Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8059-8062, October 1989 Medical Sciences Directly repeated sequences associated with pathogenic mitochondrial DNA deletions (chronic progressive external ophthalmoplegia/mutation/recombination/polymerase chain reaction/heteroplasmy) DONALD R. JOHNS*t, S. LANE RUTLEDGEt, 0. COLIN STINE§, AND OREST HURKO*§ Departments of *Neurology, tPediatrics, and WMedicine, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205 Communicated by John W. Littlefield, July 10, 1989

ABSTRACT We determined the nucleotide sequences of in embryogenesis or have been inherited from phenotypically junctional regions associated with large deletions of mitochon- normal mothers. drial DNA found in four unrelated individuals with a pheno- To delineate further the mechanisms underlying the forma- type of chronic progressive external ophthalmoplegia. In each tion of the partial deletions of mtDNA, we determined the patient, the deletion breakpoint occurred within a directly nucleotide sequences of four additional junctional regions repeated sequence of 13-18 base pairs, present in different from unrelated patients with a phenotype of chronic progres- regions of the normal mitochondrial genome-separated by sive external ophthalmoplegia, disordered oxidative phos- 4.5-7.7 kilobases. In two patients, the deletions were identical. phorylation, and abnormalities ofmitochondrial morphology.¶ When all four repeated sequences are compared, a consensus In this report we show that each of thejunctions is associated sequence of 11 nucleotides emerges, similar to putative recom- with short (13-18 nts) directly repeated sequences. Within bination signals, suggesting the involvement of a recombina- these repeated regions, there is a consensus sequence of11 nts tional event. Partially deleted and normal mitochondrial DNAs that is similar to putative recombination signals, suggesting were found in all tissues examined, but in very different these deletions may arise as a consequence of recombination proportions, indicating that these mutations originated before events. In each of these patients, we could detect partially the primary cell layers diverged. deleted mtDNA in all examined tissues (albeit in very different proportions), suggesting that such mutations in symptomatic Large deletions in mitochondrial DNA (mtDNA) have been individuals arise before the primary cell layers diverge. found in the skeletal muscle of certain patients with mito- chondrial encephalomyopathies (1-9), a clinically and bio- MATERIALS AND METHODS chemically heterogeneous group of disorders, the common Patients Studied. Biopsy specimens ofskeletal muscle were features of which are dysfunction of oxidative phosphoryla- obtained from patients of the neurology service of the Johns tion and alterations of mitochondrial morphology (10). Al- Hopkins Medical Institutions (patients 3 and 4) or the Mas- though variable in location and size in different patients, sachusetts General Hospital (patient 2) with a phenotype of approximately a third of these deletions are identical by chronic progressive external ophthalmoplegia. Routine diag- Southern analysis (7). Recently, the nucleotide sequence of nostic studies included standard histological and histochem- the junctions surrounding this common deletion has been ical staining of frozen sections and polarographic analysis of determined in several unrelated patients, demonstrating that oxygen consumption by isolated mitochondria (patients 3 and these mutations are identical and that the junctions occur 4) (12). Frozen autopsy specimens were obtained from a within a directly repeated sequence of 13 nucleotides (nts), 13-year-old girl with chronic external ophthalmoplegia and suggesting slipped mispairing or recombination (7) as an cardiac dysfunction, studied clinically and biochemically at underlying mechanism. Independently, we have determined the Wayne State University School of Medicine (13). All the nucleotide sequence of the junctional region in another studies were approved by the Joint Committee on Clinical mitochondrial myopathy patient and found that the break- Investigation of the Johns Hopkins Medical Institutions and point did not occur in a repeated sequence (9). those of the referring institutions. In all cases thus far examined [except one (6)], Southern Preparation of DNA. DNA was extracted from mitochon- analysis has shown heteroplasmy (the presence ofnormal and drial and nuclear fractions of skeletal muscle tissue that were partially deleted mtDNAs) in skeletal muscle. The proportion prepared by differential centrifugation in preparation for of the partially deleted species was different in individual oxygen electrode polarography (patients 3 and 4) (12) and patients. In contrast, Southern analysis has failed to demon- from unfractionated homogenates of frozen muscle (patients strate the presence of mtDNA with a deletion in blood cells, 1 and 2), peripheral blood (patients 3 and 4), and urinary suggesting that the deletions may have arisen as somatic sediment (patient 4). Total DNA was extracted by standard mutations, after the muscle lineage diverged. However, we proteinase K procedures (14). Samples containing 5 pg of were able to detect a minor proportion of partially deleted DNA were digested with restriction endonucleases (Be- mtDNA in blood and urinary epithelial cells in two patients thesda Research Laboratories and New England Biolabs) with mitochondrial encephalomyopathy by using a polymer- according to the manufacturers' instructions, subjected to ase chain reaction (8, 9). In the one patient from whom we had electrophoresis on 1.1% (wt/vol) agarose gels, and trans- obtained multiple tissue specimens, large proportions of the ferred to nitrocellulose. They were then hybridized with a partially deleted mtDNA were present in brain, heart, liver, cloned probe prepared from HeLa mtDNA [complementary kidney, and muscle (11). These observations suggest that the to nt 16,453-3245 of the Cambridge mtDNA sequence (15)] pathogenic mutations in these patients must have arisen early Abbreviation: nt, nucleotide. tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge IThe sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" GenBank data base (accession nos. M27283, patients 1 and 2; in accordance with 18 U.S.C. §1734 solely to indicate this fact. M27284, patient 3; and M27285, patient 4). 8059 Downloaded by guest on September 25, 2021 8060 Medical Sciences: Johns et al. Proc. Natl. Acad. Sci. USA 86 (1989) that had been radioactively labeled by the random-primer method using 32P-labeled deoxynucleotides (16) and visual- ized by autoradiography. Proportions of deleted and unde- leted mtDNA species were estimated by laser densitometry (LKB Bromma) of the exposed x-ray film. Polymerase Chain Reaction. The polymerase chain reac- _~~~~~~~~~~/Vf I00:tsus tions were performed on 100 ng of template DNA, using primer pairs (Table 1) that closely bracketed the mtDNA z _: 4142 12,427 _ deletions, but whose recognition sites on normal mtDNA were spaced too widely to permit amplification of the unde- leted species (9). Oligonucleotide primers were synthesized by Operon Technologies (San Pablo, CA). DNA Sequencing. The products of the polymerase chain reactions were collected in an ultrafiltration microconcen- trator (Centricon-30, Amicon) and sequenced directly with 32P-end-labeled oligonucleotide primers, internal to those used for amplification (Table 1), by the dideoxy chain- 4tCOI AB termination method (17).

RESULTS Clinical Studies. All four patients reported in this study FIG. 1. Partial deletions of human mtDNA in four patients. The deletions observed in our patients with mitochondrial encephalomy- were women a with phenotype ofptosis, chronic progressive opathy are indicated by the arcs. They are 4.98 kb (patients 1 and 2), external ophthalmoplegia, and elevations oflactic acid in the 4.51 kb (patient 3), and 7.67 kb (patient 4) in length. The right blood. Patients 1 and 2 demonstrated additional signs of breakpoints are clustered within 550 nts in the NADH 5 gene. The pigmentary retinopathy, cerebellar ataxia, cardiac dysfunc- genes are abbreviated as follows: 12S and 16S, rRNAs; NADH 1, 2, tion, and sensorineural deafness: clinical evidence of multi- 3, 4L, 4, 5, and 6, subunits of NADH-coenzyme Q reductase system disease of the "Kearns-Sayre" type. The family (respiratory complex I); CO I, II, and III, subunits of cytochrome was in all three oxidase (respiratory complex IV); A8 and A6, subunits of ATP history negative instances, including college- synthetase (respiratory complex V); and Cyt b, cytochrome b aged offspring of patient 3. (respiratory complex III). The origins ofreplication for the heavy and Histologic analysis of frozen muscle sections demon- light chain are indicated by OH and OL, respectively. The 22 transfer strated an excessive accumulation of mitochondria in some RNAs are represented by the small unfilled spaces. The numerals type I muscle fibers, seen as typical "ragged-red fibers" on refer to the nucleotide position according to the Cambridge sequence the Gomori trichrome stain and increased subsarcolemmal (15). deposits of reaction products on standard oxidative stains (18). Polarographic analysis of isolated skeletal muscle mi- hybridization with cloned probes specific to other regions of tochondria (patients 1, 3, and 4) demonstrated decreased the mitochondrial genome (9). Each deletion removed genes oxygen consumption with succinate, NADH-linked sub- encoding several tRNAs and components of respiratory strates, as well as with N,N,N',N'-tetramethyl-p-phenylene- complexes I and IV; the deletions of patients 1, 2, and 4 also diamine and ascorbic acid-a pattern consistent with dys- removed components of respiratory complex V (Fig. 1). function of respiratory complex IV. Polymerase Chain Reaction. We had demonstrated previ- Southern Analysis. In all four patients, Southern analysis of ously that the use of appropriate primer pairs in a polymerase total muscle DNA, after digestion with BamHI [which chain reaction leads to preferential amplification of partially cleaves only at nt 14,258 in the Cambridge human mtDNA deleted mtDNA even when the normal mtDNA is present in sequence (15)] and hybridization with cloned DNA comple- 1000-fold excess (9). Amplification by the polymerase chain mentary to a region of human mtDNA that spans the origin reaction with total DNA from skeletal muscle of patients 1 of heavy chain replication, demonstrated two populations of and 2 using DRJ-3 and -16 (Table 1) produced a 1.4-kb mtDNA. In addition to a normal 16.6-kilobase (kb) species, fragment; of patient 3 using primers DRJ-12 and -8, a 0.8-kb patients 1 and 2 demonstrated an 11.6-kb species; patient 3, fragment; and of patient 4, primers DRJ-12 and -10, a 1.3-kb a 12.1-kb species; and patient 4, an 8.9-kb species. The fragment. Identical reaction products were obtained by using partially deleted species comprised 83%, 86%, 55%, and 25% total DNA in blood (patients 3 and 4) or urinary epithelial of all mtDNA molecules in the muscle specimen of each cells (patient 4), indicating the presence of a minor population patient, respectively. of partially deleted mtDNAs. In contrast to skeletal muscle, Southern analysis of total Sequence Analysis. The amplified DNA obtained was se- DNA extracted from blood (patients 3 and 4) demonstrated quenced directly, using 32P-end-labeled oligonucleotide prim- only one species of mtDNA of normal electrophoretic mo- ers internal to those used for amplification (Table 1). Each bility after digestion with BamHI. deletion breakpoint occurs in a directly repeated sequence The approximate extent of each deletion (Fig. 1) was that is present in two widely separated regions of the normal estimated by digestion with multiple restriction enzymes and mitochondrial genome (Fig. 2). The direct repeats associated Table 1. Oligonucleotide primers used for amplification and sequencing of deletion junctions Amplification primer Patients L strand H strand Sequencing primer* 1 and 2 DRJ-3 (7407-7425) DRJ-16 (13,789-13,809) DRJ-18 (8417-8434) 3 DRJ-8 (9151-9169) DRJ-12 (14,452-14,470) DRJ-19 (9282-9299) 4 DRJ-10 (5533-5551) DRJ-12 (14,452-14,470) DRJ-17 (6291-6308) All nucleotide designations (in parentheses) refer to the Cambridge sequence (15). *Sequencing primers all complementary to the L strand. Downloaded by guest on September 25, 2021 Medical Sciences: Johns et al. Proc. Natl. Acad. Sci. USA 86 (1989) 8061

8470: including those in lacI-lacZ fusion strands of Escherichia coli (19), the human /3-globin gene (20), and some mtDNA 1&2 AC:'CTCCCTCACCAi deletions in yeast (21). In addition, directly repeated Alu > 96 found at deletion junctions of patho- &2Y L ~AC:,CTCCCTCACCA~~~~* * - 9361 sequences have been 13447: V genic mutations in several disease states, including familial 3i) 0. A -CTAACCAACACACT hypercholesterolemia (22) and Tay-Sachs disease (23). The 3**RCTA CC TMCCAACAMCT presence of direct repeats may contribute to the formation of 6325 by "slipped mispairing" during DNA replication, V ~~~~~~~~~~~13868 deletions 1J,,-CCTCCGT AGACCTAACCA 38 as has been proposed to account for the most frequently observed mtDNA deletion found in association with certain COCTCC -T:AGACCTAACCT such A mitochondrial encephalomyopathies (7). Alternatively, 13989 direct repeats could contribute to independent recombination events mediated by enzymes that recognize specific short consensus CTA CCTMCCA' sequences (19, 20). In other systems, the frequency with which deletions occur appears to correlate with the length of them, and the FIG. 2. mtDNA sequences emcompassing four independent de- repeated sequences, the distance between letionjunctions. The directly repeated sequences are numbered as in nature of neighboring sequences. Furthermore, the precise Fig. 1 and are aligned in pairs above the consensus sequence. The nucleotide sequence ofthe direct repeat itselfhas been shown direct repeats share 13 consecutive, 16/18 (10 consecutive), and to have a strong effect on the rate of formation of deletions 16/18 (11 consecutive) nts, respectively. The nucleotides in the (19). consensus are found in 4, 4, 4, 5, 6, 6, 4, 6, 6, 6, and 5 of the In our patients, the regions in which the deletion break- sequences, respectively. The mismatched nucleotides are marked points occur are similar over a stretch of 11 nts, the consensus with an asterisk. The arrows indicate the actual sequence of each sequence of which is CTACCTAACCA (Fig. 2). This se- patient's mtDNA. quence is similar to the core repeats in the hypervariable are identical at 13 minisatellite regions described by Jeffreys et al. (24), which with the deletions found in patients 1 and 2 be recombination signals in human of 13 base pairs (bp); those in patient 3, 16 or 18 bp; those in have been proposed to strands nuclear DNA (Fig. 3). Additionally, it is similar to portions of patient 4, 16 of 18 bp (Fig. 2). In each case, the two a putative recombination signal in E. coli maintain their integrity throughout the junctional region the chi sequence, cannot be (25) (Fig. 3). without any nucleotide insertions. The breakpoint We suggest that this consensus sequence may mediate determined precisely because junction occurs within a nucle- recombination in the human mitochondrial genome. How- otide sequence that is identical on both strands. When the ever, the sequence data do not distinguish between intra- and direct repeats are compared, a consensus sequence of 11 bp intermolecular recombination. An intramolecular looping can be identified (Fig. 2). mechanism has been suggested to explain the duplication and Search for mtDNA Insertions. We considered that these inversion of portions of the mitochondrial genome in lizards deletions might have arisen from unequal intermolecular (26). Alternatively, the deletions observed in our patients recombination events and predicted that the reciprocal prod- may have arisen from intermolecular recombination. The ucts would be mtDNA species with inverted insertions ofthe presence of 2-10 mtDNA molecules per human mitochon- same size as the corresponding deletion. No mtDNA species drion (27) eliminates any obvious physical barrier to inter- of a size appropriate to such inserted species was found in molecular recombination. Such intermolecular recombina- Southern blots of skeletal muscle in any of the four patients. tion between two misaligned molecules would produce a In patient 1, the search was extended further through the use deleted molecule and a molecule with a corresponding in- of the polymerase chain reaction with primers designed to verted insertion. Our initial attempts to identify the corre- overlap the junction of the predicted insertion species: DRJ- sponding insertions were unsuccessful. However, the puta- 20, complementary to nt 8539-8520 of the H strand of the tive larger, inserted species would be expected to suffer a Cambridge sequence (15), and DRJ-21, complementary to nt replicative disadvantage, and thus its absence is not unex- 13,360-13,379 of the L strand. No reaction product was pected. detected when 100 ng oftotal DNA from skeletal muscle was There is ample genetic (28) and physical (29) evidence for used as a template. intermolecular recombination of mtDNA in yeast, where it may contribute to the formation of certain rho- deletions DISCUSSION (30). Intermolecular recombination is generally assumed not Our results demonstrate that directly repeated sequences lambda 33.11 TGG GC AGGAAG present in widely separated regions ofmtDNA are commonly ** * in deletions associated with genetic disease. Such junction consensus involved 1 ** 2* repeated sequences occur frequently in the mitochondrial AGGTGG genome: a computerized search (MICROGENIE, Beckman) of lambda 33.15 TGGGC the published human mtDNA sequence (15) reveals 198 duplicated sequences of at least 10 consecutive matching bases, which lie within a segment bracketed by the origins of chi GCTGGTGG2 replication for the light and heavy strands. (Repeated se- , 1 quences that straddle either origin would be predicted to give 2 rise to partially deleted species that would be incapable of from consideration.) FIG. 3. Comparison of the sequences associated with partial replication. Hence, they were excluded deletions of mtDNA to the heavy (opposite) strand of the consensus The inappropriate junctions in patients 1 and 2 occur within sequence associated with deletionjunctions in mtDNA. The mtDNA one of two such duplications that contain 13 consecutive deletion sequence shares 8 of 11 nts with the core region from two identical nucleotides. This deletion junction has been found minisatellite clones of Jeffreys et al. (24). A 33.11 and 33.15. The independently in several other patients (7). mismatched nucleotides are marked with an asterisk. In addition, the Deletions in other systems appear to be promoted by the first four (1) and last five (2) bases of the consensus are similar to the presence of flanking short, directly repeated sequences, chi sequence (25), a known recombination signal in E. coli. Downloaded by guest on September 25, 2021 8062 Medical Sciences: Johns et al. Proc. Natl. Acad. Sci. USA 86 (1989)

to occur in animal mtDNA (31), but two lines of evidence 6. Saiffuddin-Noer, A., Marzuki, S., Trounce, I. & Bryne, E. suggest that it may indeed occur. (i) In human population (1988) Lancet Hi, 1253-1254. studies, two polymorphic restriction sites have been found in 7. Schon, E. A., Rizzuto, R., Moraes, C. T., Nakase, H., Zevi- are to ani, M. & DiMauro, S. (1989) Science 244, 346-349. all combinations in individuals who known differ only 8. Johns, D. R., Drachman, D. B. & Hurko, 0. (1989) Lancet i, at those two sites (32). This observation could be explained 393-394. by the highly unlikely occurrence of two independent muta- 9. Johns, D. R. & Hurko, O., Genomics, in press. tions in a single site (with a probability of <10-10) or, 10. Petty, R. K. H., Harding, A. E. & Morgan-Hughes, J. A. alternatively, it could be explained by recombination in (1986) Brain 109, 915-938. heteroplasmic individuals. Heteroplasmy in humans occurs 11. Hurko, O., Johns, D. R., Rutledge, S. L., Stine, 0. C., Drach- with mtDNA deletions [as shown by these data and those of man, D. B., Brown, R. H., Martens, M. E., Peterson, P. L. & others (1-5, 7)] and with restriction site polymorphisms in Lee, C. P., Pediatr. Res., in press. patients with sickle cell anemia (O.C.S. and K. D. Smith, 12. Moreadith, R. W., Batshaw, M. L., Ohnishi, T., Kerr, D., hybrid cell Knox, B., Jackson, D., Hruban, R., Olson, J., Reynafarie, B. unpublished data). (ii) In 19 of 34 rodent-human & Lehninger, A. L. (1984) J. Clin. Invest. 74, 685-697. lines, the mtDNA banded at a density intermediate to that of 13. Martens, M. E., Peterson, P. L., Lee, C. P., Nigro, M. A., rodent and human mtDNAs (33). In these gradients, radio- Hart, Z., Glasberg, M., Hatfield, J. S. & Chang, C. H. (1988) active complementary RNAs from each species gave identi- Ann. Neurol. 24, 630-637. cal hybridization profiles that differed from those obtained 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular when pure mtDNAs from each species were mixed (33). The Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold authors concluded that the mtDNAs in the interspecific Spring Harbor, NY). hybrids must have recombined (33). 15. Anderson, S., Bankier, A. T., Barrel, B. G., De Brujn, Our data indicate that the deletions must have occurred M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, of the myogenic lineage. Partially D. P., Roe, B. A., Sanger, F., Scrier, P. H., Smith, A. J. H., before the divergence Staden, R. & Young, I. G. (1981) Nature (London) 290, 457- deleted mtDNA was found in all tissues of the patients 465. regardless ofwhether they had clinical evidence of multisys- 16. Feinberg, A. P. & Vogelstein, B. (1983) J. Mol. Biol. 26, tem disease or only dysfunction of the extraocular muscles. 365-369. The proportions ofpartially deleted and normal mtDNA were 17. Higuchi, R., Von Beroldingen, C. H., Sensabaugh, G. F. & very different in various tissues from a given patient. The Erlich, H. A. (1988) Nature (London) 332, 543-546. partially deleted mtDNA was always found in highest pro- 18. Dubowitz, V. & Brooke, M. H. (1973) Muscle Biopsy: A portion in skeletal muscle and was barely detectable in blood Modern Approach (Saunders, London). and urinary epithelial cells. This may reflect our selection of 19. Albertini, A. M., Hofer, H., Calos, M. P. & Miller, J. H. (1982) disease. Alternatively, there Cell 29, 319-328. patients with presumed muscle 20. Efstratiadis, A., Posakony, J. W., Maniatis, T., Lawn, R. M., may be stronger selection against cells with higher propor- O'Connell, C. O., Spritz, R. A., DeRiel, J. K., Forget, B. G., tions of partially deleted mtDNA in mitotically active cells Weissman, S. M., Slightom, J. L., Blechl, A. E., Smithies, O., than in mitotically inactive ones. Baralle, F. E., Shoulders, C. C. & Proudfoot, N. J. (1980) Cell We have sequenced previously a mtDNA deletionjunction 21, 653-668. from another patient with chronic progressive external oph- 21. Ahne, A., Muller-Derlich, J., Merlos-Lange, A. M., Kanbay, thalmoplegia and found that the deletion breakpoint did not F., Wolf, K. & Lang, B. F. (1988) J. Mol. Biol. 202, 725-734. occur in a repeated sequence (9). This may be the result of an 22. Lehrman, M. A., Russell, D. W., Goldstein, J. L. & Brown, exchange mediated by a repeated sequence accompanied by M. S. (1987) J. Biol. Chem. 262, 3354-3361. Biol. Chem. or may have arisen a 23. Myerowitz, R. & Hogikyan, N. D. (1987) J. 262, gene conversion (34), it through 15369-15399. different mechanism. In conclusion, direct repeats are fre- 24. Jeffreys, A. J., Wilson, V. & Thein, S. L. (1985) Nature quently associated with pathogenic deletions of human (London) 314, 67-73. mtDNA, and recombination may occur in human mtDNA. 25. Smith, G. R., Kunes, S. M., Schultz, D. W., Taylor, A. & K. L. Cell 429-436. We are grateful for patient referrals from Drs. Patti L. Peterson, Triman, (1981) 24, Margaret E. Martens, C. P. Lee, Robert H. Brown, Daniel B. 26. Moritz, C. & Brown, W. M. (1987) Proc. Natl. Acad. Sci. USA and David S. Zee. Muscle biopsies and histologic anal- 84, 7183-7187. Drachman, 27. Hauswirth, W. W. & Laipis, P. J. (1985) in Achievements and yses were performed by Drs. Daniel B. Drachman and Paul Tuttle; eds. were performed by Dr. Perspectives ofMitochondrial Research: Biogenesis, Qua- isolation of mitochondria and polarography E. C. & Kroon, Tatiana Chechik. We acknowledge many valuable and pleasurable gliarello, E., Slater, C., Palmieri, F., Saccone, discussions with Drs. Haig Kazazian, Stylianos Antonarakis, Victor A. M. (Elsevier, New York), Vol. 2, pp. 49-59. and Richard T. Johnson. These studies 28. Coen, D., Deutsch, J., Netter, P., Petrochilo, E. & Slonimski, McKusick, Pamela Talalay, P. P. Soc. Biol. 449-496. were supported in part by grants from the Muscular Dystrophy (1970) Symp. Exp. 24, Institutes of Health (AR38231). 29. Michelis, G., Petrochilo, E. & Slonimski, P. P. (1973) Mol. Association and the National Gen. Genet. 123, 51-65. 1. Holt, I. J., Harding, A. E. & Morgan-Hughes, J. A. (1988) 30. Lewin, B. (1981) Gene Expression: Eukaryotic Chromosomes Nature (London) 331, 717-719. (Wiley-Interscience, New York), Vol. 2, pp. 603-614. 2. Lestienne, P. & Ponsot, G. (1988) Lancet i, 885. 31. Moritz, C., Dowling, T. E. & Brown, W. M. (1987) Annu. Rev. 3. Holt, I. J., Cooper, J. M., Morgan-Hughes, J. A. & Harding, Ecol. Syst. 18, 269-292. J. A. (1988) Lancet i, 1462. 32. Scozzari, R., Torroni, A., Semino, O., Sirugo, G., Brega, A. & 4. Ozawa, T., Yoneda, M., Tanaka, M., Ohno, K., Sato, W., Santachiara-Benecerti, A. S. (1988) Am. J. Hum. Genet. 43, Suzuki, H., Nishikimi, M., Yamamoto, M. & Horai, S. (1988) 534-544. Biochem. Biophys. Res. Commun. 154, 1240-1247. 33. Horak, I., Coon, H. G. & Dawid, I. B. (1974) Proc. Natl. Acad. 5. Zeviani, M., Moraes, C. T., DiMauro, S., Nakase, H., Bonilla, Sci. USA 71, 1828-1832. E., Schon, E. A. & Rowland, L. P. (1988) Neurology 38, 34. Klein, H. L. & Petes, T. D. (1981) Nature (London) 289, 1339-1346. 144-148. Downloaded by guest on September 25, 2021