Relationship of genotype to phenotype in fibroblast-derived transmitochondrial cell lines carrying the 3243 mutation associated with the MELAS encephalomyopathy: shift towards mutant genotype and role of mtDNA copy number
Relationship of genotype to phenotype in fibroblast-derived transmitochondrial cell lines carrying the 3243 mutation associated with the MELAS encephalomyopathy: shift towards mutant genotype and role of mtDNA copy numberHerman A. C. M. Bentlage+,* and Giuseppe Attardi
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
Received September 11, 1995;Revised and Accepted November 28, 1995
Transmitochondrial cell lines were isolated by fusing mtDNA-less [rho]o206 cells with enucleated fibroblasts derived from four members of a pedigree carrying in their muscle varying proportions of the mutation at position 3243 in the tRNALeu(UUR) gene associated with the MELAS encephalomyopathy. The mitochondrial transformants derived from an asymptomatic individual were all homoplasmic for wild-type mtDNA. The proportion of wild-type transformants derived from clinically affected members of the pedigree appeared to decrease in correspondence with an increase in severity of the clinical symptoms of the cell donor. Furthermore, the average proportion of wild-type mtDNA in the transformants derived from each member of the pedigree was very similar to that found in mtDNA from the fibroblasts of that individual, suggesting that the distribution of genotypes in the transformants reflected fairly closely that in the fibroblasts. The genotype and phenotype of ten transformants derived from one severely affected individual were investigated during continuous culture up to 17-24 weeks after the transformation step. Six heteroplasmic clones showed a progressive increase in the proportion of mutant mtDNA, whereas the mitochondrial genotype remained constant in four clones apparently homoplasmic for wild-type mtDNA or nearly homoplasmic for mutant mtDNA. An analysis of the rate of repopulation of [rho]o206 cells with fibroblast-derived mtDNA revealed a large variability among different transformants, with the full re-establishment of the control ratio of mtDNA to nuclear DNA being observed between ~6 weeks and more than 22 weeks after the transformation step. An increase in rate of O2 consumption generally accompanied the increase in mtDNA copy number of the transformants, pointing to the important role of the mtDNA copy number in determining the phenotype of a cell. The observation that a very small amount of wild-type mtDNA (2 to 5% of the control level), coexisting with strongly predominant mutant mtDNA, conferred upon the transformants a substantial respiratory capacity (50% or more) and the evidence of proportionality between O2 consumption rate and mtDNA copy number, which occurred at widely different mutant to wild-type mtDNA ratios, strongly suggest a contribution of the mutant mtDNA to the cell respiratory competence.
Deletions, point mutations and depletion of mtDNA have been described in association with myopathies, encephalomyopathies and a variety of other disorders (for reviews, see refs 1 and 2 ). The phenotypic manifestation of the so-called `common mtDNA deletion' (4977 base pairs) associated with chronic progressive external ophthalmoplegia (CPEO) and with Pearson syndrome, and of some point mtDNA mutations has been studied in clonal cell lines derived from patient's cells (3 -5 ) and in human mtDNA-less ([rho]o) cells (6 ) repopulated with mutant mitochondria through fusion with enucleated patient-derived cells (7 -13 ). The point mutations investigated included those in the tRNALys gene (at positions 8344 and 8356) associated with the MERRF (myoclonic epilepsy and ragged red fiber) syndrome (3 ,7 ), those in the tRNALeu(UUR) gene (at positions 3243 and 3271) associated with the MELAS (myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome (4 ,9 ,10 ,11 ), that in the same gene (at position 3260) associated with myopathy and cardiomyopathy (12 ), and that in the ATPase 6 gene (at position 8993) associated with the Leigh syndrome (13 ). These studies have demonstrated that the mtDNA mutations mentioned above cause defects in mitochondrial protein synthesis and cell respiration; in particular, the biochemical defect underlying the protein synthesis impairment in the MERRF syndrome caused by the 8344 mutation has been elucidated (14 ). Furthermore, it has been shown that these mutations are functionally recessive, since wild-type mtDNA, even when present in relatively small proportion, can protect a cell against the phenotypic effects of the mtDNA mutation (3 ,8 ,10 ). A useful extension of the [rho]o cell repopulation approach has involved the use of platelets, which are naturally occurring anucleate derivatives of megakariocytes, as mitochondria donors (15 ).
A propagative advantage of mutant mtDNA molecules over wild-type molecules, leading to a segregation of the genotype towards the pure mutant type, has been observed in genotypically unstable transmitochondrial cell lines carrying in heteroplasmic form the `common' 4977 bp deletion (8 ), or the 3243 tRNA Leu(UUR) mutation (16 ,17 ), or the 8344 tRNALys mutation (18 ). However, evidence has also been reported indicating that the direction of segregation towards the mutant or the wild-type genome is influenced by the nuclear background (17 ). Skin fibroblast cultures from a patient with Pearson syndrome have been reported to exhibit a loss of the 4977 bp deletion when cultured in the absence of uridine, and amplification of the deleted mtDNA in the presence of uridine (5 ). A loss of the deletion dependent on the absence of uridine was, on the contrary, not seen in lymphoblastoid cell lines from two Pearson syndrome patients (5 ).
In the present study, a shift of the mitochondrial genotype towards higher levels of the tRNALeu(UUR) (3243) mutation was found in all genotypically unstable heteroplasmic transmitochondrial cell lines derived from fibroblasts of one member of a previously described MELAS pedigree (19 ). However, the rate of this shift was much slower than previously observed in unstable transformants derived from myoblasts of MELAS patients (16 ,17 ). The re-establishment of the parental mtDNA copy number was in general slow in the [rho]o cell transformants and accompanied by an increase of rate of O2 consumption. Very low proportions of wild-type mtDNA (possibly as low as 2%) were able to protect against the deleterious effects of the mutation. Thus, both the proportion of wild-type mtDNA and the mtDNA copy number play a role in the phenotypic manifestation of the mutation.
Figure 1 a shows the MELAS pedigree investigated here (19 ). This included a 56 year old woman (I2) and her five daughters (II1 to II5). The clinically unaffected mother had 35% mutated mtDNA in her muscle, but no mutated mtDNA in her blood and fibroblasts. The daughter II1 only suffered from deafness, whereas the daughters II3 and II5 both exhibited the MELAS syndrome: the latter showed higher percentages of mutated mtDNA in their tissues than the former one. The other two daughters (II2 and II4) were clinically unaffected, and showed no or only minor proportions of mutated mtDNA in her tissues (19 ).
The ratio of mtDNA to nuclear DNA (reflecting the mtDNA copy number) in mitochondrial transformants from three family members (I2, II1 and II5) was measured, at different times after fusion, by slot blot analysis, using a mtDNA probe and a 28S rRNA probe, respectively. They are expressed relative to the mtDNA/nuclear DNA ratio in 143B.TK- cells, which has 9700 molecules mtDNA per cell (6 ) and is the parental line from which the [rho]o206 cells were derived. As shown in Figure 3 , the relative mtDNA copy number of the transformants, surprisingly, varied greatly, when measured 6 to 11 weeks after fusion. The variation covered a range between 6 and 178% of the 143B.TK- mtDNA/nuclear DNA ratio (set equal to 1) in the 12 wild-type transformants derived from the asymptomatic mother (I2), the mtDNA copy number being >75% of the 143B.TK- level in the majority of them. Two transformants derived from patient II1, C3 and C10, exhibited a mtDNA copy number corresponding to 85 and 97% of the relative 143B.TK- mtDNA level at 102 days after fusion, having a proportion of 15 and 100% wild-type mtDNA, respectively. In eleven transformants derived from II5 (named A1, A4, A5, A7, A10, B1, B3, B4, B6, B11 and B12), the mtDNA copy number varied between ~2 and ~130% of the relative 143B.TK- mtDNA level, without any obvious relationship to their proportion of mutant mtDNA. One transformant (A7), which had a mtDNA copy number corresponding to only 2% of the 143B.TK- level, with 2% of the mtDNA molecules being wild-type, grew only in the presence of 50 µg/ml uridine. This cell line resembles the previously described human cell mutants, which are severely depleted of mtDNA (20 ). During continued growth in medium not supplemented with uridine, the mtDNA copy number of all the II5-derived transformants below the 143B.TK- level showed a tendency to increase slowly and to approach or slightly surpass that level between 90 and 150 days after fusion (set to 1 in Fig. 3 ). The only exception appeared to be clone II5-B6, whose mtDNA copy number rose from 1.3 times the 143B.TK- level, measured 51 days after fusion, to 1.9 times that level, measured 130 days after fusion.
In previous work (16 ), it was found that, among 13 myoblast-derived [rho]o206 cell transformants that were heteroplasmic for the MELAS 3243 mutation, five underwent a rapid shift of their mitochondrial genotype towards the pure mutant type, whereas the other eight cell lines, which included six exhibiting nearly homoplasmic mutant mtDNA and one carrying nearly homoplasmic wild-type mtDNA, maintained a stable genotype. In no case, a shift of mtDNA towards the wild-type form was observed. We have reinvestigated this behavior in ten transformants derived from fibroblasts of patient II5. The proportion of mtDNA with the MELAS mutation was determined with the `last radioactive cycle PCR', which excludes the possibility of heteroduplex formation between wild-type and mutant mtDNA, followed by ApaI digestion. The autoradiogram of one such experiment is shown in Figure 4 . A weak band, with an intensity proportional to the level of mutant mtDNA, was seen moving slightly faster than the expected 158 bp band. We believe that this extra band is derived from the band above it, probably by partial denaturation in the polyacrylamide gel, due to its lower GC content as compared to the 214 bp band (39.9 vs. 44.9%). The shift towards mutant mtDNA can be clearly seen in clones B4 and A1 (Fig. 4 ). Two clones (A4 and A10) exhibited apparently pure wild-type mtDNA, and two clones (B1 and B6) exhibited nearly homoplasmic mutant DNA. The other six clones were heteroplasmic for the mutation. The proportion of mutant mtDNA was quantified by a Phosphorimager. As shown in Figure 5 , all heteroplasmic transformants exhibited a decrease in the proportion of wild-type mtDNA, whereas this remained constant in clones with apparently homoplasmic wild-type or nearly homoplasmic mutant mtDNA. In no case was a shift towards the wild-type mtDNA observed. Furthermore, it was noted that the shift to the mutant genotype in the heteroplasmic clones was remarkably slower, almost by a factor of 7, than previously observed in myoblast-derived [rho]ocell transformants (16 ,17 ), as exemplified by the behavior of clone 94B in Figure 5 .
Eleven transformants derived from patient II5 maintained in culture over a long time, were investigated in detail to determine their phenotype to genotype relationship. The respiratory capacity of nine transformants was measured both rather early (41-80 days) and late (94-148 days) after the mitochondria transfer into [rho]o cells. A diagram illustrating the relationship between proportion of mtDNA carrying the MELAS mutation and degree of respiratory activity in the [rho]o cell transformants derived from patient II5 is shown in Figure 6 a. It appears that the O2 consumption rate rises sharply with the percentage of wild-type mtDNA until it reaches 2 fmol/min/cell, i.e. ~50% of the normal level, around 2% wild-type genomes, as observed in two different stages of the B1 transformant. The O2 consumption values of the B6 transformant, which are presumably high due to the significant increase in its mtDNA copy number (1.3 to 1.9 times the 143B.TK- level) (18 ), after correction for this factor, would also fit a threshold around 2% of wild-type mtDNA. The O2 consumption rates continue to rise, with increasing proportions of wild-type mtDNA, to reach ~4 fmol/min/cell by ~10% wild-type mtDNA, remaining thereafter more or less constant up to 100% wild-type genomes. Note that the early clones of A5, B4 and A10, having mtDNA copy numbers of 57, 41 and 26% of the 143B.TK- level, respectively, and a proportion of wild-type mtDNA of 53, 76 and 100%, respectively, have O2 consumption rates below 2 fmol/min/cell. The relatively low number of transformants with very high proportions of mutant mtDNA makes it difficult to define precisely the threshold level for protection against the effects of the MELAS mutation. However, it would appear that, in the fibroblast-derived [rho]o206 cell transformants investigated here, such threshold is lower than previously reported for myoblast-derived transformants carrying the MELAS mutation (~6%, ref. 10 ). This difference is possibly due to methodological factors. In fact, in the present work, the mutant mtDNA proportions were determined with a phosphorimager using the `last radioactive cycle PCR' method (see Materials and Methods, below), whereas, previously, correction for heteroduplex formation was made by laser densitometry of ethidium bromide-stained gels on the basis of a mixed-template standard curve (10 ).
The present work has provided insights into the multiple factors that play a role in determining the phenotype of transmitochondrial cell lines constructed by mitochondria transfer into mtDNA-less cells. In particular, the relationship of genotype to phenotype has been analyzed in transformants obtained by using fibroblasts from members of a family carrying the MELAS A to G transition at position 3243 of mtDNA as mitochondria donors, and the 143B [rho]o206 cell line as recipient. Three main conclusions have been derived from these studies:
(i) The transformants carrying the MELAS mutation in heteroplasmic form, with 15% to 85% wild-type mtDNA, exhibited a consistent genotype shift towards mutant mtDNA during prolonged culture; by contrast, the transformants carrying apparently pure wild-type mtDNA or mutant mtDNA in nearly homoplasmic form exhibited a stable genotype over a period of 13 to 18 weeks after fusion. The mtDNA shift towards the mutant type in the unstable heteroplasmic transformants was much slower than previously observed in myoblast-derived mitochondrial transformants of [rho]o206 cells (16 ,17 ).
(ii) The re-establishment of the normal mtDNA/nuclear DNA ratio with fibroblast-derived mtDNA in the [rho]o cell transformants occurred at a variable rate, but in general more slowly than previously observed in [rho]o206 cell mitochondrial transformants derived from established cell lines (6 ), and was accompanied by a parallel increase in O2 consumption.
(iii) A very small absolute amount of wild-type mtDNA (2-5% of the control level) in [rho]o cell transformants carrying strongly predominant mutant mtDNA conferred upon them a substantial respiratory capacity (50% or more). This observation, as well as the evidence of a proportionality between mtDNA copy number and O2 consumption rate, which occurred at widely different ratio of mutant to wild-type mtDNA, strongly suggested that the mutant mtDNA gives an essential contribution to the respiratory competence of the transformants.
In the present work, the near-identity of the average percentages of mutant mtDNA molecules in the transformants derived from each member of the pedigree and in the fibroblasts of that individual may point to a similar distribution of genotypes in the transformants and in the parental fibroblasts. If this interpretation is correct, the correlation between severity of the clinical phenotype of the cell donors, on one hand, and increase in proportion of heteroplasmic or nearly homoplasmic mutant transformants derived from those donors, on the other, suggests that the segregation towards the mutant genotype observed in the transformants may reflect a phenomenon occurring in vivo in the original fibroblasts. In other studies, among [rho]o206 transformants constructed using fibroblasts from a family exhibiting a non-insulin-dependent diabetes mellitus associated with the tRNA Leu(UUR) mutation (21 ), only one of seven heteroplasmic clones showed a stable genotype, while the others segregated, at a similar rate as that observed here, towards mutant mtDNA (22 ).
A mitochondrial genotype shift towards the mutant type has been previously observed in a number of transformants of the [rho]o206 cell line derived from myoblasts of individuals belonging to five different pedigrees carrying the MELAS 3243 mutation (16 ,17 ). In these studies, the majority of the [rho]o206 cell transformants derived from the same MELAS patients maintained a stable genotype, whereas no transformant exhibited a shift of the mitochondrial genome towards the wild-type. Interestingly, in one of the studies mentioned above (17 ), experiments utilizing myoblasts from the same MELAS patient as mitochondria donors revealed, in a subset of heteroplasmic transformants, a genotype shift towards either mutant mtDNA or wild-type mtDNA, depending on whether the [rho]o206 cell line or, respectively, a different [rho]o cell line ([rho]oA549) was used as recipient. These results clearly pointed to the role of the nuclear background in determining the direction of genome segregation. Recently it was claimed that the segregation process for the MELAS 3243 mutation in heteroplasmic myoblast-derived [rho]o cell transformants grown under non-selective conditions is stochastic in nature (23 ). However, differences in experimental procedures are likely to account for the different outcomes of this and our study.
The mechanism(s) of the rapid segregation towards the mutant or wild-type genome is unknown, and it is quite possible that two different mechanisms, i.e. replicative advantage and intercellular selection, respectively, operate in the shift towards the mutant and wild-type mtDNA. In particular, in previous work from this laboratory, a detailed analysis of myoblast-derived unstable [rho]o206 cell transformants shifting towards the mutant genotype has provided evidence suggesting that a replicative advantage of the mutant molecules was mainly responsible for the genotype shift (16 ). A plausible model for this replicative advantage involves a feedback phenomenon that induces the selective replication of mtDNA in organelles functionally compromised by the presence in them of mutated mtDNA in pure form, i.e., under conditions excluding intramitochondrial complementation (16 ,24 ). Replicative advantage has been suggested before to explain the preferential accumulation of deleted or otherwise grossly altered mtDNA in yeast and filamentous fungi (25 ,26 ). The observation that only a minority of the transformants obtained by using cells from the same patient as mitochondria donors for transfer to a given [rho]o cell line exhibited an unstable genotype can be explained by possible differences in nuclear background among cells in these transformants. Evidence for such variation among cells of the same [rho]o cell line has been previously presented (15 ). Thus, the more abundant stable transformants may represent cells whose nuclear background does allow extensive intramitochondrial complementation of the mutation, due, for example, to a slower rate of mitochondrial division relative to organellar growth, which would favor mutant and wild-type gene product mixing.
In the present work, no direct evidence has been obtained concerning the mechanism underlying the mitochondrial genomic shift towards the mutant type in the fibroblast-derived [rho]o206 transformants. An analysis of the doubling times of these transformants failed to reveal any correlation with their proportion of mutant mtDNA or with their genotype stability or genomic shift towards the mutant type (unpublished data). These results would argue against a significant role of intercellular selection of faster growing mutant mtDNA-enriched cells, suggesting that a replicative advantage of the mutant genomes, due to the same mechanism occurring in the myoblast-derived [rho]o206 transformants, may also operate here. As to the slower rate of genomic shift towards the mutant type in the fibroblast-derived transformants analyzed here, as compared to myoblast-derived transformants, an intriguing possibility is that it reflects the nature of the cell type utilized as a mitochondria donor. In particular, differences in the cytoplasts derived from distinct cell types may affect the reintegration of the foreign mitochondria into the general organization of the recipient cell, influencing the segregation behavior of the mutant mtDNA. However, on the basis of the available knowledge, a more likely possibility is that the particular mtDNA haplotype of the MELAS pedigree investigated in this work played an important role in the difference in behavior of the fibroblast-derived transformants.
There is ample evidence of the existence of a high degree of variation in human mtDNA (27 ,28 ). Although it has been generally assumed that mtDNA variants, other than those carrying clearly pathogenic mutations, are well tolerated, an increasing amount of evidence points to mtDNA polymorphism as a possible contributor to respiratory dysfunction (29 ). Thus, the synergistic effect of secondary mutations in mtDNA genes encoding subunits of NADH dehydrogenase in the pathogenesis of the Leber's hereditary optic neuropathy (LHON) has been suggested (28 ,30 ), and the role of the mtDNA haplotype as contributing to the pathogenesis of certain neurodegenerative diseases has been proposed (28 ,31 ), although there is no direct evidence yet to support these suggestions (32 ). Recently, an increased sequence variation in certain Complex I mtDNA-encoded subunits has been reported in patients affected by a variety of mtDNA linked diseases, and it has been proposed that this variation may contribute to the Complex I defect in these patients (29 ). MtDNA polymorphism can also conceivably affect the segregation of mutant and wild-type genomes. Thus, in human cell cybrids isolated by transfer of fibroblast mtDNA into HeLa cells, preferential loss of HeLa cell mtDNA has been reported (33 ). In S. cerevisiae, evidence has been obtained indicating that the expression of the mitochondrial genome is required for maintenance of the mitochondrial genomes (34 ). It is, therefore, possible that a difference in the rate of synthesis or stability of some mitochondrial translation products or in their functional efficiency, due to the occurrence of mtDNA polymorphism, influences the segregation behavior of this genome. Since, as mentioned above, the nuclear background can determine the direction of segregation of the MELAS 3243 mutation, it is conceivable that the mtDNA haplotype, by interfering with or facilitating the mechanism that underlies the shift towards the mutant or wild-type genome, can influence the rate of this shift. Further work is needed to clarify the mechanism which determines the slower segregation of the mitochondrial genome towards the mutant type in the fibroblast-derived (this study and ref. 22 ), as compared to the myoblast-derived transformants (16 ,17 ) carrying the MELAS 3243 mutation. In particular, it would be important to compare the segregation behavior of mtDNA in [rho]o cell transformants derived from cloned myoblasts and fibroblasts of the same MELAS patient.
The slow rate of reestablishment of the normal mtDNA copy number in the fibroblast-derived transformants analyzed here, as compared to the previously investigated transformants derived from established cell lines (6 ), may also be due to the donor cell mtDNA haplotype affecting the [rho]o cell repopulation process. In another context, the increase in O2 consumption observed during prolonged culture of the fibroblast-derived transformants, which paralleled in general the increase in their mtDNA copy number, clearly illustrates the role of the mtDNA level as a limiting factor in the mitochondrial respiratory phenotype. This phenomenon has been seen previously in cultured cells from patients with mtDNA depletion, during restoration of the mtDNA level by nuclear complementation (35 ,36 ). On the other hand, the observation of an increase in O2 consumption rate which was seen, upon prolonged culture, in clones (A4 and B3) already exhibiting at early times nearly wild-type respiratory activity, and which was independent of an increase in mtDNA content, suggests the intriguing possibility that a physiological adaptation may occur, which increases the respiratory capacity of the transformants by a mechanism not involving a change in mtDNA level. As concerns the contribution of mutant mtDNA to the respiratory competence of the transformants, which is strongly suggested by the evidence obtained in the present work, it could be the result of a complementation of the MELAS mutation by wild-type genes and/or of a residual functional capacity of the mutant molecules.
The bromodeoxyuridine (BrdU)-resistant 143B.TK- cell line and [rho]o206 cell line, an mtDNA-less derivative of the latter obtained by long-term exposure of the cells to low concentrations of ethidium bromide, were grown as described elsewhere (6 ). Human skin fibroblast lines from members of a family with the MELAS tRNALeu(UUR) 3243 mutation (19 ) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 50 µg uridine per ml and 20% fetal bovine serum (FBS).
Cytoplast fusion was carried out as described previously (6 ). In particular, enucleated fibroblasts (~2 × 104 cells) were fused to 106 [rho]o206 cells, and the cell mixture was subsequently plated into 96-well (I2, II1,II3, and II5) or 24-well (II3) plates at ~0.26 or ~5.2 original fibroblasts per well, respectively. Transformants were isolated from wells containing single clones and maintained in DMEM with 5% dialyzed FBS and 100 µg BrdU per ml.
Sixteen transformants of patient II5 were analyzed for the presence of mutant mtDNA between 27 and 53 days after fusion. Of these, eleven were maintained further in culture for analysis of their mtDNA copy number and their genotype to phenotype relationship. One transformant (A7) grew only in medium supplemented with uridine (50 µg/ml), while another one (A1) was only analyzed late after fusion.
All cell lines described in this report tested negative for the presence of mycoplasma by Hoechst 33258 (bisbenzimide, Sigma) fluorescence.
Cells were arrested in metaphase phase by treatment with colcemid (0.1 µg/ml). Approximately twenty-five air-dried metaphase spreads were examined according to standard procedures.
For quantitation of mtDNA, total DNA samples were isolated from unlabeled cultured cells with an Applied Biosystems 340A DNA extractor and analyzed in triplicate by slot blot hybridization, as described (16 ). Possible quantitative variations among different DNA samples were corrected by normalizing the data for the amount of nuclear 28S rRNA genes, using a 28S rRNA probe (18 ). Quantification of the hybridization reactions was carried out by analyzing the slot blots in a Phosphorimager (Molecular Dynamics).
The presence and proportion of mutated mtDNA in total cell DNA samples, prepared by proteinase K digestion of 0.5% Tween 20 cell lysates (18 ), were determined by testing for the presence of the ApaI site created by the MELAS mutation (37 ) at position 3243 (38 ) in a PCR-amplified fragment. The amplified fragments were digested with ApaI in the presence of pBluescript II KS(+) (Stratagene) DNA linearized with XmnI, as an internal marker for completion of digestion, and analyzed by agarose gel electrophoresis (10 ). Two primer pairs were used for the PCR reaction. The first pair consisted of two oligodeoxynucleotides corresponding to positions 3031-3050 and 3341-3360 in the Cambridge mtDNA sequence (38 ), yielding a PCR product of 330 bp, which was digested by ApaI into two fragments of 214 and 116 bp. Digestion with ApaI was performed for 10 h at 25oC by adjusting the PCR reaction mixture to the pH and salt conditions recommended by the supplier. The proportion of digested and undigested molecules, separated by electrophoresis at room temperature in a 1.4% agarose gel in Tris-borate-EDTA in the presence of ethidium bromide, was determined by laser densitometry (using an LKB Pharmacia instrument), and corrected for the resistance to digestion of heteroduplexes of wild-type and mutant mtDNA on the basis of a mixed-template standard curve (16 ). Alternatively, oligodeoxynucleotides corresponding to positions 3031-3050 and 3384-3402 were used for PCR, yielding a product of 372 bp, which is digested by ApaI into two fragments of 214 and 158 bp. In this case, heteroduplex formation was avoided by adding 5 µCi of [[alpha]-32P]dATP (3 000 Ci/mmol) only in the last (26th) cycle (39 ). After digestion with ApaI, the radioactive products were electrophoresed at 4oC through an 8% nondenaturing polyacrylamide gel, and quantified with a Phosphorimager. Completion of digestion of the internal marker was checked by electrophoresing portions of the samples at room temperature through a 1.8% Metaphor agarose (FMC BioProducts, Rockland) gel in the presence of ethidium bromide.
Rates of O2 consumption were measured (in duplicate) with a Gilson 5/6 oxygraph on samples of ~5 × 106 cells in 1.85 ml of DMEM lacking glucose, supplemented with 5% dialyzed bovine serum (6 ).
We are grateful to Drs J. Begeer and P. Smit (University Hospital Groningen, the Netherlands) for sending us tissues from their patients. We thank H. Janssen (Department of Human Genetics, Nijmegen, The Netherlands) for chromosome analysis, and A. Drew, B. Keeley, S. T. Lai and L. Tefo for technical assistance. These investigations were supported by a fellowship from the Ter Meulen Fund (Royal Dutch Academy of Sciences) and a travel grant from the Netherlands Organization for Scientific Research (NWO) (both to H.A.C.M.B.) and by National Institutes of Health Grant GM-11726 (to G.A.).
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*To whom correspondence should be addressed
+Present address: Department of Pediatrics, University Hospital Nijmegen St. Radboud, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
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