1. Mitochondria isolated from hamster tumors induced by adenovirus type 12 possessed circular DNA fibers.
2. The mean value of the length of the highest frequency group of the circular DNA molecules was 4.92 ±0.38 &#956;.
3. Catenated dimer DNA molecules and small (less than 2 &#956; in length) circular DNA molecules were observed.</p

Nishida, Shigeru

Oda, Takuzo

English

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 24, Issue 6 1970 Article 1DECEMBER 1970Mitochondrial DNA from hamster tumorsinduced by adenovirus type 12Shigeru Nishida∗ Takuzo Oda†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Mitochondrial DNA from hamster tumorsinduced by adenovirus type 12∗Shigeru Nishida and Takuzo OdaAbstract1. Mitochondria isolated from hamster tumors induced by adenovirus type 12 poeed circularDNA fibers. 2. The mean value of the length of the highest frequency group of the circular DNAmolecules was 4.92±0.38 µ. 3. Catenated dimer DNA molecules and small (le than 2 µ in length)circular DNA molecules were observed.∗PMID: 4258713 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 24, 551-557 (1970)MITOCHONDRIAL DNA FROM HAMSTER TUMORSINDUCED BY ADENOVIRUS TYPE 12Shigeru NISHIDA and Takuzo aDADepartment of Biochemistry, Cancer Institute, Okayama University Medical School,Okayama, Japan (Director: Profesjor T. Oda)Received for publication, May 21, 1970Circular DNA molecules have been observed in mitochondria fromvarious tissues (1-17). And many reports have revealed that mitochon-drial DNA differs from nuclear DNA in many properties 08-29).Recently, investigations on the differences of mitochondrial DNAs bet-ween cancer cells and normal cells have been done in the field of carcino.genesis (6, 7, 11-17).This report describes the electron microscopic observations on themitochondrial DNA extracted from adenovirus Type 12-induced hamstertumors.MATERIALS AND METHODSVirus and animals: Adenovirus type 12 (Ad 12) was originally provided byDr. KATSUO OGAWA of Department of Pathology, Okayama University MedicalSchool. The stockvirus was propagated in our laboratory in HeLa cells. Theinfectivity titer of the virus was 102.5TCID50/O. 1 ml.0.05 ml of the stockvirus was injected intraperitoneally into syrian hamsterswithin 24 hours after birth. Forty-eight days after Ad 12 inoculation, tumorswere excised, minced and very loosely homogenized. 0.5 ml of the cell suspensionwas subcutaneously inoculated into unconditioned hamsters of 45 to 60 days ofage. Two weeks later, the hamsters were sacrificed, and the tumors were excised.Necrotic masses in the center of the tumors were elaborately excluded.Isolation of mitochondria: Mitochondria were isolated according to the methodof HOGEBOOM and SCHNEIDER (30). The crude mitochondrial fraction was suspen-ded in 0.25M sucrose [with O.lmM EDTA and 2mM Tris buffer, pH 7.6 andlayered on 0.34M sucrose. It was centrifuged at 700xg for 10 min. and approx-imately three fourths of the upper layer was carefully pipetted ;out. The mito-chondrial fraction was washed four times with the sucrose solution (12,000 xgfor 10 min.).Isolation of DNA from mitochondria: DNA was isolated by MARMUR'S method(31) with a slight modification. Since mitochondrial DNA fibers could not bespooled on a glass rod, the DNA-ethanol mixture was stored at -20°C overnightand it was centrifuged. The pellet was dissolved in dilute SSG. The absorption5511Nishida and Oda: Mitochondrial DNA from hamster tumors induced by adenovirus typeProduced by The Berkeley Electronic Press, 1970552 S. NISHIDA and T. ODAspectrum of the suspension utilizing a Hitachi EPS.3T automatic spectrophoto-meter revealed that the wave length at the maximal optical density was 260 m,u.Rotary shadowing and electron microscop)!: Rotary shadowing of DNA fibers withplatinum-palladium was performed following KLEINSCHMIDT and FREIFELDER'Stechnique (32). The specimens were observed with a HITACHI HU.IIC electronmicroscope. Contour length of DNA fibers was measured on electron micro-graphs.RESULTSIn the electron micro3copy of the specimens, considerably many,small linear DNA fragments (usually shorter than 2 fl. in length) andnumerous circular DNA fibers were observed. Long, linear fibers (longerthan 5 fl. in length) were far less frequently recognized. Circular DNAfibers were arranged in various forms; open, catenated and twisted.Highly twisted circles were occasionally observed. They were, however,excluded from measuring of the contour length, because of the difficultyin accurate measurement. Circular DNA molecules ranging in lengthfrom 4.0 to 5.5 ,f.!. occupied about 70 % of the total number of the circular-form DNA fibers, while those shorter than 2,0. in length amounted toapproximately 18 %. The mean value of the length of the highest fre.quency group was 4.92±0.38 p..18Length (!()Fig. 1 A histogram of the contour length distributions of circular mitochon-drial DNA molecules isolated from hamster tumors induced by adenovirus type12. Total number of circular DNA molecules: 57 Mean value of the length ofthe highest frequency-group: 4.92±O.38ttDISCUSSIONIn mitochondrial DNA molecules from hamster tumors induced byAd 12, the mean value of the length of the highest frequency group was2Acta Medica Okayama, Vol. 24 [1970], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss6/1Mitochondrial and from Hamster Tumors Induced by Adenovirus Type 12 553Fig. 2 An open circular mitochondrial DNA molecule isolated from hamster tumorsinduced by Ad 12. A few short linear DNA fibers are also observed.Fig. 3 Two twisted circular mitochondrial DNA fibers3Nishida and Oda: Mitochondrial DNA from hamster tumors induced by adenovirus typeProduced by The Berkeley Electronic Press, 1970554 S. NISHIDA and T. GDAFig. 4 Catenated circular mitochondrial DNA fibers. Two circular DNA fibers ofthe same length are catenated with each other.Fig. 1 Small circular mitochondrial DNA fibers. (a)O.3f! (b)O.4~ (c)O·.9~ (d)I.OfL(e)1.3~4.92 ± 0.38 ,II.. A report (10) described that the mean value of the lengthin mitochondrial DNA molecules from normal hamster liver was 5.13±0.28 p.. Another recent report (11) showed similar data on the differencein length of mitochondrial DNA molecules between human normal liverand human hepatoma. The significance of the difference in length ofcircular DNA fibers between tumor and normal tissues is unknown.The presence of small circular DNA in tumor cell mitochondriaaroused the interest of many workers because small circular DNA viruseswere highly oncogenic in some animals and cultured cells. However,4Acta Medica Okayama, Vol. 24 [1970], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss6/1Mitochondrial and from Hamster Tumors Induced by Adenovirus Type 12 555BENJAMIN (33) showed the absence of homology between DNA from smallcircular DNA viruses and mitochondrial DNA from virus-induced tumors.It still remains unknown whether the difference in length of circular DNAfi bers between normal and tumor tissue mitochondra is of any significancein oncogenicity.The most probable explanation on short linear DNA fibers is thatsome mitochondrial DNA molecules had been broken into small piecesthrough the procedure of DNA extraction. A small number of the linearDNA fibers might be originally present in mitochondria. As to the longlinear fibers, there may have been a little nuclear contamination in themitochondrial fraction.SUMMARY1. Mitochondria isolated from hamster tumors induced by adeno-virus type 12 possessed circular DNA fibers.2. The mean value of the length of the highest frequency group ofthe circular DNA molecules was 4.92 ±O.38 fl..3. Catenated dimer DNA molecules and small (less than 2 fl. inlength) circular DNA molecules were observed.ACKNOWLEDGEMENTWe wish to express our hearty gratitude to all the staff members of Department ofBiochemistry, Cancer Institute of Okayama University Medical School.This investigation was supported by a Scientific Research Grant from the Ministry ofEducation of Japan.REFERENCES1. LUCK, D. J. L., and REICH, E.: DNA in mitochondria of Nenrospora crassa. Proc. Natl.Acad. Sci. 52, 931, 19642. RABINOWITZ, M., SINCLAIR, J., DESELLE, L., HASELKORN, R. and SWIFT, H. H.: Iso-lation of deoxyribonucleic acid from mitochondria of chick embryo heart and liver.: Proc.Natl. Acad. Sci. 53, 1126, 19653. NAss, M. M. K., NAss, S. and AEZELIUS, B. A.: The general occurrence of mitochon-drial DNA. Exptl. Cell Res. 37, 516, 19654. SCHNEIDER, W. C. and KUFF, E. L.: The isolation and some properties of rat livermitochondrial deoxyribonucleic acid. Proc. Natl. Acad. Sci. 54, 1650, 19655. TEWARI, K. K., JAYARAMAN, J. and MAHLER, H. R.: Separation and characterizationof mitochondrial DNA from yeast. Biochem. Biophys. Res. Commun. 21, 141, 19656. INABA, K.: Nucleic acids and protein synthesis in cancer cell mitochondria. I. Nucleicacids in rat hepatoma mitochondria. Acta Med. Okayama 21, 297, 19677. CLAYTON, D. A. and VINOGRAD, J.: Circular dimer and catenate forms of mitochon-5Nishida and Oda: Mitochondrial DNA from hamster tumors induced by adenovirus typeProduced by The Berkeley Electronic Press, 1970556 S. NISHIDA and T. GOA'drial DNA m human leukemia leucocytes. Nature 216, 652, 19678. KALF, G. E. and Gd:GE, A.: The isolation of deo:<yribonucleic acid from lamb heartmitochondria. j. Biol. Chem. 241, 1019, 19669. CORNES, G., M.JRE, C., SANADI, D. R., GROSSMAN, L. I. and MARMUR, J.: Mitochon-drial D_\JA in yeast and some mammalian species. Science 151, 687, 196610. INOHARA, R.: Electron microscopy of mitochondrial D.\JA molecules from rat andhamster livers. Ohayama Igakkai Zasshi 80, 1199, 196311. TAKE, S.: D~A's from human hepatoma and gastric cancer mitochondria. Acta Med.Okayama 23, 465, 196912. DDA, T.: Genetic substances and membrane systems in the cytoplasm of cancer cells.Proc. .lap. Cancer Ass., 26th Ann. Meeting, p.ll, 1967; Saishin Igaku 23, 464, 195813. DDA, T.: Circular D~A's from tumor cell mitochondria and nuclei. j. Cell Biol. 39,l73a, 196814. DDA, T.: Mitochondrial D:'JA of cancer cells. jap. j. Clin. Med. 27, Supple. 67, 196915. GOA, T.: Mitochondrial membrane. In "Biochemistry of Biological Membrane" ed. T.DDA, M. NAKAO, and R. SATO, p.2l4, Asakura Publisher, 196916. DDA, T.: l'\ucleic acids in mitochondria. In "Nulceic Acids", ed. by Y. MIURA andT. TAKAGI, p.69, ASAKURA Publisher, 197017. YAMAMOTO, G. and GOA, T.: Studies on nucleic acids in Rous sarcoma virus-inducedmouse ascites cells: Distribution and electron microscopy of nucleic acids in subcellularfractions and circular D~A in mitochondrial fraction. Acta Med. Okayama 24, 287, 197018. SCHATZ, G., HASLSRUNNER, E. and Tuppy, H.: Deoxyribonucleic acid associated withyeast mitochondria. Biochem. Biophys. Res. Commun. 15, 127, 196419. SLONIMISKI, P.: Biochemical aspects of the biogenesis of mitochondria, edited by E. B.SLATER, J. M. TAGERS, S. PAPA, and E. QUAGLIARIELLO, BARI: Adriatica Editrice,196720. NASS, A., NASS, M. M. K. and HENNIX, U.: Deoxyribonucleic acid in isolated ratliver mitochondria. Biochem. Biophys. Acta 95, 426, 196521. DuBuy, H. G., MATTERN, C. F. T. and RILY, F. L.: Comparison of the DNAs obtainedfrom brain nuclei and mitochondria of mice and from the nuclei and kinetoplasts ofLeishmania enteriettii. Biochem. Biophys. Acta 123, 298, 196622. DAVID, I. B.: Evidence for the mitochondrial origin of frog egg cytoplasmic D:'JA. Proc.Natl. Acad. Sci. 56, 269, 196623. SINCLAIR, J. H. and STEVENS, B.J.: Circular DNA filaments from mouse mitochondria.Proc. Nat!. Acad. Sci. 56, 503, 196624. NASS, M. M. K.: The circularity of mitochondrial D'JA. Proc. Natl. Acad. Sci. 56, 1215,196625. VAN BRUGGEN, E. F.J., BORST, P., RUTTENBERG, G.J. C. M., GRUBER, M. and KROON,A. M.: Circular mitochondrial D:'JA. Biochem. Biophys. Acta 119, 437, 196626. KROON, A. M., BORST, P., VAN BRUGGEN, E. F. J., and RUTTENBERG, G. J. C. M.:Mitochondrial D:'JA from sheep heart. Proc. Natl. Acad. Sci. 56, 1836, 196627. RADLOFF, R., BAUER, W., and VINOGRAD, J.: A dye-bouyant-density method for thedetection and isolation of closed circular D'JA in HeLa cells. Proc. Natl. Acad. Sci. 58,1514, 196728. SINCLAIR, J. H., STEVENS, B.J., SANGHAVI, R., and RABINOWITZ, M.: Mitochondrial-satellite and circular D:'JA filaments from yeast mitochondria. Science 156, 1234, 196729. AVERS, C. J.: Heterogenous length distribution of circular D:'JA filaments from yeastmitochondria. Proc. Natl. Acad. Sci. 58, 620, 196730. HOGEBOOM, G. H., SGHNEIDER, W. Coo and PALLADE, G. E.: Cytochemical studies ofmammalian tissues. 1. Isolation of intact mitochondria from rat liver: some biochemical6Acta Medica Okayama, Vol. 24 [1970], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss6/1Mitochondrial and from Hamster Tumors Induced by Adenovirus Type 12 557properties of mitochondria and submicroscopic particulate meterial. j. Bioi. Chern. 172,619, 194331. MARMUR, J.: A procedure for the isolation of deo >yribonucleic acid from micro-orga-nisms. j. Mol. Bioi. 10, 208, 196132. FREIFELDER, D. and KLEINSCHMIDT, A. K.: Single-strand breaks in duplex D~A ofcoiiphage T7 as demonstrated by electron microscopy. j. Mol. Bioi. 14, 271, 196533. BENJAMIN, T. L.: Absence of homology between polyoma or SV40 viral DNA and mito-chondrial D;'JA from virus-induced tumors. Virology 36, 685, 19687Nishida and Oda: Mitochondrial DNA from hamster tumors induced by adenovirus typeProduced by The Berkeley Electronic Press, 1970

A dye-bouyant-density method for the detection and isolation of closed circular D'JA in HeLa cells.

A procedure for the isolation of deo >yribonucleic acid from micro-organisms.

Absence of homology between polyoma or SV40 viral DNA and mitochondrial D;'JA from virus-induced tumors.

Biochemical aspects of the biogenesis of mitochondria, edited by

Circular D~A's from tumor cell mitochondria and nuclei.

Circular dimer and catenate forms of mitochon5 Nishida and Oda: Mitochondrial DNA from hamster tumors induced by adenovirus type Produced by The Berkeley Electronic Press,

Circular DNA filaments from mouse mitochondria.

Circular mitochondrial D:'JA.

Comparison of the DNAs obtained from brain nuclei and mitochondria of mice and from the nuclei and kinetoplasts of Leishmania enteriettii.

Cytochemical studies of mammalian tissues. 1. Isolation of intact mitochondria from rat liver: some biochemical Acta Medica Okayama,

D~A's from human hepatoma and gastric cancer mitochondria.

Deoxyribonucleic acid associated with yeast mitochondria.

Deoxyribonucleic acid in isolated rat liver mitochondria.

DNA in mitochondria of Nenrospora crassa.

Electron microscopy of mitochondrial D.\JA molecules from rat and hamster livers.

Evidence for the mitochondrial origin of frog egg cytoplasmic D:'JA.

Genetic substances and membrane systems in the cytoplasm of cancer cells.

Heterogenous length distribution of circular D:'JA filaments from yeast mitochondria.

Isolation of deoxyribonucleic acid from mitochondria of chick embryo heart and liver.:

l'\ucleic acids in mitochondria. In &quot;Nulceic Acids&quot;,

Mitochondrial D_\JA in yeast and some mammalian species.

Mitochondrial D:'JA from sheep heart.

Mitochondrial D:'JA of cancer cells.

Mitochondrial membrane. In &quot;Biochemistry of Biological Membrane&quot;

Mitochondrialsatellite and circular D:'JA filaments from yeast mitochondria.

Nucleic acids and protein synthesis in cancer cell mitochondria. I. Nucleic acids in rat hepatoma mitochondria. Acta Med.

Separation and characterization of mitochondrial DNA from yeast.

Single-strand breaks in duplex D~A of coiiphage T7 as demonstrated by electron microscopy.

Studies on nucleic acids in Rous sarcoma virus-induced mouse ascites cells: Distribution and electron microscopy of nucleic acids in subcellular fractions and circular D~A in mitochondrial fraction.

The circularity of mitochondrial D'JA.

The general occurrence of mitochondrial DNA.

The isolation and some properties of rat liver mitochondrial deoxyribonucleic acid.

The isolation of deo:<yribonucleic acid from lamb heart mitochondria.

Mitochondrial DNA from hamster tumors induced by adenovirus type 12

Mitochondrial DNA from hamster tumors induced by adenovirus type 12

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