genes controlling the assembly of COX are responsible for some of them. Examples are mutations of SURF-1, leading to LS (2,3), SCO2, causing severe infantile cardiomyopathy (4), and COX10, associated with progressive leukoencephalopathy (5). In contrast, in only a few reports have mutations been described in mtDNA-encoded COX structural genes (http://www.gen.emory.edu/mitomap.html ) (6), whereas no mutations are known in nuclear genes encoding COX subunits. We have found a novel frameshift mutation in the mtDNA COIII gene, predicting the synthesis of a prematurely truncated COX subunit III. This mutation was virtually homoplasmic in both muscle and skin fibroblasts of a child affected by a progressive neurological disorder characterized by symmetric necrotic lesions of putamina, similar to those observed in LS (7). This is the first case of a Leigh-like syndrome due to a mutation in a structural COX gene. The clinical features of our patient were those of a slowly progressive disorder dominated by spastic/dystonic tetraparesis and ophthalmoparesis, with little impairment of muscle trophism and a modest intellectual impairment. The relatively mild course of the disease is in contrast with the marked biochemical defect of COX activity and with the severity of the genetic lesion. The presence of the mutation in fibroblasts offered the unique opportunity to generate COX IIInull cell lines to study the role of COX III in the assembly and function of human COX. Qualitative and quantitative abnormalities of COX assembly were investigated by western blot analysis of protein fractions resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and blue-native gel electrophoresis (BNE).
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RESULTS |
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Morphology and biochemistry
The presence of severe lactic acidosis and symmetrical lesions of the posterior part of putamina (Fig. 1A) prompted us to perform muscle and skin biopsies. In both we found the virtual absence of the histochemical reaction to COX (Figs 1B and 2A), whereas the reactions to NADH dehydrogenase and succinate dehydrogenase (SDH) were both normal in muscle (data not shown). Ragged-red fibres (RRF) were absent (Fig. 1C). The profound, isolated defect of COX activity was confirmed biochemically in both muscle homogenate and digitonin-treated fibroblasts (Table 1). As shown in Table 1, the activities of the other respiratory enzymes, as well as of citrate synthase (CS), a Krebs-cycle enzyme, were all within the normal range.
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In a second set of experiments, we measured the kinetics of COX for cytochrome c in muscle homogenate of the patient and three controls. The apparent Km was 10.4 µM in the patient’s sample and 11.8 ± 2.1 µM in the control samples.
Studies on hybrids and cybrids
To establish whether the gene mutation underlying the disease was carried by a nuclear or a mitochondrial gene, we performed a complementation assay based on the fusion of the proband’s cytoplasts with mtDNA-less (rho-zero, 
0) derivatives of 143B.206 human osteosarcoma cells (143B.206-0) (Fig. 2B). We collected 20 cybrid clones that were later shown to contain 100% mutant mtDNA (see below). One clone, called Cy7, was used for all the subsequent studies. Cy7 did not show a correction of the COX defect histochemically (Fig. 2C). Biochemical analysis demonstrated that the defect was specific to COX (Table 1). The amount and integrity of Cy7 mtDNA was demonstrated to be normal by Southern blotting, indicating that the COX defect in Cy7 was not due to gross abnormalities of mtDNA (e.g. mtDNA depletion). In contrast to the fusion of the proband’s cytoplasts with 143B.206-0 cells, full restoration of COX activity was obtained in cybrids (Fig. 2F) of patient-derived 0 fibroblasts (Fig. 2D) fused with cytoplasts derived from HeLa cells (Fig. 2E). Taken together, the results pointed to the presence of a deleterious mutation in a mitochondrial COX gene.
Identification of an mtDNA 9537Cins mutation
Sequence analysis of the three mtDNA-COX genes revealed the presence of an insertion of an extra C at nucleotide position 9537 (9537Cins) in the midportion of the COIII gene in both muscle and fibroblast mtDNA (Fig. 3A). The frameshift produced by this mutation creates a stop codon two codons downstream of the insertion, leading to the synthesis of a prematurely truncated polypeptide of 110 amino acids. The normal protein is composed of 260 amino acids. Restriction fragment length polymorphism (RFLP) analysis of an allele-specific PCR fragment using the restriction endonuclease BslI confirmed the results obtained by nucleotide sequence analysis. The mutation was virtually homoplasmic in mtDNA from skeletal muscle, fibroblasts and lymphocytes (Fig. 3B). The mutation was also homoplasmic in mtDNA derived from oral and urinary-tract epithelial cells (data not shown). However, after exposure for 48 h, a wild-type mtDNA band could be detected in the lymphocyte sample, but not in the other samples of the patient (data not shown). The amount of heteroplasmy was quantified as <1% of the total amount of lymphocyte mtDNA. No mutation was detected in mtDNA extracted from lymphocytes of both her brother and maternal grandmother, or from lymphocytes, hair and oral epithelial cells of the mother.
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Northern blot analysis and mtDNA translation in vivo
To investigate whether the loss-of-function mutation of the COIII gene was associated with instability of the corresponding transcript, we performed northern blot analysis on total RNA extracted from Cy7 cybrids, patient’s fibroblasts, control fibroblasts and the original 143B.206 human osteosarcoma cells used to create the
0 derivative 143B.206-0. A PCR fragment corresponding to the entire COIII gene was used as a probe. As shown in Figure 4A, three hybridization bands were obtained in all lanes. According to previous reports (8–10), a high molecular weight band was identified as an uninterrupted polycistronic transcript of the entire mtDNA. A second band of intermediate size was considered as a partially processed RNA species composed of COIII + ATPase 6/8. A third band, 
1000 nucleotides in size, was identified as the mature COIII mRNA. Neither qualitative nor obvious quantitative abnormalities were present in the mutant transcripts compared with the controls.
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To evaluate the consequence of the mutation on mtDNA translation, an in vivo assay was performed with Cy7 and control (original 143B.206) cells. As shown in Figure 4B, the mtDNA-specific translation pattern obtained from the COX IIInull mutant cell line was identical to that of the control cell line, except that the band corresponding to the COX III polypeptide was absent.
Studies on COX assembly
To verify the presence and amount of COX subunits in mutant cell lines, we performed western blot analysis on mitochondrial proteins from Cy7- and control 143B-derived isolated mitochondria, separated by one-dimensional (1D) SDS–PAGE. As shown in Figure 5A, the same amount of mitochondrial protein was loaded in each sample, as confirmed by the comparable intensity of a band immunostained by a monoclonal antibody against the 70 kDa flavoprotein of SDH. Bands with identical electrophoretic mobility, corresponding to each polypeptide, were obtained in both samples using antibodies against COX subunits I, II, IV, Va, Vb, VIa (isoform L), VIb and VIc. The amount of crossreacting material (CRM) in the patient’s samples was comparable with that in the control samples for subunits IV, Va and VIc; it was slightly reduced for subunits I, II, Vb and VIb and markedly reduced, but not absent, for subunit VIa.
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To understand the consequence of the mutation on the assembly of the holoenzyme, western blot analysis was carried out on equal amounts of mitochondrial proteins, extracted from the same number of Cy7 cells, patient’s fibroblasts and HeLa cells and separated by two-dimensional (2D) BNE. Four subcomplexes (S1, S2, S3 and S4) have previously been defined by 2D-BNE as COX-assembly intermediates of increasing molecular weight (11). In normal conditions, S1 corresponds to COX subunit I on its own. The incorporation of COX subunit IV, forms a second intermediate, S2. The third intermediate, S3, is the result of the association of S2 with a large series of subunits, including COX II, III, Va, Vb, Vc, VIb, VIc, VIIb, VIIc and VIII. Finally, assembly of the holoenzyme, corresponding to S4, is completed by the incorporation of two final subunits, COX VIa and VIIa. As shown in Figure 5B, in HeLa cells most of the COX I-specific CRM was confined to S4, corresponding to fully assembled COX. In contrast, most of the CRM in the 9537Cins mutant Cy7 clone was present in subcomplex S1, corresponding to COX I alone, and in two intermediate subcomplexes, identified as S2A and S3. The fully assembled complex S4 was absent. Subcomplex S2A had an electrophoretic mobility different from subcomplex S2 identified in pulse–chase control experiments, suggesting a qualitative abnormality of COX assembly. Western blot analysis performed with an anti-COX II antibody showed the presence of specific CRM in S2A and S3 subcomplexes (Fig. 5B), indicating that this subunit had been incorporated in the early steps of COX assembly, as also seen in controls. Identical results as those obtained with Cy7 were also obtained using patient’s fibroblasts (data not shown). Immunostaining of 2D-BNE western blots using antibodies against subunits COX Vb and VIc failed to detect the corresponding CRM in Cy7, whereas a specific signal was clearly present in the control cell lines (Fig. 5C). Identical results were obtained using patient’s fibroblasts. Compared with control cells, a very faint signal, corresponding to S2a, was detected by an antibody against COX Va, whereas the signal obtained with an antibody against COX IV was comparable with that for control cells. In the latter experiment, the signal in the mutant cell line was distributed in the intermediate subcomplexes (S2, S2a, S3), whereas most of the CRM for all of the subunits of the control cell line was concentrated in S4. In addition, we measured by densitometry the integrated area and intensity of the spots corresponding to assembled enzyme in 2D-BNE western blots from gels loaded with identical amounts of proteins from digitonin-treated cells. The densitometrically estimated amount of COX III-defective assembled enzyme (S2a +S3 in cell line Cy7) was
15–25% of the amount of wild-type COX (S4 in cell line 143B.206), a percentage similar to that of the residual activity of the mutant enzyme relative to the control mean. |
DISCUSSION |
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Clinical considerations
This is the third mutation of the gene encoding COX subunit III to be reported in patients affected by mitochondrial disorders. In contrast to the previous cases (12,13), in which the COX activity defect was confined to mature skeletal muscle, in our patient the defect was detected in both skeletal muscle and fibroblasts. Again in contrast with the previous cases, which were both characterized by isolated myopathy of adulthood, our patient presented with an early-onset neurological syndrome that had some features resembling those found in LS. A profound and generalized defect of COX activity is indeed commonly observed in LS. Most of the cases of LSCOX are due to mutations of SURF-1 (2,3,14), a gene encoding a COX assembly factor (15,16), whereas no mutations of genes encoding structural components of COX have ever been associated with LS. This is the first case of a Leigh-like syndrome due to a mutation in a structural COX gene. The neuropathology in our patient was confined to the neostriatum, with hardly any involvement of the thalamus, subthalamus and brainstem structures, as typically found in LSCOX cases associated with SURF-1 mutations (G. Uziel and M. Zeviani, manuscript in preparation). In addition, the clinical course was milder than the rapidly progressive impairment of brainstem functions of typical LS. Our patient suffered from a slowly progressive neurological syndrome dominated by impairment of the pyramidal and extrapyramidal motor systems, but with rapid, partially reversible worsenings caused by life-threatening attacks of lactic acidosis.
The relatively static clinical course of our patient is difficult to reconcile with the presence of a homoplasmic mutation destroying one major component of complex IV. One possibility is that the mutation, which was ostensibly homoplasmic in muscle and fibroblasts, may be heteroplasmic in brain and other tissues. This possibility is supported by the demonstration of a minimal amount of wild-type mtDNA (<1%) detected in lymphocytes. A ‘protective’ effect due to the presence of a proportion of wild-type mtDNA could explain the limited extension of the brain lesions and the absence of clinical symptoms attributable to impairment of other organs frequently involved in mitochondrial disorders, such as the heart or the inner ear. A second related observation is that the activity of the COX III-defective enzyme was not completely abolished. A 15–20% residual activity was measured in muscle, fibroblasts and cybrid Cy7, where the mitochondrial genotype was entirely composed of the 9537Cins mutant mtDNA species. This finding could explain the relatively mild impairment of skeletal muscle function in our patient. In addition, such a residual activity may perhaps be sufficient to maintain cellular respiration at levels adequate for function in organs and tissues, such as the liver, the kidney or the heart, that appear to be clinically spared in our patient.
Biochemical considerations
How can this residual activity be reconciled with the destruction of a highly conserved, large subunit of the enzyme? In prokaryotes, COX complexes are much smaller than the 13 subunit mitochondrial COX, sharing only three conserved polypeptides: subunits I, II and III (17). In these systems, subunit I contains haems a and a3; the latter forms a Fe-Cu binuclear center that binds O2, catalyzes its reduction to water and couples this process to proton pumping. Subunit II contains two other Cu atoms, binds and oxidizes cytochrome c and transfers the electron to haem a on subunit I. As far as subunit III is concerned, in spite of its high interspecific conservation, its function is still unknown. Similar to our case, the deletion of subunit III of the Paracoccus denitrificans enzyme resulted in inhibition and inactivation of electron transport (18). A role of subunit III of the mitochondrial COX in proton pumping has been suggested by some authors who observed that removal or proteolytic digestion of subunit III greatly inhibits proton pumping. In addition, it was shown that DCCD, which reacts specifically with a conserved acidic residue in subunit III (homologous to E98 in P.denitrificans) also inhibits proton pumping (19–21). However, loss of proton pumping on removal of subunit III or binding of the bulky DCCD must be ascribed to non-specific secondary perturbations of subunits I and II, as COX from P.denitrificans has been isolated in a fully functional form that contains only subunits I and II (22) and site-directed mutants with substitutions for the conserved E98 and D259 residues in P.denitrificans retain wild-type proton translocation activity (23). Deletion of the gene for subunit III in P.denitrificans leads to accumulation of assembly intermediates of COX, including a complex of subunits I and II, as well as the haem-containing free subunit I and the free subunit II (24). This null mutant has only residual COX activity, but the activity is coupled to proton pumping (23). In conclusion, experiments on simpler organisms suggest that subunit III does not play an important direct role in energy conversion by COX, but is rather involved in assembly and stabilization of the entire complex.
Our findings are in agreement with this conclusion, for the following reasons. First, the relative amount of residual COX activity measured in 9537Cins cell lines is 15–25% compared with control cell lines. This figure is similar to the percentage of ‘assembled’ COX (S2a + S3) in COX III-defective cell lines, relative to fully assembled COX in control cell lines, quantified densitometrically on 2D-BNE western blots. Secondly, the apparent Km of the mutant COX for cytochrome c was comparable to that of the wild-type enzyme, suggesting no difference in the affinity for its natural substrate. Both observations suggest that the decrease of COX activity in the mutant specimens is mainly due to a proportional reduction of ‘assembled’ COX, i.e. to a quantitative defect of COX holoprotein, rather than to a qualitative abnormality of the catalytic rate of the COX III-defective enzyme.
Disruption of COX assembly has been proposed for a recently discovered stop mutation of the COI gene (25) and for one of the previously described COIII gene mutations (26), a heteroplasmic 15 bp deletion associated with myopathy and myoglobinuria (12). However, in contrast to our observations, cybrid cell lines homoplasmic for the 15 bp deletion had no detectable COX activity (26). The 15 bp deletion is predicted to remove the central region of the third N-terminal membrane-spanning helix (helix III) of subunit III, whereas the 1 bp insertion in our patient is predicted to truncate subunit III just C-terminal of helix III (27). Helices I and III of subunit III are associated with subunit I, whereas the other five membrane-spanning helices of subunit III show no direct contact with subunit I (27). Our experiments do not fully rule out the presence of low levels of truncated subunit III, below the detection limit of the in vivo translation experiment shown in Figure 4B. Therefore, whereas the 15 bp deletion is likely to result in an impairment of the interaction between subunits I and III, we cannot exclude in principle that the truncated COX III polypeptide predicted by the 1 bp insertion is synthesized and possibly associated with subunit I to form a partially active complex.
The 1D-SDS–PAGE western blots showed that most of the smaller COX subunits were present in equal amounts in both control and mutant cell lines, with the exception of subunit VIa. Interestingly, studies based on crystallized bovine COX have demonstrated that the transmembrane region of subunit VIa interacts with helix IV of subunit III (27). Our 2D-BNE western blot experiments clearly showed that despite the normal steady-state levels of nuclear-encoded subunits, the COX III-defective enzyme not only lacked COX VIa, but also several, if not most, of the other nuclear-encoded subunits of complex IV. A notable exception was the nuclear-encoded COX subunit IV, which is one of the first subunits to be incorporated in the nascent complex, at a step earlier than incorporation of COX III. In contrast, the unassembled subunits were the ones whose incorporation follows that of COX III. This observation suggests that the presence of COX III is critical to the completion of the later steps of COX assembly.
The presence of non-assembled subunits, as demonstrated by 1D-SDS–PAGE, is surprising, since unassembled proteins would be expected to be degraded rapidly. One possibility is that these proteins might be associated with chaperones (e.g. hsp-60) in a relatively ‘protected’ and ‘stable’ condition. Another possibility is that the defective assembly of COX, its impaired activity, or both, could trigger a compensatory mechanism, such as increased synthesis rate and/or protein import of the nuclear-encoded COX subunits.
Taken together, our findings indicate that the COIII mutation does not influence the synthesis rate, or import and maturation of most of the nuclear-encoded subunits, but rather prevents them from being incorporated into, or remain integrated within, the holoenzyme. COX III can therefore be considered as an ‘intrinsic organizer’, necessary for the incorporation of the late subunits of COX.
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MATERIALS AND METHODS |
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Case report
The proband is an 11-year-old girl with a negative family history and an apparently healthy younger brother. She was born at term after an uneventful pregnancy. A difficult labour that was attributed to entrapment of the umbilical cord led to an episode of perinatal cyanosis but with no obvious consequences. The birth weight was 3.6 kg. Psychomotor development was normal during the first months of life. However, at 1 year of age she suffered an episode of metabolic coma associated with severe metabolic acidosis, hyperchaetonuria and hypoglycaemia. A second similar episode occurred at 3 years of age. She walked independently at 2 years. At the age of 4 years she suffered of a third episode of extreme metabolic acidosis (pH 7.2, pCO2 = 31.6, HCO3– = 12.9, base excess = –13.3). Blood lactate was 11.1 mM [normal values (n.v.) 0.5–1.8] and pyruvate was 0.14 mM (n.v. 0.04–0.13); blood glucose was 47 mg%. She was subsequently reported as suffering from severe progressive tetraparesis, associated with ophthalmoparesis, convergent strabismus, reduced visual acuity and moderate mental retardation.
At first admission to our Institute, at the age of 9 years, she could not walk independently and presented a severe kyphoskoliosis associated with bilateral retraction of the flexor muscles of the hip and a plastic hypertonus which was more severe in the lower limbs. A moderate intellectual deficit was noticed, but with a good social attitude and appropriate verbal skills.
Laboratory analysis revealed the presence of a severe metabolic acidosis (pH 7.33, pCO2 = 16.9, HCO3– = 9, base excess = –13.5) due to very high levels of blood lactate (5.01 mM) and less elevated levels of pyruvate (0.14 mM) and alanine (0.53 mM, n.v. 0.15–0.36). Elevated levels of these metabolites were also detected in the cerebral spinal fluid. Blood glucose concentration was normal. Nuclear magnetic resonance (NMR) examination of the brain revealed the presence of bilateral lesions in the putamina (Fig. 1A) and mild atrophy of the brain and cerebellum, whereas the brainstem structures appeared as normal. The brainstem auditory evoked potentials, visual evoked potentials and electroretinogram were all normal, but the electroencephalogram revealed the presence of an 
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background rhythm, with bursts of spike-wave abnormalities, more abundant in the frontal regions. A muscle computerized tomography scan showed the presence of mild hypotrophy of the posterior thigh muscles. Eletrocardiogram was normal. Her clinical conditions have remained unchanged.
Morphological and biochemical analyses
Morphological analysis of skeletal muscle and biochemical assays of the individual respiratory complexes on muscle homogenate and digitonin-treated fibroblasts were carried out as described (28). Specific activities of each complex were normalized to that of CS, an indicator of the number of mitochondria.
COX kinetic curves for cytochrome c were obtained by measuring COX activity in the presence of increasing concentrations (8, 16, 20, 40, 80 and 120 µM) of horse heart cytochrome c (fraction V; Sigma, St Louis, MO) in 1 mg/ml bovine serum albumin, 10 mM K+ phosphate, pH 7.0. Lineweaver–Burk linearization was used to obtain the apparent Km and Vmax.
Sequence analysis
Suitable overlapping DNA fragments encompassing the three mtDNA genes encoding COX subunits I, II and III were generated by PCR amplification of total genomic DNA using pairs of 25mer sense and antisense oligonucleotides as primers, following standard cycles at an annealing temperature of 56°C. Each fragment was sequenced in both directions using the big dye-terminator Ready Reaction mix and read on an ABI 373A automated sequencer (Perkin-Elmer, Foster City, CA).
RFLP analysis
A 200 bp fragment encompassing nucleotides 9370–9570 of the mtDNA Cambridge sequence (29) was PCR-amplified using a 25mer sense oligonucleotide (from nucleotide 9370 to 9394) and the following 37mer antisense oligonucleotide: 5'-CCCCAAGGAAGTAGGGCACTGGCCCCCAACATGCA-TCA-3'. The underlined GG doublet replaces a wild-type TT doublet in order to create a diagnostic BslI restriction site specific to the mutant mtDNA species. The underlined Ts replace wild-type Gs in the modified oligonucleotide in order to eliminate naturally occurring BslI restriction sites. After an initial denaturation at 96°C for 2 min, each of the 33 PCR cycles were carried out as follows: denaturation at 94°C for 45 s, annealing at 58°C for 30 s, extension at 72°C for 45 s; followed by a final extension at 72°C for 5 min. An aliquot of 0.2 µCi/sample [32P]dCTP was added in the last PCR cycle. In the presence of the 9537Cins mutation, the modified antisense oligonucleotide creates a BslI-specific restriction site (CCNNNNNNNGG) which was used for RFLP analysis. After digestion with BslI, the cleaved 167 and 33 bp fragments, corresponding to the mutant species, were differentiated from the 200 bp uncut fragment, corresponding to the wild-type species, by non-denaturing polyacrylamide–TBE gel electrophoresis. After drying, the gel was exposed for 6 h, or overexposed for 48 h, in a BioRad (Hercules, CA) screen cassette. The autoradiography was visualized in an FX Personal Molecular Imager (BioRad). The proportion of mutant versus total mtDNA was calculated by densitometry.
Fibroblast and cybrid cell cultures
Immortalized fibroblast cell lines were created using pBABE40 (30), a vector co-expressing the gene conferring resistance to puromycin and the major T antigen of SV40. Fibroblasts were continuously cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and 0.5 µg/ml puromycin.
Transmitochondrial cybrids were obtained by polyethylene glycol fusion followed by selection in a uridine-free medium, as described (31). In a first set of experiments, cytoplasts derived from cytochalasin-B-treated patient’s fibroblasts were fused with 143B.206-0, a 0 derivative of the human osteosarcoma cell line 143B.206. In a second set of experiments, cytoplasts derived from cytochalasin-B-treated HeLa cells were fused with a 0 derivative of our patient’s puromycin-resistant fibroblast transformants. The absence of mtDNA in rho-zero cell lines and its presence in transmitochondrial cybrids was confirmed by PCR using pairs of primers that amplify the D-loop region, as described (28,31). The presence of the appropriate nuclear genotypes in transmitochondrial cybrids was confirmed by fragment length polymorphism analysis using the highly polymorphic marker D11S533, as described (32).
Northern blot analysis
Total RNA was isolated from control fibroblasts, patient’s fibroblasts and in the 100% mutant cybrid clone Cy7, using the Ultraspec RNA Isolation System (Biotecx, Houston, TX). Approximately 15 µg/lane of total RNA was separated through a 1.0% agarose–formaldehyde gel, transferred to a Hybond-N+ nylon membrane (Amersham, Piscataway, NJ) in 50 mM NaOH, prehybridized for 2 h at 60°C in 5x SSPE/5x Denhardt’s solution, 0.5% SDS, 100 µg/ml sonicated herring sperm DNA. The filter was hybridized overnight at 60°C with a PCR fragment corresponding to the COIII gene, radiolabelled by the random primer method (Boehringer, Mannheim, Germany) in the presence of [32P]dCTP. After washing, the filter was exposed for 6 h in a BioRad screen cassette. The autoradiography was visualized in an FX Personal Molecular Imager (BioRad).
In vivo mtDNA translation
Pulse-labelling of exponentially growing cells was carried out for 30 min in the presence of [35S]methionine (1000 Ci/mmol, 150 µCi/ml) and 100 µg/ml emetine. Translation products were electrophoresed through a denaturing 15–20% exponential gradient polyacrylamide gel, as described previously (32). The gel was fixed for 30 min in 30% methanol, 10% acetic acid, soaked for 60 min in DMSO and for 120 min in 22% PPO in DMSO. After washing in distilled water for 60 min, the gel was dried and exposed for 3–4 days in a screen cassette. The autoradiography was visualized in an FX Personal Molecular Imager (BioRad).
Western blot analysis of protein fraction resolved by 1D-SDS–PAGE and 2D-BNE
One-dimensional SDS–PAGE was performed according to Rahman et al. (33), on a mitochondrial fraction prepared from digitonin-treated cells, as described (34).
For 2D-BNE, crude mitochondrial pellets, obtained as described (15), were resuspended in 100 µl of 1.5 M 6-aminohexanoic acid, 50 mM Bis–Tris, pH 7.0. Twenty microlitres of 10% ß-lauryl maltoside were added and the samples were incubated for 15 min on ice. Clearing of the samples was performed by centrifugation at 12 000 g for 20 min at 4°C. The supernatant was removed and 10 µl of 5% Serva Blue G in 1 M 6-aminohexanoic acid was added prior to loading. Blue-native 2D-PAGE was performed as described (35). Gels were used for western blot analysis as previously described (15).
The blots were immunostained using mouse monoclonal antibodies raised against bovine COX subunits I, II, IV, Vb, VIc (Molecular Probes, Eugene, OR), Va (36), VIa (37) and VIb (36) and the flavoprotein subunit of SDH (38).
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ACKNOWLEDGEMENTS |
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We are indebted to Ms B. Geehan for revising the manuscript. We thank Prof. R. Bisson for critical discussion and Dr Marco Rimoldi, Dr Marina Mora and Mr Murtada H. Farhoud for technical advice and help. This work was supported by Fondazione Telethon-Italy (grant no. 1180 to M.Z.), Ricerca Finalizzata Min. San. ICS 030.3/RF98.37 and EU Human Capital and Mobility network grant on ‘Mitochondrial Biogenesis in Development and Disease’.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +39 02 239 4388; Fax: +39 02 266 4236; Email: zeviani@tin.it
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REFERENCES |
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