Electron microscopic observation of replicating SV 40 DNA has revealed the existence of two types of RF, e form and (1 form. The frequency of RF at 54 hours after infection was 8.9% for the e form and 4.3% for the (1 form. Morphological evidence exhibits that in (1 form
RF the tails are, predominantly, shorter than the viral genome and double length SV-40 genomes are also capable of replication in SV-40
infected VERO cells.</p

Oda, Takuzo

Yamamoto, Shinichiro

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 25, Issue 4 1971 Article 2AUGUST 1971Electron microscopic studies of replicating SV40 DNAShinichiro Yamamoto∗ Takuzo Oda†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Electron microscopic studies of replicating SV40 DNA∗Shinichiro Yamamoto and Takuzo OdaAbstractElectron microscopic observation of replicating SV 40 DNA has revealed the existence of twotypes of RF, e form and (1 form. The frequency of RF at 54 hours after infection was 8.9% for thee form and 4.3% for the (1 form. Morphological evidence exhibits that in (1 form RF the tails are,predominantly, shorter than the viral genome and double length SV-40 genomes are also capableof replication in SV-40 infected VERO cells.∗PMID: 4343049 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 25, 237-243 (1971)ELECTRON MICROSCOPIC STUDIES OF REPLICATINGSV 40 DNAShinichiro YAMAMOTO and Takuzo ODADepartment of Biochemistry, Cancer Institute, Okayama University Medical School,Okayama, Japan (Director: Prof. T. ODA)Received for publication, June 12, 1971The existence of replicating form (RF) of circular DNA moleculeswith one linear single tail (0' form) was reported in epx 174 (I), E. coli 15(2), colicin factor E I (3) and lamda phage (4). Previous studies of thebiophysical properties of SV 40 DNA revealed the existence of RF ofcircular DNA molecules with two branch points and no free end (8 form)(5). In the present report we have isolated 0' form RF of SV 40 DNA fromvirus-infected VERO cells, applying the method of HIRT (6). In additionto e form RF single tailed circular molecules were observed not only inmonomers but also in double length SV 40 DNA.MATERIALS AND METHODSTissue cultureVERa cells, an established cell line from African green monkey kidney cells,were used for the propagation of virus. The cells were grown in a dilution bottle(5.5x 11.0 em) using medium YLE containing 0.1% PVP, 10% heat-inactivatedbovine serum. Two bottles of monolayer culture of VERa cells at the 450thpassage were used at the same time, one is for noninfected control and the otherfor SV 40 infection.Labeling of SV 40 DNA with 3H.thymidineMonolayer culture of VERa cells were infected with 1 ml SV 40 virus hav-ing the infectivity of 106 PFU Iml, absorbed for two hours at 37°C. After infec-tion SV 40 virus was removed and supplemented with 10 ml YLE mediumcontaining 10% bovine serum. At 16 hours after infection 5,uc/ml 3H-thymi-dine (20.6 c/mmole) was added to the bottle. At 53 hours after infection radio-active medium was removed and replaced by fresh medium containing 10 %bovine serum and kept for one hour to exhaust thymidine pool. Infected cellswere washed with phosphate buffered saline (PBS (-), pH 7.6) three times andlysed according to the method of HIRT (6). Uninfected controls were mockinfected with PBS (-) instead of SV 40 virus and other treatments were the sameas in SV 40-infected cells.Selective extraction of SV 40 DNASV 40-infected cells were lysed by adding 5 ml of 0.6% sodium lauryl sulfate2371Yamamoto and Oda: Electron microscopic studies of replicating SV 40 DNAProduced by The Berkeley Electronic Press, 1971238 S. YAMAMOTO and~T. QDAcontammg 0.01 M EDTA, pH 7.5 and the lysate was treated with 2 mg/mlpronase (Nagarse) at 37°C for one hour. Five M NaCl was added to make the finalconcentration of 1 M. Most of the cellular DNA was precipitated by centrifuga-tion of l7,000xg for 30 minutes and the supernatant was treated with 0.3mg/mlRNase at 37°C for one hour.Equilibrium density gradient centryugationSolid CsCl (3.60 g) was added to 4 ml above supernatant (mean density 1.55g/cm3) and centrifuged at 45000 rpm for 20 hours in an RPS 65 TA rotor of aHitachi ultracentrifuge. Eight drop fractions (0.15 ml) were collected from thebottom successively. 0.5 ml SSC (0.15 M NaCl +0.015 M Na-citrate, PH 7.6)was added to each fraction. 0.05 ml of each fraction was assayed for radio-activity by a Packard liquid scintillation counter.Electron microscopyDNA was prepared for electron microscopy essentially as described byKLEINSCHMIDT et al. (7). A solution containing l5,ug/ml DNA, 1 M ammoniumacetate, 0.01% cytochrome c and 0.5% formalin were spread over a 0.3 M am-monium acetate solution containing 0.5% formalin. The film was picked up on acarbon coated copper grid and the grid was fixed by submersion in 95 % ethanoland air-dried. The grid was rotary shadowed with platinum palladium andobserved in a Hitachi 11 D electron microscope. Magnification calibration wasdone with carbon grating replicas. Contour lengths of DNA were measured witha map ruler.RESULTSCsCl density gradient centrifugationThe sedimentation behaviour of replicating SV 40 DNA was analyzed(Fig. 1). Both infected and noninfected cells had one major peak at frac.tion 6 or 7, but in the noninfected cells no middle peak was noted andinfected cells had two more minor peaks at fractions 11 and 17. Thesethree peaks (fractions 7, 11 and 17) of the infected cells were examined byelectron microscopy. Fractions 7 and 11 consisted of cellular DNA,namely, long linear fiber and no circular DNA but a heterogeneous popula.tion of molecules was observed from fraction 17 of the infected cellsconsisting of three types; twisted or open circular molecules, circularmolecules with two branch points (0 form) and circular molecules with asingle tail ((1' form). The latter two were thought to be RF of SV 40 DNA.Fractions 16-20 of the infected cells were collected and examined. Outof 370 molecules of SV 40 DNA, e form and (1' form existed by 33 and 16molecules, the frequency of which corresponded to 8.9 % and 4.3 %respectively.2Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/2EM Etudies on Replicating SV~4O DNA 239400(A) NONINFECTED1200800400'?:·s~E::l0~::c:(B) INFECTED"" 120080010 20 30Fraction NumberFig. 1 esCI density gradient centrifugation of DNA from (A) noninfectedVERO cells and (B) SV 40 infected VERO cells.Centrifugation was done at 5,400 rpm for 20 hours in an RPS 65 TArotor of Hitachi ultracentrifuge. Eight drops were collected from the tubebottom and each fraction was added with 0.5 ml SSC. 3H count is shown. Noteone major peak at fraction 7 and minor peaks at fractions 11 and 17 of theinfected cells.RFSV 40 DNAOf the circular molecules with tails, sixteen were spread well enoughto be measured. Of these sixteen molecules, eleven possessed rings ofviral DNA length and five possessed rings twice this length (Fig. 2). Allof the molecules with tails (except one) had tails of less than viral length.One molecule had a tail 19% longer than the ring to which it was attached(Fig. 2, No. 11). The rate of circle length to tail length had considerablyuniform distribution. The replication rate ranged from 10% to 95%almost uniformly. Representative molecules are shown in Fig. 3. A doublelength ring with a tail is shown in Fig. 3.d. We have found from this3Yamamoto and Oda: Electron microscopic studies of replicating SV 40 DNAProduced by The Berkeley Electronic Press, 1971240'"u"5uu-0E5.1'-'-'-'-'..~~. YAMAMOTO and T.ODAt=:::::l ci rc leJ'lZZZ:1ZZm tailFig. 2 Distribution of taillengthsTail length is compared withthe circle length. One molecule(No. 11) has a longer tail thanthe circle. The replication rate(ratio of tail to circle) is uniformlydistributed.1.0Contour length (fl.)Fig. 3 Tailed molecules in DNA from fractions 16 to 20 of SV 40infected VERO cells (fig. I). d. shows a double length DNA with a tail.The bar indicates 0.5 p.4Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/2EM Studies 'On Replicating SV.40 DNA 241experiment and former experiment (8) the existence of much deletedmolecules which undergo the replication. As shown in Fig. 2, the smallestmolecule of RF was I. 0l~ and from the former experiment we have founde form RF of the contour length of O. 71 to I. O,,-tDISCUSSIONRF of SV 40 DNA were isolated from virus.infected VERa cells.Two kinds of RF, e. g. e form and t7 form RF were noted at 8.9 % and4·.3% of all the viral DNA examined, respectively. Concerning t7 formRF, the replication rate, namely, the ratio of tail to circle seems to be inuniform distribution, which was also noted in e form RF of SV 40 DNA(8). In lambda phage RF in an early stage was found more than that inlate stage (4) and they pointed out the possibility that the rates of DNAreplication vary, dependent upon the extent of replication; namely, thefirst portion of the molecules might be replicated more slowly than theremainder, giving rise to a pool of incomplete intermediates possessing ashort tail. On the contrary LEVINE et ai. (9) reported that about 75 % ofthe replicating molecules had completed 90 to 65% of the replication andascribed the skew toward the late stage of replication to a rate.limitingstep in the replication process. In our experiment the skew of RF toneither early nor late stage of replication was observed. The speed ofreplication of SV 40 DNA in VERa cells seems to be uniform frombeginning to end of replicating process.The tail length in t7 form RF, is generally shorter than the circle,which were verified in ¢x 174 (l) and colicin factor E I (3). One moleculepossessing a tail longer than the circle was noted in our experiment.KIGER and SINSHEIMER (4) observed the same phenomena and supposedthe cause that if maturation of phage DNA and DNA replication are notcoordinated, conditions may exist under which DNA replication couldoccur at a greater rate than maturation. Replication might then continuepast the point of initiation on the ring and generate a tail longer thanone phage DNA. This assumption might l:e applied to SV 40 DNAreplication.As pointed out in Fig. 2, the existence of double length rings withtails is an intermediate of replication. In lambda phage (4) morphologicalevidence was observed showing double length rings to which tails areattached. The fact that double length rings of viral DNA could replicatewas demonstrated not only in la'mbda phage but in SV 40. Anotherfindings are the existence of small circular RF of SV 40 DN.A,. The5Yamamoto and Oda: Electron microscopic studies of replicating SV 40 DNAProduced by The Berkeley Electronic Press, 1971242 ~. YAMAMOTO and T.OOAcontour length of SV 40 DNA was reported to be 1.4 to I. 7 fl. (5, 10, II),while molecules smaller than 1.0 ,f}. were noted to replicate in our experi-ment. YOSHIIKE (II) reported that successive undiluted passage causedthe shortening of DNA approximately by 15 % from plaque formers andthe shorter DNA was thought to be defective virus DNA. We havedemonstrated RF of SV 40 DNA smaller than 1.0 p. (5, 8) and ascribedthe cause to the deletion of DNA. Small RF not only in e form (5, 8) butin (1 form observed in our experiment suggests that greatly deleted defec-tive SV 40 DNA are capable of replication as in case of plaque formers.SUMMARYElectron microscopic observation of replicating SV 40 DNA hasrevealed the existence of two types of RF, e form and (1 form. Thefrequency of RF at 54 hours after infection was 8.9% for the e form and4.3% for the (1 form. Morphological evidence exhibits that in (1 formRF the tails are, predominantly, shorter than the viral genome anddouble length SV-40 genomes are also capable of replication in SV-40infected VERO cells.ACKNOWLEDGEMENTSThis investigation was supported by a Grant-in-Aid for Scientific Reseach from theMinistry of Education of japan.REFERENCES1. KNIPPERS, R., WHALLEY, J. M. and SINSHEIMER, R. M.: The process of infection withbacteriophage flSx 174. XXX. Replication of double-stranded l'6x DNA. Proc. Natl. Acad.Sa. lJ. S. 64, 275, 19692. LEE, C. S. and DAVIDSON, S.: Physicochemical studies on the minicircular DNA in Esc-herichia coli 15. Biochim. Biophs. Acta 204, 285, 19703. INSELBURG,j. and FUKE, M.: Replicating DNA: ~tructure of colicin factor E 1. &ience169, 590, 19704. KIGER,jR,j. A. and SINSHEIMER, R. L.: DNA of veg(tative bacteriophage lambda, VI.Electron microscopic studies of replicating lambda DNA. Proc. Nat!. Acad. Sci. lJ. S.68, 112, 19715. YAMAMOTO, S., OMURA, S. and OOA, T.: Relationship between molecular length andbiological activity of SV 40 DNA. Acta Med. Okayama 24, 273, 19706. HIRT, B.: Selective extraction of polyoma DNA from infected mouse cell cultures. j.Mol. Bioi. 26, 365, 19677. KLEINSCHMIDT, A. K.. LANG, D., jACHERTS, D. and SAHN, R. E.: Darstellung und Lan-genmessungen des gesamten deoxyribonucleinsiiureinh~ltesvon T 2-bacteriophagen. Bio-chim, BioPhs. Acta 61! 153, 19626Acta Medica Okayama, Vol. 25 [1971], Iss. 4, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss4/2EM Studies on Replicating SV-40 DNA 2438. YAMAMOTO, S. and ODA, T.: Electron microscopic studies on replicating form of defectiveSV 40 virus DNA. (in preparation)9. LEVINE, A.]., KANG, H. S. and BILLHEIMER, R. M.: DNA replication in SV 40 infectedcells. 1. Analysis of replicating SV 40 DNA. j. Mol. Bioi. 50, 549, 197010. ANDERER, F. A., SCHLUMBERGER, H. D., KOCH, K. A., FRANK, H and EGGERS, H.].:Structure of simian virus 40. II. Symmetry and components of the virus particle.Virology 32, 511, 196711. YOSHIIKE, K.: Studies on DNA from low-density particles of SV 40. I. Heterogeneousdefective virions produced by successive undiluted passages. Virology 34, 391, 19687Yamamoto and Oda: Electron microscopic studies of replicating SV 40 DNAProduced by The Berkeley Electronic Press, 1971

Electron microscopic studies of replicating SV 40 DNA

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