Circular DNA isolated from human kidney mitochondria was studied by electron microscopy.
I. Mean contour length of monomers of the mitochondrial DNA was 4.96 ± SE 0.28 /&#956;
2. The complex molecules (oligomers) of mitochondrial DNA were observed in frequency of 6.2 per cent. Among them circular dimers accounted for two per cent of all circular DNA molecules.
3. Circular DNA fibers with an intermediate perimeter between the&#12288;monomer and dimer, and with a contour length shorter than 3 &#956; were occasionally observed.
4. Some discussions were made on the emergence of the circular dimer.</p

Hirata, Seiichi

Oda, Takuzo

Seki, Shuji

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 25, Issue 4 1971 Article 3AUGUST 1971Electron microscopic observation ofmitochondrial DNA isolated from a humankidney: circular dimers in histologicallynormal organ mitochondriaShuji Seki∗ Takuzo Oda†Seiichi Hirata‡∗Okayama University,†Okayama University,‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Electron microscopic observation ofmitochondrial DNA isolated from a humankidney: circular dimers in histologicallynormal organ mitochondria∗Shuji Seki, Takuzo Oda, and Seiichi HirataAbstractCircular DNA isolated from human kidney mitochondria was studied by electron microscopy.I. Mean contour length of monomers of the mitochondrial DNA was 4.96 ± SE 0.28 /µ 2. Thecomplex molecules (oligomers) of mitochondrial DNA were observed in frequency of 6.2 per cent.Among them circular dimers accounted for two per cent of all circular DNA molecules. 3. CircularDNA fibers with an intermediate perimeter between the&#x3000;monomer and dimer, and with acontour length shorter than 3 µ were occasionally observed. 4. Some discuions were made on theemergence of the circular dimer.∗PMID: 4263558 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Merl. Okayama 25, 245-253 (971)ELECTRON MICROSCOPIC OBSERVATION OF MITOCHON-DRIAL DNA ISOLATED FROM A HUMAN KIDNEY:CIRCULAR DIMERS IN HISTOLOGICALLYNORMAL ORGAN MITOCHONDRIAShuji SEKI, Takuzo aDA and Seiichi HIRATADepartment of Biochemistry, Cancer Institute, Okayama University Medical School,Okayama, Japan (Director: Prof. T. Oda)Received for publication, July 9, 1971The DNA in mitochondria of many higher organisms IS m the formof double-stranded circular molecules with a contour length of 4.5 to5.5l-~ corresponding to a molecular weight of9 to 10 million daltons 0-5).Besides the simple circular molecules, there were observed complex circularmolecules of twice or several times the size of 5 p. (dimers, trimers, etc,)(6-19). The naturally occurring dimers are tentatively classified in twoforms, unicircular double length molecules (circular dimers) and catenanes(catenated dimers) made up of interlocked 5-p. monomers (6-8). Amongthem, the circular dimers have not been observed in normal tissues (9, 10,20). In human, especially, the circular dimers have been observed only inleukemic leukocytes (7,20,21) and solid tumors (21).In this report, electron microscopic data on the mitochondrial DNAisolated from a histologically normal human kidney are presented. Thecircular dimers, which have been reported only in leukemic leukocytes(7, 20, 21) and solid tumors (21), are observed in about 2 per cent of thenon-tumorous kidney mitochondrial DNA.MATi-RIALS AND METHODSPreparation of mitochondria: Mitochondria were isolated 2 hr post-mortemfrom the kidney of a 54-year old patient, died of hepatoma associated with livercirrhosis, which was provided by a pathologist of our school. Mitochondria wereprepared essentially according to the method of HOGEBOOM (22) as reported ina previous paper (23). Namely, the renal cortex, obtained after removing thecapsule, pelvis, calices and medulla from the kidney, was minced and washedwith 0.25M sucrose solution (containing 10 mM Tris-Hel and 0.1 mM EDTA,pH 7.6). The minced cortex was homogenized in the 0.25M sucrose solutionwith a teflon homogenizer, and the pH of the homogenate was adjusted to 7.4by adding 5 N KOH. The homogenate was centrifuged at 60xg for 10 min, andthe supernatant was superimposed on a equal volume ofO.34M sucrose solution2451Seki et al.: Electron microscopic observation of mitochondrial DNA isolatedProduced by The Berkeley Electronic Press, 1971246 S. SEKI, T. QDA and S. HIRATAand centrifuged at 700xg for 10 min. Upper, three fifths of the supernatant wascollected and centrifuged at 7,000 X g for 10 min. The supernatant was decanted.The residue consisted of two layers, greenish-brown top layer and light brown,trace bottom layer. The greenish-brown top layer, constituting bulk of the pellet,was carefully separated from the bottom layer and suspended in a 0 .25M sucrosesolution (free of Tris-HGI and EDTA). The suspension was centrifuged at7, OOOxg for 10 min, and the greenish-brown layer was carefully separated. Theabove process of suspension and centrifugation was repeated a few times untilthe residue became a homogeneous greenish-brown pellet.The final preparation was suspended in saline-EDTA (0.15M NaGl, O.lMEDTA, pH 8.0).Isolation cif mitochondrial DNA: Mitochondrial DNA was isolated essentiallyaccording to Marmur's method (24). Outline of the procedures is presented asfollows. Mitochondria suspended in saline-EDTA was centrifuged at 10,000xgfor 10 min. The residue was suspended in saline-citrate (0.15M NaGl, 0.015Msodium citrate). Twenty-five per cent sodium lauryl sulfate was added to makea final concentration of 2 %. The mixture was incubated at 60° for 10 min andthen cooled to room temperature. Five-M perchlorate (neutralized) was addedto a final concentration of 1 M, and the whole mixture was shaken with an equalvolume chloroform-isoamyl alcohol (24: 1) for 30 min and centrifuged at 3,000rev. /min for 15 min. Upper aqueous phase containing nucleic acids, wascarefully pipetted off into a tube. Two volumes of ethanol were layered on theaqueous phase, gently mixed, and left standing overnight at 0.....4°. Nucleic acidscollected by centrifugation at 3,000 rev. /min for 20 min was dissolved in saline-citrate. The solution was shaken again with an equal volume of chloroform-isoamyl alcohol for 30 min, centrifuged, and the aqueous phase was collected.The above deproteinization process was repeated a few times. The nucleic acidsprecipitated with ethanol after deprotinization were dissolved in saline-citrateand treated with heat-treated RNase 1 A (50,ug/ml) at 37° for 30 min. Aftercooling to 0°, the mixture was deproteinized. The DNA so purified was precipi-tated from the deproteinized upper aqueous phase with two volumes of ethanol,and solubilized in a small volume of saline-citrate.Electron microscopic observation: Protein monolayer technique of KLEINSCHMIDT(25) modified by FREIFELDER and KLEINSCHMIDT (26) was mostly used for spreadingmitochondrial DNA. Namely, DNA dissolved at a concentration of about 2 ,ugper ml in a solution of 1 M ammonium acetate and 0.01 % cytochrome c andadded with formalin in final concentration of 0.5 % ran down an inclined cleanglass slide onto a hypophase of 0.3 M ammonium acetate containing 0.5 % for-malin. The protein-DNA film was picked up on carbon-coated collodion filmsmounted on copper grids. Rotary shadow-casting was conducted on the gridswith platinum-palladium (80: 20, about 20 mg in weight) at a shadow angle of6..... 10° at a mean distance of about 7 cm in a Hitachi evaporating unit (11,25).The grids were then examined in HU-ll G electron microscope (Hitachi Ltd.).Micrographs were taken at 75 KV on Fuji film (FG), at an optical magnificationof 10,000. Electron optical magnification was checked by simultaneous observa-2Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/3Electron Microscopy of Mitochondrial DNA 247tion of standard carbon replicas of diffraction grating with I.051-t spacing. Con-tour length of DNA molecules was measured on the four- or five-time enlargedprints with a map-measuring device (24).RESULTSPathological findings: The kidney used for the preparation of mito-chondrial DNA did not show any noteworthy pathological change. Nometastasis of liver carcinoma was detected.Electron microscopic observation: Electron micrographs of the mitochon.drial DNA.preparation showed circular DNA fibers in about 84 per centof the sum-total length of the entire DNA fibers and linear fibers in about16 per cent. The length of each linear fiber was mostly shorter than 5 l'..Even considering involuntary selection of circular DNA on photography,the above numerical values mean that contamination of nuclear DNA andsplitted mitochondrial DNA are of little significance. Complete opencircular DNA molecules were less than 22 per cent of the total circularDNA molecules. The histogram of the distribution of lengths of the circularDNA molecules are illustrated in Fig. 1. Circular dimers, trimers, andcircular DNA fibers with an intermediate perimeter and with a contour50,..---------,-------..,--------,.--..,40U>Gl3~ 30"0E~ 20E:>z10Of-U---""-----'"o 5 10 15Length (p.lFig. I Distribution of lengths of circular DNA molecules isolatedfrom human kidney mitochondria. White columns indicate circulardimers. Hatched columns include monomers, catenated dimers, trimersand intermediate length circul~r DNA molecules,3Seki et al.: Electron microscopic observation of mitochondrial DNA isolatedProduced by The Berkeley Electronic Press, 1971248 S. SEKI, T. aDA and S. HIRATAFig. 2 Electron micro~raphs of circular DNA molecules isolated from human kidney mito-chondria. Rotary shadowin~ with platinum-palladium (80 : 20). a. Monomers. b. A catenateddimer and monomers. (x 40,000.)4Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/3Electron Microscopy of Mitochondrial DNA 249length shorter than 3,11. were occasionally observed. Mean contour lengthsof the monomers, dimers and trimers were 4.96 ± SE 0.28 /'-, 9.85 ± SE0.62,1'- and 15.0/,-, respectively. Scoring the frequency of complex mito-Fig. 3 Electron micro.sraphs of circular DNA molecules isolated from human kidney mito-chondria. Rotary shadowins with platinum-palladium. a. A catenated dimer and trimer, con-sisting of an open circular climer interlocked with a monomer. b. A circul~r dimer. (X 40, 000.)5Seki et al.: Electron microscopic observation of mitochondrial DNA isolatedProduced by The Berkeley Electronic Press, 1971250 S. SEKI, T. aDA and S. HIRATAchondrial DNA was systematically performed at the microscope by directobservation on the fluorescent screen, according to the method of CLAY.TON et al. (9). The result is illustrated in Table 1. Circular dimers accoun.·ted for 2 per cent of circular DNA molecules isolated from human kidneyFig. 4 Electron micrograr-hs of circular DNA molecules isolated from human Lidney mito-chondria. a. A catenated dim~r. b. A trimer, consisting of three interlocked monom~TS. c. ADNA circle (contour kngth, 7. 2:~) having an intermediate length between monomer and dimer.d. A small size DNA circle (contour length, 2. Ill). ('./ 40, 000. )6Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/3Electron Microscopy of Mitochondrial DNA 251mitochondria. Electron micrographs of monomers, oligomers, and circularDNA fibers with an intermediate perimeter and with a contour lengthshorter than 3 fl. were illustrated in Fig. 2,..,,4.TABLE 1 DISTRIBUTION OF CIRCULAR DNA FORMS OF MITOCHONDRIA ISOLATEDFROM THE HUMAN KIDNEYDNA formsMonomersOligomersCatenated dimersCircular dimersTrimersHypertwisted, ambiguous formsDISCUSSIONSNumber585442814277Frequency (%)83.06.24.02.00.310.9Electron microscopic data on mitochondrial DNA isolated from humankidney were presented in this paper. Mean contour length of the mito-chondrial DNA was 4.96/1 in the monomer form, which is about the sameas that of mitochondrial DNA previously observed in the same organ ofthe other mammals (4, 9) or in the other organs, tissues or cells 0-19).Complex forms of the mitochondrial DNA (6-10) were also observed inthe preparation.As reported in preceding papers, catenanes are most commonly observ-ed in the complex forms and occur at a frequency of 2 to 9 per cent ofmitochondrial DNA molecules (9, 10). The circular dimers, on the otherhand, have been observed only in the mitochondrial DNA isolated fromcultured mouse fibroblasts (L cells) 00, 28, 29) and neoplastic cells such ashuman leukemic leukocytes (7, 21, 27), rat ascites hepatoma (AH 130strain) (4), C3H mouse ascites tumor induced by Schmidt-Ruppin strainof Rous sarcoma virus 05, 18), and human solid tumors (21), but notfrom normal tissues (9). CLAYTON et al. (20) referred the circular dimer toan "abnormal" form, 1:'ecause it occurred in leukemic leukocytes and didnot occur in the appropriate controls. Recently, they suggested a signifi.cant relationship between the presence of the abnormal mitochondrialDNA form and neoplasia in man (21),We revealed in the present study that the circular dimer occurred inabout 2 per cent of the total number of circular DNA molecules in the mitochondria isolated from the histologically normal kidney of a patient, whodied of hepatoma associated with cirrhosis, but extrahepatic metastasis7Seki et al.: Electron microscopic observation of mitochondrial DNA isolatedProduced by The Berkeley Electronic Press, 1971252 S. SEKI, T. QDA and S. HIRATAwas not detected anywhere. From the data presented in this paper, abnor-mality of the emergence of the circular dimer in human mitochondria andrelatively high emergence of the dimer in neoplasia cannot be denied, butthe possibility of a direct correlation of the emergence of it with neoplasiain man may be ruled out. This supposition can also be supported by occa.sional emergence of the circular dimer in the mitochondria isolated fromadult rat liver (14,15) and from beef liver (30). NAss (10) reported that thecircular dimer and higher oligomers were reversibly generated in mouseL cell mitochondria by keeping cells in stationary phase of growth, bytreatment of cells with cycloheximide, and by amino acid starvation. Inthe present case, as the liver functions were markedly disturbed a fewmonth before death, metabolic disorder of the patient whose kidney wasused in this experiment was thought to be remarkable. The patient wasgiven 2.5 g rolitetracycline nitrate (pyrrocycline N) for 5 days in totalbefore death. No other antibiotics and antitumor drugs were administered.Referring to the above Nass' data, it might be thought that the metabolicdisorder of the patient or administered rolitetracycline nitrate had a bear.ing on the emergence of the circular dimer, though it should be backedby direct experimental data.It has been reported that small circular DNA's are present in neoplasticcells (HeLa cells (6, 31)) and neoplastic cell mitochondria (rat ascites hepa.toma (11), C3H mouse ascites tumor (14, 18) and human hepatoma (17)).In our experiment, too, there were occasionally observed unusual circularDNA such as DNA with intermediate perimeter between the monomerand dimer, and with a contour length shorter than 3 p., though the signi.ficance of their presence is not clear.SUMMARYCircular DNA isolated from human kidney mitochondria was studiedby electron microscopy.I. Mean contour length of monomers of the mitochondrial DNA was4.96 ± SE 0.28 /1..2. The complex molecules (oligomers) of mitochondrial DNA wereobserved in frequency of 6.2 per cent. Among them circular dimersaccounted for two per cent of all circular DNA molecules.3. Circular DNA fibers with an intermediate perimeter between themonomer and dimer, and with a contour length shorter than 3 p_ wereoccasionally observed.4. Some discussions were made on the emergence of the circular dimer.8Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/3Electron Microscopy of Mitochondrial DNAACKNOWLEDGEMENTS253The authors are indebted to Dept. Pathology, Okayama Univ. Med. School, for supply-ing the kidneys of autopsy cases and the a utopsy records, and to the members of our labo-ratory for their valuable discussions and advices. Acknowledgement is also due to Mr. T.NAKAMURA for his skillful technical assistances, and to Mrs. N. SEKI for aid in the prefa-ration of the manuscript. This work was supported by research grants from the Ministry ofEducation, Japan.REFERENCES1. VAN BRUGGEN, E. F., BORST, P., RUTTENBERG, G.]. C. M., GRUBER, M. and KROON,A. M.: Biochim. Biophys. Acta 119, 437, 19662. SINCLAIR, J. H. and STEVENS, B. J.: Proc. Natl. Acad. Sci. U. S. 56, 508, 19663. NASS, M. M. K.: Proc. Nat!. Acad. Sci. U. S. 56, 1215, 19664. SINCLAIR, J. H., STEVENS, E. J., GROSS, N. and RABINOWITZ, M.: Biochim. Biophys. Acta145, 528, 19675. NASS, M. M. K.: Science 165, 25, 19696. RADLOFF, R., BAUER, W. and VINOGRAD, J.: Proc. Nat!. Acad. Sci. U. S. 57, 1514,19677. HUDSON, B. and VINOGRAD, ].: Nature 216, 647, 19678. CLAYTON, D. A. and VINOGRAD, ].: Nature 216, 652, 19679. CLAYTON, D. A., SMITH, C. A., JORDAN, J. M., TEPLITZ, M. and VINOGRAD, J.: Nature220, 976, 196910. NASS, M. M. K.: Nature 223, 1124, 196811. INABA, K.: Acta Med. Okayama 21, 297, 196912. ODA, T.: Proc. Jap. Cancer Assoc. 26th Ann. Meetin5, p. 11, 196713. ODA, T.: Saishin Igaku 23, 464, 196814. ODA, T.: jap. j. Clin. Med. 27, 67, 196915. OOA, T.: j. Cell BioI. 39, 173a, 196816. INOHARA, R.: Okayama Igakkai Zasshi 80, 1189, 196317. TAKE, S.: Acta Med. Okayama 23, 465, 196918. YAMAMOTO, G. and OOA, T.: Acta Med. Okayama 24, 287, 197019. NISHIDA, S. and ODA, T.: Acta Med. Okayama 24, 417, 197020. CLAYTON, D. A., DAVIS, R. W. and VINOGRAD, J.: j. Mol. BioI. 47, 137, 197021. CLAYTON, D. A., SMITH, C. A. and VINOGRAD, J.: Fed. Proc. 28, 532, 196922. HOGEBOOM, G. P.: Methods in Enzymology (:3. P. COLOWICK and N. O. KAPLAN, eds.) Vol.1, p.16, 195523. IWATA, S., SEKI, ~. and ODA, T.: Acta Med. Okayama 21, 191, 196724. MARMUR, J.: j. Mol. BioI. 3, 203, 196125. KLEINSCHMIDT, A. K., LANG, D., JACHERTS, D. and ZAHN, R. K.: Biochim. Biophys. Acta61, 857, 196226. FREIFELDER, D. and KLEINSCHMIDT, A. K.: j. Mol. BioI. 14, 271, 196927. CLAYTON, D. A. and VINOGRAD, J.: Proc. Nat!. Acad. Sci. U. S. 62, 1077, 196928. NASS, M. M. K.: j. Mol. BioI. 42, 529, 196929. NASS, M. M. K.: j. Mol. BioI. 42, 521, 196930. SEKI, S. and ODA, T.: un~utlished data31. ODA, T., O.\lURA, S., YAMAMOTO, S. and NISHIDA, S.: Acta Med. Okayama 24, 405,19709Seki et al.: Electron microscopic observation of mitochondrial DNA isolatedProduced by The Berkeley Electronic Press, 1971

Electron microscopic observation of mitochondrial DNA isolated from a human kidney: circular dimers in histologically normal organ mitochondria

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