Ring DNA from rat liver mitochondria has been examined by circular dichroism (CD) in the region of the 225 to 320 m/~ and the followings have been clarified. The ring DNA gives a CD spectral curve somewhat different from linear DNA from nuclei, showing a big positive peak at 266 m/~ and a small negative band at 243 m!~. That is, the positive CD band of ring DNA shifted by about 7 m/~ to the shorter wavelength side from the band of the ordinary nuclear DNA, 273 m!~. Negative band appeared at the same region as that of linear DNA but reduced in depth. Heat denaturation of the ring DNA induced a red shift of the positive band, by about 4 mp., but no change in negative band. From these experimental results it has been concluded that the ring DNA has highly twisted
conformation and high in G.C contents, both of which are responsible for the blue shift of the CD spectrum.</p

Ito, Nobutaka

Miyahara, Masanobu

Seno, Satimaru

Okayama University Digital Information Repository

Conformational studies of mitochondrial DNA

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 25, Issue 1 1971 Article 3FEBRUARY 1971Conformational studies of mitochondrial DNANobutaka Ito∗ Masanobu Miyahara†Satimaru Seno‡∗Okayama University,†Okayama University,‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Conformational studies of mitochondrialDNA∗Nobutaka Ito, Masanobu Miyahara, and Satimaru SenoAbstractRing DNA from rat liver mitochondria has been examined by circular dichroism (CD) in theregion of the 225 to 320 m/∼ and the followings have been clarified. The ring DNA gives a CDspectral curve somewhat different from linear DNA from nuclei, showing a big positive peak at266 m/∼ and a small negative band at 243 m!∼. That is, the positive CD band of ring DNA shiftedby about 7 m/∼ to the shorter wavelength side from the band of the ordinary nuclear DNA, 273m!∼. Negative band appeared at the same region as that of linear DNA but reduced in depth. Heatdenaturation of the ring DNA induced a red shift of the positive band, by about 4 mp., but nochange in negative band. From these experimental results it has been concluded that the ring DNAhas highly twisted conformation and high in G.C contents, both of which are responsible for theblue shift of the CD spectrum.∗PMID: 4258664 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Merl. Okayama 25, 29-35 (970)CONFORMATIONAL STUDIES OF MITOCHONDRIAL DNANobutaka ITO, Masanobu MIYAHARA and Satimaru SENODepartment of Pathology, Okayama University Medical School,Okayama, Japan (Director: Prof Satimaru Seno)Received for publication, April 20, 1970Recent observations of mitochondria and chloroplasts revealed thatthey have DNA, the ring DNA, similar to that of some microorganisms.Each ring DNA molecule should has the complete set of gene needed forthe construction of the organella or the microorganism 0--3). It appearsas a ring on the spread and negatively stained specimen, and its duplicat-ing pattern has also been observed under electron microscope. But themorphologic pictures of the DNA appearing as ring on the section ofthe organellae or the microorganism suggest some complicated threedimentional structure of the DNA in the living cell. It may undergo someconformational changes according to the changed functional state, resting,duplicating or transcription stage. At present we have no informationconcerning how it assumes the three-dimentional structure of ring DNA andits possible conformational changes. However, the recent developmentof the analytical method, the spectrography by circular dichroism oroptical rotatory dispersion, makes it possible to reveal the three-dimentionalconformation of large melecules, like polypeptide and protein. And it hasbecome quite promising to obtain a reliabe information on the threedimentional structures of DNA by the same method. In the present paperthe authors present the spectral pattern of the circular dichroim (CD) ofthe ring DNA of rat liver mitochondria, which suggests a highly twistedconformation of the DNA in its native state or in the living cell.MATERIALS AND METHOD3Isolation ojmitochondria: The mitochondria were isolated from the liver of 25-to 35-day old wister rats by the modified method of HOGEBOOM (4). Immediatelyafter sacrifice of the animals livers were removed and homogenized by glasshomogenizer adding about 5 volumes of 0.25 M sucrose containing 50 I-tM EDTAand I mM Tris-HCI buffer (pH 7.4). The homogenate was centrifuged at 50 x gfor 10 min. The upper half of the supernatant was taken carefully and superim-posed on the equal volume of 0.34 M sucrose containing 50 I-tM EDTA and I mMTris-HCI (pH 7.4), centrifuged at 700 x g for 10 min. and the precipitant wasremoved. Repeating the same process the contaminated nuclei were eliminated as291Ito et al.: Conformational studies of mitochondrial DNAProduced by The Berkeley Electronic Press, 197130 N. ITO, M. MIYAHARA and S. SENDprecipitate. The supernatant obtained through three centrifugations was furthercentrifuged at 5000 x g for 10 min, and the mitochondrial fraction was obtainedas the precipitate. The mitochondria were washed twice with the homogenizingmedium and twice again with 0.15 M NaCl (pH 8.0) containing 0.1 M EDTAby the repeated centrifugation and stored at -20°C being suspended in anappropriate volume of the washing medium.Isolation rif DNA from the mitochondria: The stored mitochondrial suspensionwas frozen-thawed and the mitochondrial pellet was obtained by centrifugationat 7,000 x g for 10 min. By using the pellet DNA was isolated by the modifiedmethod of MARMUR (5). That is, the pellet was deproteinized by shaking withisoamylalcohol-chloroform vigorously, the denatured protein was removed bycentrifuging at 800 X g for 10 min, and DNA was precipitated by treating theaqueous layer with 95 % cold ethanol. The DNA was dissolved with citrate-salinesolution and the process of the deproteinizaiotn was repeated further three times.The final citrate.saline solution was treated with RNase in a final concentrationof 50 ,Ltg/ml for 30 min at 37°C. The digest was subjected to the deproteinizationprocess as just described, repeating two to three times. Finally the sample wassuspended in citrate-saline, and added with an equal volume of 80 % phenol bythe method of MIURA, and washed with ether. The DNA suspension obtainedwas used for the physical analyses.Isolation of DNA from hamster thymus nuclei: Hamster thymus nuclei wereisolated by the method of CHAUVEAU et al. (6) and the DNA was purified by themethod of MARMUR as described above.Measurement of the CD sjJecrta of DNA: With the DNA suspension just men-tioned the spectrogram of circular dichroism of the DNA was obtained by usingthe JASCO spectropolarimeter model ORD/UV.5 having CD attachment.Measurement was carried out with the samples in a quartz cell on the light pathsvarying from O. 1 to 1.0 em. The circular dichroism was expressed in terms of thedifference of the molar extinction coefficient for left- and right.handed circularlypolarized light(~r~R) at the frequency II. The molar ellipticity can be found fromthose as un = 3300(~J.-~R)'Absorption spectra of the DNA were obtained by using the Hitachi auto-recording spectrophotometer.RESULTSThe DNA solution from rat liver mitochondria, which was carefullyprepared by the method just described, shows the typical absorptionspectra of DNA (Fig. 1). The absorption ratios were A z60 : A 23o = 1.77 andA 260 : A 28o = 2.61, and indicate that the sample contains neither phenol norprotein. This suggests that the DNA is of double strand and splits into twosingle strands by heating. The mitochondrial DNA shows a typical circulardichroic spectrum in the 0.15 A1 NaCI containing 0.015 M sodium citrate2Acta Medica Okayama, Vol. 25 [1971], Iss. 1, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss1/3CD of Mitochondrial DNA 310·80·6>l-t)Z~ 0·4~oC(ui=~0 0·2220 280Fig. I UV-absorption spectrum of the isolated liver mitochondrial DNAat pH 7.0 (SSC solution). It displays one peak and one trough; a peakhaving a broad base with the summit at 266 mp., and a small trough ataround 243 ml-t (Fig. 2, curve a). By heating at 90°C for 10 min andrapid cooling, the DNA was denatured and the CD spectrum showed320310300290280210260250+5+4+3+2w"'l +1, 0o-'lW-1-2-3-4-5220 230 240WAVELBliGTH ( ml' )Fig. 2 Circular dichroism of isolated liver mitochondrial DNAa: mitochondrial DNA, b: mitochondrial DNA, heated at 90°C for 10 min andcooled rapidly, c: nucelar DNA from hamster thymus. For details refer to the text.3Ito et al.: Conformational studies of mitochondrial DNAProduced by The Berkeley Electronic Press, 197132 N. ITO, M. MIYAHARA and S. SENOsome change in its positive peak, i. e. a slight red shift of the summit from266 to around 270 mp. with decrease in height (Fig. 2, curve b). Thedecrease in the height of the peak means the loss of secondary structure.The nuclear DNA from hamster thymus dissolved in sse solution at pH7.0 gave also one peak having the summit at 273 m/" and one trough ataround 243 m/~ (Fig. 2, curve c). The curve represents the typical patternof CD spectrum of nuclear DNA of linear type as reported on the DNAfrom the nucleus of calf thymus, fowl erythrocytes, rat liver as well asE. coli in aqueous saline solution. The data indicate clearly that the CDspectra of ring DNA of rat liver mitochondria is largely different fromthat of nuclear linear DNA, i. e. the native ring DNA has a positive CDband at 266 mt" while the nuclear DNA at 273 m,a, though the troughsaJVpeared at around 243 mt" in both DNAs. By heat denaturation the CDband of the ring DNA shifted by about 3 mt" toward the blue side.The optical rotatory dispersion (ORD) of the nuclear DNA has beenextensively studied by YANG and his associates (7, 8) and it has been revealedthat Cotton effect appearing around 260 m/" reflects the conformation ofDNA. Recently CD method has been also applied to the study of theconformation of nuclear DNA and the ribonucleoside by BRAHMS and hisassociates (9-11) and HASHIZUME and IMAHORI (2). Generally, theoreticalevaluation of spectroscopic evidence has led to an assignment of electrictransition in purines and pyrimidines 03-16). The strong absorption ataround 260 m,a is ascribed to a rr ---t rr* transition and another transition isthe region of 280 m,n which is attributable to an rr---trr* transition. Besidesthese, DNA shows negative CD peak at 243 to 245 m/" but RNA does notgive the similar peak. For the characteristic negative CD band by DNAHASHIZUME and IMAHORI (2) explain as follows: (I) The dA+dT showsthe characteristic big negative CD band at 243 mt", and dG+dC givesthe positive CD band around 255 m/" which is rather week. So the formernegative band is retained without neutralization. (II) The absence of2'OH in the ribose ring of the DNA reflects week negative band of CDspectra at around 245 m/". Therefore, it is reasonably deduced that thebroad positive band of ring DNA from 250 to 29rJ ml" as revealed in thepresent experiment is the integrated feature of 2 to 3 bands appearing inthe region.The most important point of this experiment is the blue shift of posi-tive band of mitochondrial DNA in CD spectra as compared to that ofnuclear DNA; This blue shift may be explained by the two possible ways.The one possibility is the high G-C contents of mitochondrial DNA,because the maximum positive band of the CD spebtra of double strand4Acta Medica Okayama, Vol. 25 [1971], Iss. 1, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss1/3CD of Mitochondrial DNA 33poly (G+C) helix appears at 261 m/~. As is well known, high G-C contentsmean the increase of Tm. Actually, it has been reported that the G-Ccontent of mitochondrial DNA is slightly higher than that of nuclearDNA (1) and also it was found that the Tm of mitochondrial DNA isslightly higher than that of nuclear DNA (17). The other possibility isthat the blue shift of the positive CD peak of ring DNA is due to its highlytwisted conformation. The electron microscope observation of monolayerpreparation of mitochondrial DNA from rat liver revealed three kinds ofmolecular conformation. The first one is the highly twisted ring, thesecond is an open ring and the third is the linear form probably ofartifact (18-30). HASHIZUME and IMAHORI (12) reported a close relationbetween molecular conformation and the CD spectra in several naturaland synthetic polynucleotides. Namely, the peak of CD band showeda red shift when base pairing between two strands of polynucleotides wasruptured. Essentially the similar results were obtained on hypochromicCotton effect as observed by the ORD (8, 31). From these facts it isdeduced that the blue shift of the positive band of ring DNA from mito.chondria reflects the increase in the G-C contents in certain region of theDNA molecule and the highly twisted molecular conformation. Probably,in the highly twisted condition of the molecule the stacking of the baseswill become closer. Explanation of the decrease in the depth of thenegative CD band in ring DNA seems to need further experiments andinformations.SUMMARYRing DNA from rat liver mitochondria has been examined by circulardichroism (CD) in the region of the 225 to 320 m/~ and the followingshave been clarified. The ring DNA gives a CD spectral curve somewhatdifferent from linear DNA from nuclei, showing a big positive peak at266 m/~ and a small negative band at 243 m!~. That is, the positive CDband of ring DNA shifted by about 7 m/~ to the shorter wavelength sidefrom the band of the ordinary nuclear DNA, 273 m!~. Negative bandappeared at the same region as that of linear DNA but reduced in depth.Heat denaturation of the ring DNA induced a red shift of the positiveband, by about 4 mp., but no change in negative band. From these experi-mental results it has been concluded that the ring DNA has highly twistedconformation and high in G.C contents, both of which are responsible forthe blue shift of the CD spectrum.5Ito et al.: Conformational studies of mitochondrial DNAProduced by The Berkeley Electronic Press, 197134 N. ITO, M. MIYAHARA and S. SENOACKNOWLEDGEMENTThe authors wish to express their sincere thanks to Dr. Kozo UTSUMI, Department ofBiochemistry, Cancer Institute, Okayama University Medical School for his helpful discus-sions.REFERENCES1. VINOGRAD, ]., LEBOWITZ, ]., RADLOFF, R., WATSON, R. and LAIPIS, P.: The twistedcircular from of polyoma viral DNA. Proc. Natl. Acad. Sci. 53, 1l04-, 19652. SAGER, R. and ISHIDA, M. R.: Chlroplast DNA in chlamidomonas. Proc. Natl. Acad.Sci. 50, 725, 14633. SINCLAIR, ]. H., STEVENS, B. ]., GROSS, N. and RABINOWITZ, M.: The constant sizeof circular mitochondrial D:'-JA in several organisms and different organs. Biochim. Biophys.Ada 145, 528, 19674. 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Chern. 240, 2088, 19657Ito et al.: Conformational studies of mitochondrial DNAProduced by The Berkeley Electronic Press, 1971

Conformational studies of mitochondrial DNA

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