Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4496-4500, November 1975 Cell Biology Specific cleavage analysis of mammalian mitochondrial DNA (restriction endonuclease/gel electrophoresis/interspecies comparisons/intraspecies differences) S. STEVEN POTTER, JOHN E. NEWBOLD, CLYDE A. HUTCHISON III, AND MARSHALL H. EDGELL Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, N.C. 27514 Communicated by Jerome Vinograd, September 5, 1975 ABSTRACT Mitochondrial DNA from several mamma- fresh from local slaughterhouses; dog, rabbit, rat, and ham- lian species has been digested with a site-specific restriction ster (Syrian golden) specimens were obtained from conven- endonuclease (HaeIII) from Haemophilus aegyptius. A quan- titative analysis of the resulting specific fragments indicates tional suppliers, and sacrificed for fresh tissues in the labora- that the mtDNA of any individual mammal is predominantly tory; mice were obtained from Dr. G. Haughton; African a single molecular clone. green monkey livers were purchased from Flow Laborato- Gel analysis of specific cleavage products has proven quite ries; chimpanzee livers were provided by Dr. R. Metzgar of sensitive in detecting differences in mtDNA: mtDNAs from Duke University; buffalo (Bos bison) liver was obtained the more distantly related mammals studied (e.g., donkey from the Buffalo Ranch, Concord, N.C.; human hearts and and dog) are found to have few bands in common, and very closely related mammals (e.g., donkey and horse) share only livers were obtained as autopsy samples from the N.C. Me- about 50% of their bands. This procedure has detected sever- morial Hospital, Chapel Hill; leukocytes (removed as part of al intraspecies mtDNA differences. Six distinct human pat- the treatment) from a patient with chronic myelogenous leu- terns have been found, with one pattern usually differing kemia (CML) were provided by Dr. A. Huang of Duke Uni- from another in two or three bands. versity. mtDNAs from different organs of single individuals have Preparation of mtDNA. Mitochondria were prepared at also been analyzed, and no differences have been found. 40 either directly from fresh tissues or cells, or from fresh Our understanding of the organization and regulation of material stored frozen at -20°. Large livers were first prokaryotic genes is ultimately based on the fine structure minced with a razor and then an equal volume of S solution analysis of a relatively few specific genetic regions in Esche- (0.25 M sucrose, 1 mM EDTA, pH 7.4) was added. Aliquots richia coli. Hopefully, fine structure mapping of eukaryotic of about 15 ml of this material were homogenized quickly genes will allow similar insight into the structure and func- (about 3 sec) with a Teckmar Tissumizer, model SDT (48 V). tion of eukaryotic genetic material. This homogenate was then diluted to 10 tissue volumes with Specific cleavage of small DNA virus genomes by restric- S solution. Small livers (mouse, hamster, and rabbit) were tion enzymes (1, 2) allows the construction of physical maps minced, washed in S solution, diluted to 10 tissue volumes of DNA molecules which have many of the useful features with S solution and then homogenized with a Potter-El- of recombination maps. The specific fragments produced by vehjem homogenizer fitted with a motor driven teflon pes- enzymatic cleavage are readily isolated, and have been used tle. Kidney tissue was treated as were small livers. Heart to map a number of sites of genetic significance. These in- tissues were disrupted according to procedure 3 of Smith clude origins of DNA replication (3), RNA polymerase bind- (10). Tissue culture cells were homogenized as described by ing sites (4), type 1 restriction recognition sites (5), regions Kasamatsu et al. (11). coding particular RNA species (early and late mRNA) (6, 7) Nuclei and unbroken cells were removed from the ho- and also sites of genetic mutations (8). These techniques are mogenate by centrifuging twice (500 X g, 10 min). Mito- potentially extendable to the mapping of ribosomal binding chondria were then pelleted (10,000 X g, 10 min) and sites and regions that code for specific proteins (in conjunc- washed once with S solution. tion with in vitro protein synthesizing systems). The washed mitochondrial pellet was resuspended in This strategy for the production of a genetic map is not 0.075 M NaCl, 0.05 M EDTA, 0.025 M Tris-HCl at pH 7.4, dependent on the isolation and analysis of mutants and is and made 2% for sodium dodecyl sulfate. This material was therefore directly applicable to any simple DNA molecule. then extracted twice with phenol, dialyzed against SSC (0.15 The mammalian mitochondrial genome provides a simple M NaCl-0.015 M sodium citrate, pH 7) and the supercoiled system which is susceptible to specific cleavage analysis. All DNA purified by centrifugation in a CsCl ethidium bromide mammalian mitochondrial DNAs are double-stranded and gradient. Ethidium bromide was removed from the mtDNA circular with molecular masses of close to 107 daltons (see by three extractions with isopropanol. The mtDNA was di- ref. 9 for a review). In this paper we show the results of spe- alyzed against SSC and collected by pelleting at 33,000 rpm cific cleavage of mtDNA from a variety of mammalian for 10 hr in a SW50.1 rotor at 40; the pellet was dissolved in sources. Analysis of the characteristic fragment patterns so a small volume of TBS (0.15 M NaCl, 0.05 M Tris-HCl at pH produced provides an extremely sensitive method for distin- 7.4) and stored at -20°. guishing closely related mitochondrial genomes. Digestion. The HaeIII (endonuclease Z) restriction endo- nuclease (12) from H. aegyptius was prepared as described MATERIALS AND METHODS by Smith (13). Enzyme was used as eluted from the phos- phocellulose column or after subsequent ammonium sulfate Source Materials. Organs from domestic farm animals, precipitation; no difference in these two enzyme fractions namely, cow, pig, sheep, horse, and donkey, were obtained has been detected. A single batch of enzyme sufficed for all Abbreviations: SSC, 0.15 M sodium chloride-0.015 M sodium ci- digestions described in this study. The digestion mixture trate, pH 7; SV40, simian virus 40. used has been previously described (14). The HaeIII enzyme 4496 Downloaded by guest on October 2, 2021 Cell Biology: Potter et al. Proc. Nat. Acad. Sci. USA 72 (1975) 4497 (a ) (1 Al), as eluted from the phosphocellulose column, com- 2 34 56 7 8910 11 1213 14 151617181920 pletely digested 10 Mg of simian virus 40 (SV40) DNA in 10 W6 hr at 370. mtDNA (10-20 Mg) was digested with 5 Ml of en- 1*O9*@* 9 &'' zyme for a minimum of 10 hr and then 5 Al more enzyme was added and the digestion continued for 2 hr. Electrophoresis. Fractionation of DNA fragments by electrophoresis (4 V/cm) in polyacrylamide gels has been described previously (2). Slot gels (30 cm long) were used when the gels were to be dried down on filter paper and were used to expose x-ray film (2). For ethidium bromide a- staining (1 Mg/ml), cylindrical 3.5% acrylamide gels (7 mm u) by 30 or 60 cm, no agarose) were loaded with about 10-20 Mg of mtDNA. Gels were photographed on Polaroid type 55 film. The photographs (Figs. 2-5) displayed in this paper were printed as negatives to enhance the contrast. RESULTS FR ACT 0 N The specific limit fragments of small viral DNAs produced (b) 1 234 56 7 8910 11 1213 141516171820 22122 24 25 2627 28 29 30 by digestion with a site-specific restriction endonuclease are i + + +4 + + 19 + 23 resolved into a characteristic pattern of bands upon electro- 1000 phoresis in acrylamide gels (1, 2). The bands of DNA frag- 900 ments can be detected either by staining techniques or by radioisotope label. The characteristic band patterns for 70C mammalian mtDNA obtained in this way were found to be very reproducible. For example, three preparations of ham- CL 50c ster liver mtDNA were made, each of which involved pool- 40C ing the livers from 20 to 30 inbred animals. Separate diges- 30C tions of these mtDNA preparations were performed and the SOC resulting band patterns were identical. Similar results were IOC obtained in the analysis of large equine livers. Several inde- 1015 20 25 30 35 40 45 50 55 60 6 707808 90g lo 5'o 5 2 5 335 pendent preparations of mtDNA have been made from 300 FR ACT IO N g portions of individual equine livers. The band patterns FIG. 1. Autoradiographs and radioactivity distributions of from any single organ are found to be invariant. Some prep- 32P-labeled mtDNA from HeLa S3 digested with HaeIII and elec- arations of mtDNA were contaminated with nuclear DNA. trophoresed on gels containing (a) 2.5% and (b) 3.5% polyacrylam- The contaminating nuclear DNA was seen in these restric- ide. Each band resolved in either (a) or (b) is assigned a number. Cells at 50% confluency were labeled for 48 hr in minimum essen- tion analyses as a background smear and never as additional tial medium containing 1o-4 M phosphate, 10% fetal calf serum bands of DNA fragments. (dialyzed against Hank's balanced salt solution without phos- To investigate the homogeneity of a mammalian mtDNA phate), and 5 gCi/ml Of 32p. Arrows in (b) point to the relative po- preparation, we did a detailed quantitative analysis of the sitions of SV40 size markers (see Results). HaeIII digested Vogt- HaeIII digestion products of HeLa S3 mtDNA. Fig. 1 shows Dulbecco small plaque strain SV40 DNA was used (13). autoradiograms of uniformly 32P-labeled HeLa mtDNA that had been digested and analyzed on 2.5% and 3.5% polyac- rylamide gels.