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JOURNAL OF VIROLOGY, May 1977, p. 340-345

Copyright©1977 AmericanSocietyforMicrobiology Printed inU.S.A.

Mitochondrial DNA Synthesis

in Adenovirus Type 2-Infected

HeLa

Cells

PAUL B. FISHER' AND MARSHALL S. HORWITZ*

Departments ofMicrobiology-Immunology,*Cell Biology, and Pediatrics, Albert Einstein College of

Medicine,

Bronx,

NewYork 10461

Receivedforpublication17November1976

Mitochondrial DNA synthesis inadenovirus type 2-infected HeLa cellswas

measuredat various timesfrom0 to 24 hpostinfection. Althoughviral infection

effectively turned off host chromosomal DNA synthesis, mitochondrial DNA

synthesiswasnotinhibited. Thesefindingsindicateadissociation between the

regulation of host andmitochondrial DNA synthesis after infection with

adeno-virustype2.

Anasymmetrical model of bidirectional

rep-lication has beenproposed for adenovirus DNA

(5, 11, 12, 29, 31). This model of displacement

replication is similartomodels proposed for the

replication ofmitochondrial DNA (1, 17, 27).

Adenovirusandmitochondrial DNAsyntheses

have also been found to be similar in their

resistance to the effect of protein inhibitors (13,

30). Individual molecules ofadenovirus DNA

arebothinitiated and completely replicatedin

the presence of concentrations of cyclohexi-mide, which inhibit up to 97% of HeLa cell

proteinsynthesis (13).Similarly,mitochondrial

DNA synthesis in HeLa cells is within 75%

of normal for45minand approximately50%of

theratesofcontrols foranadditional3hafter

addition of cycloheximide (30). In contrast,

HeLa cell chromosomal DNA synthesis is

re-duced more than 90% within 10 min of the

addition of cycloheximide (13). This

dissocia-tionbetween protein synthesis and DNA

syn-thesisdemonstrated by both mitochondria and

adenovirus is notfound invarious other DNA

viruses infecting eucaryotic cells. Inhibition of

proteinsynthesisincells infected with polyoma

virus (3), simian virus 40 (SV40) (19), rabbit

poxvirus(18), orpseudorabiesvirus (16) results

inarapid cessation of viral DNA synthesis.

The effects of three DNA viruses, SV40 (21), polyoma (33), and herpes simplex type 1 (25,

26), onmitochondrial DNA synthesis have been

studied. SV40 infection of confluent African

green monkey kidney or mouse embryo 3T3

cultures results in a stimulation of both

nu-clearand mitochondrialDNAsynthesis; in

con-trast, SV40 infection of BSC-1 cells does not

stimulate nuclear or mitochondrial DNA

syn-1Presentaddress: Institute of Cancer Research, Colum-biaUniversity College of Physicians and Surgeons, New York,NY10032.

thesis (21). A stimulatory effect on

mitochon-drial DNA synthesis also results when

con-fluent 3T3 cultures are infectedwiththe DNA

tumor virus, polyoma (33). In type 1 herpes

simplex-infected HeLa cells, chromosomal

DNA synthesis is inhibited during 1 to 5 h

postinfection (p.i.), whereas mitochondrial

DNAsynthesis wasstimulated (26). Similarly,

aherpes-inducedenhancement of DNA

synthe-sis in isolated HeLa mitochondria has been

demonstrated (25).

Because of the similar mechanism of

dis-placement synthesis and resistance to protein

inhibitorsduring the replication of both

adeno-virusandmitochondrialDNA,wehave

investi-gatedthe effect of adenovirus type 2 (Ad2)

in-fection on mitochondrial DNA synthesis in

HeLacells.

MATERIALS AND METHODS

Cells and virus. Thesourcesof HeLa S3 cellsand

Ad2 have beenpreviously described(22). Cellswere grown in suspension culture with Eagle medium

supplementedwith 5%fetalcalfserum. Cellswere infected withpurifiedvirionat aninputmultiplicity

of4,000 particles/cell as previously described (10). Mock-infectedcells weresimilarly washedand con-centrated, butno virus wasadded.

Isolation of mitochondria. Mitochondria were isolated fromcells that were washed twiceinEagle

medium without serum and suspended in hypotonic bufferconsisting of 50mMNaCl,1.5mMMgCl2,and 10 mMTris-hydrochloride,pH8.0(9). Thecells (5x 106/ml) were ruptured by homogenization (16

strokes) with a Dounce homogenizer. Approxi-mately95% of the cells were disrupted, as monitored by phase-contrast microscopy. The cytoplasmic su-pernate was freed of nuclei and debris by three

centrifugationsat2,000 rpm for5mininthe inter-national PRJ centrifuge. Mitochondria were sedi-mented from the supernate by centrifugation at 10,000 rpm for30 min in the angle30 rotorof the 340

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tracting the aqueous phase twice with chloroform-isoamyl alcohol, as described by Schildkraut and Maio (28). The RNase step included in their proce-dure (28) was omitted because Hela cell mitochon-drial DNA containsa small number of ribonucleo-tides and is, therefore, nicked by RNase A andT, (34).

Characterization of mitochondrial DNA. Mito-chondrial DNApurifiedfromAd2-infected cultures ormock-infected HeLa cell controls wasanalyzedby sedimentation in neutral sucrose (12) and cesium

chloride-ethidium bromide (CsCl-EtBr) (24)

gra-dients. Foranalysis of DNA in neutral sucrose, a 0.5-mlsample ofpurified mitochondrial DNA was

layered on a 16.5-mlgradientcontaining 5 to 20% neutral sucrose in 1 M NaCl, 0.01 M phosphate buffer, and 0.01 M EDTA with a 0.5-ml cushion of 60%sucrose. Sampleswere centrifugedfor 18hat 24,000 rpm inthe SW27.3 rotor of the L350Beckman

ultracentrifuge. Gradients were fractionated by

pumping equal portions (approximately 0.6 ml)

throughaprobeplaced1.5 cmabove the bottom of the tube. Determination ofacid-precipitable radio-activity was performed by methods previously

re-ported(10). ForCsCl-EtBranalysisofpurified mito-chondrialDNA,a0.75-mlsamplewasaddedto3.2 g ofCsCl,0.2mlof EtBr solution (10mg/ml),41Lgof calf thymusDNA, and2.25 ml of0.Olx SSC. The refractive index was adjusted to 1.3888 (density,

1.580 g/cm8) with 0.01x SSC. Samples were then

centrifugedinaSpincoangle40rotor at36,000rpm for 40to48h,and0.1-mlfractionswerecollectedby

puncturing the bottom of the tube. Refractive in-dexes of every fifth samplewere determined, and

acid-precipitablematerialwasquantitated.

Band sedimentation of DNA on alkalinesucrose gradients. Whole cells wereplaced onalkaline su-crosegradientsandsedimentedat24,000rpmfor14 h withanSW27.3 rotor,aspreviously described (10).

Gradientswere fractionated, and the incorporated radioactivity in the fractions and the sonically

treatedpellet (2ml)wasquantitated. Under these

conditions,cell DNAwaspelletedintothecushion,

and viral DNAappearedinthegradient.

Radioactivelabelingof DNA.Marker HeLa

mito-chondrialDNAwaslabeled with['4C]thymidine by

growingcells for3daysinmediacontaining3,uCiof

[I4C]thymidine (54 mCi/mmol) per 100 ml.

Mito-chondrial DNA from mock- andAd2-infected cells wasisolated from1 x 108to1.5x 108cells grown for 4 h with 1.5 mCi of [3Hlthymidine (15 Ci/mmol)

addedattime0, 4, 8,12, 16,or20hp.i.Ad2-infected cells (1.25x 106),used forquantitationof viral DNA at various times, were labeled with 25 ,ACi of

[3H]thymidine (16Ci/mmol)for1hfollowedbya 30-minchase withunlabeledthymidine(10-sM).

and relaxed-circular (form II) mitochondrial

DNA, whichsedimentat37Sand 26S,

respec-tively (14), were separated from mock- and

Ad2-infected HeLacells by centrifugation ina

5 to 20%neutral sucrose gradient (Fig. 1). By

using 14C-labeled uninfected cells mixed with

3H-labeled mock- or Ad2-infected cells, it was

possibletoquantitatemitochondrial DNA

syn-thesis until 12h p.i. Total mitochondrial DNA

synthesis, monitoredfor 4-hperiods,wasfound

tobe similar in4-, 8-, and 12-h Ad2- or

mock-infected cultures (Fig. 1 and Table 1). Theuse

ofneutral sucrose gradients to monitor

mito-chondrialDNA synthesisbeyond 12 hp.i. was

not possible, because large amounts of Ad2

DNA(31S)contaminated the mitochondrial

re-gion of gradients. We and others have found

that most of theencapsidatedadenoviralDNA

leaksfrom the intact nucleiprepared by 0.5%

Nonidet P-40lysis of cells and isfound inthe

cytoplasm (M. S. Horwitz, personal

observa-tion; reference 4). Unencapsidated viral DNA

remainsinthenucleus. Since thereis

approxi-mately a 6-h lag between the onset of viral

DNA synthesis and its encapsidation into

virion, we were ableto use the sucrose

gradi-entmethod until12hp.i. The DNAinvirions,

whichcontaminated the cytoplasm, sedimented

with themitochondriaat10,000 xgfor30min.

Virion could notbe separated from

mitochon-dria by sedimentation through a 15 to30%

su-crosegradientat24,000 rpm for30min(Fisher,

personal observations). The inability to

subse-quently separate mitochondrial from

adeno-virus DNA on sucrose gradients necessitated

the useofCsCl-EtBrequilibrium gradientsto

separate mitochondrial DNA from adenovirus

DNA when the cells were infected for more

than 12h.

CsCI-EtBr gradient analysis of

mitochon-drial DNA synthesis. Form I mitochondrial

DNAwas, therefore,quantitatedincells either

mock or Ad2 infected from 4 to 24 h earlier

(Fig. 2and Table 1). Between0and16h,

mito-chondrial DNA synthesis in both mock- and

Ad2-infectedcultures wassimilar, whereas

be-tween 16 to 20 and 20 to 24 h, mitochondrial

DNA synthesis in mock-infected cells was

ap-proximately twice that found in Ad2-infected

cultures. This apparent decrease in

mitochon-4

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0 1

3-

2-0 10 20 30 0 10 20 30

FRACTION NUMBER

FIG. 1. Neutral sucrosegradientanalysis ofmitochondrial DNA synthesis in mock-infectedand Ad2-infectedHeLa cells. (A) Mitochondrial DNA wasisolatedfromamixtureof108mock-infectedHeLa cells labeled with[3H]thymidinebetween 0 and4hand 5 x 107[14C]thymidine-labeledHeLa cells(seetext).A 0.5-ml sampleofpurifiedDNAwas run on a5to20%neutralsucrosegradientfor18hat24,000rpm.Gradients werefractionated,andacid-precipitable radioactivity fromeachfractionwasdetermined. (B)Mitochondrial DNA wassimilarly isolatedfromamixtureof108Ad2-infectedcells labeled with[3HWthymidine between 0 and4 hand 5 x 107[14C]thymidine-labeled HeLa cells. Thepurified DNA was run on neutralsucrose

gradientsas describedabove.(Thepeak of formImitochondrial DNA is infraction 8,andformII isin

fraction13.Thepeak oftheAd2 DNAmarker(31S)sedimented infraction10.Sedimentationisfrom

right

to left in allgradients.)

80 A B 9

0101.61021

60 -30 -.9

jl.57

40-20

.-a550

10-~~~~

xT

5-4 4

3-3I

2 -2

0 10 20 30 40 0 10 20 30 40 FRACTION NUMBER

FIG. 2. CsCl-EtBrgradient analysis ofmitochondrial DNA synthesis in mock- andAd2-infectedHeLa cells.(A) Mitochondrial DNAwasisolatedfrom108[3Hthymidine-labeledmock-infectedHeLacells labeled for4-hperiods between 0 and24 hp.i.The cellsweremixed with5 x 107[14C]thymidine-labeledHeLacells

beforepurificationofthe mitochondria and the DNA. Thesampleswere runon4-mlCsCl-EtBrgradients for

48hat36,000 rpm.Onehundred-microliterfractionswerecollectedbypuncturing the bottomofthetube,and

acid-precipitable material was determined. Refractive indexes of everyfifth sample were measured. The

samplelabeledfrom0 to 4hp.i. is shown.(Thepeak of formImitochondrial DNA isinfraction10;other linear DNAs bandinthe upperfractions.) (B) Mitochondrial DNAwasisolatedfromAd2-infectedcells and processedidenticallyto(A). Thesample labeled from 0to 4hp.i.isshown,andthe resultsfromother time pointsaresummarizedinTable1.

342

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8 to 12 1.06 0.98

12 to 16 - 0.97

16 to 20 - 0.46c (0.93)

20 to 24 _ 0.49" (0.96)

aAtotal of 108to1.5 x 108mock-orAd2-infected

HeLa cells were labeled with 1.5 mCi of

[3H]thymidine(15Ci/mmol)for4-h periods.

bMitochondrial DNA synthesis was expressed as the ratio of [3H]thymidine-labeled mitochondrial DNA from adenovirus-infected cells divided by

[3H]thymidine incorporated into HeLa mitochon-drial DNAinuninfected cells. Both numbers were

corrected for losses during processingby addinga constant amount of ['4C]thymidine-labeled HeLa cells to each sample. The "4C-labeled mitochondrial

DNA served as a reference point for the recovery of

purified DNA. Only form I DNA (37S in neutral sucrose or1.595g/cm3inCsCl-EtBrgradients) was used for the calculations. Numbers reflected the average ratios ofAd2-infected to mock-infected cells from two experiments.

cThese numbers reflect the valuesobtainedwhen

the "control" wasmock-infected cells. However, by

16 hafter mock infection, the cell number had dou-bled inthe uninfected cell culture but was

essen-tiallyunchangedinAd2-infected cells. If the values wereeithercorrected for cell numberorcompared

withcells mockinfectedforlessthan 16 h, the val-uesshown inparentheseswereobtained.

drial DNA synthesis in the infected culture

appears toresult fromanincreasedcell number

inthe uninfected controls that had dividedby

16 to 20 h after mock infection. Due to the

feedingschedule ofourHeLacells,theytendto

becomepartially synchronized and divideover

a4-to5-hperiod. This results inanabrupt rise

incell number rather thanagradual doubling

over 18 to 24h (Table 1). Thus, the amountof

[3H]thymidine-labeled mitochondrialDNA

dur-inga4-hpulse increasesat 16hinuninfected

HeLa cells, but remains constant in

adenovi-rus-infectedcells.

Figure 3 compares the species of DNA

syn-thesized and associatedwiththemitochondrial

pelletinadenovirus-infectedandmock-infected

HeLa cells from 16 to 20h p.i. The

mitochon-drial DNAs from both cellsaresuperimposable

(fraction 12), but thereis ashift ofdensity in

thenonmitochondrial DNA atthetopsofthese

gradients. The densityof the DNAfrom

adeno-virus-infected cells (fraction 31) is 6 mg/cm3

greaterthan that ofuninfected cells.Thus,the

C.,

0

Ix

rI

100-

10-

5-I

1 10 20 30 40

FRACTION NUMBER

FIG. 3. CsCl-EtBrgradient analysis of mitochon-drial DNAsynthesisinHeLacellsinfected for16to 20h.Mitochondrial DNAwas isolatedfroma

mix-ture of 108 Ad2-infected HeLa cells labeled with

[3H]thymidine between 16 and 20 h p.i. andmixed

with 5 x 107[14C]thymidine-labeleduninfectedHeLa cells.Samples wereassayedonCsCl-EtBrgradients

asdescribedinFig.2and thetext.

greater density of native adenovirus DNA in

comparisontoHeLaDNAismaintainedinthe

presenceofethidium bromide.

Rates of viral, chromosomal, and

mito-chondrial DNA synthesis. Ad2 DNA can be

separatedfrom intact HeLa cell DNAas a

con-sequence of differences in the sizes of these

molecules (8). With alkaline sucrosegradients

(10), Ad2 DNA isfound inthegradient (34S),

and HeLa cell DNA is largely found in the

pellet (_77to 80S). Theeffect of Ad2 infection

on host chromosomal and mitochondrial DNA

is shown in Fig. 4. Host DNA synthesis was

markedly reduced by10hp.i. andcontinuedto

decline during the course of Ad2 infection,

whereas Ad2 DNAwasdetectedat6hp.i. and

was at a maximum rate at 10 to 14 h p.i. In contrast to thesechanges, mitochondrial DNA

synthesis remainedat aconstantrate

through-outthecourse(0to24h) of Ad2infection (Fig.4

and Table1).

DISCUSSION

Mitochondrial DNA replicationwas

investi-gated in adenovirus-infected cells because of

several unusual similaritiesin the

replication

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344

IN

LU

50

I-I "

\NVIRUS

,

HOST-2 6 10 14 18 22 24

TIME (hours)

FIG. 4. Mitochondrial, Ad2, and host chromo-somal DNA synthesis in Ad2-infected HeLa cells. Mitochondrial DNA synthesis was determined by

both neutralsucroseand CsCl-EtBrgradient

analy-sis between0 and 24 hp.i.asdescribed in thetext

and Table 1.Ad2 andtotalHeLa cell DNAsynthesis after infection withAd2wasdeterminedby alkaline sucrose gradient analysis. 1.25 X 106Ad2-infected

cellsatdifferent times p.i.werelabeled with 25 uCi [3H]thymidine for1 h andchased with cold thymi-dine (10-5 M) foranadditional 30min. Cells were

pelleted, suspended, and centrifugedasdescribedin thetext.One hundredpercentsynthesis for mitochon-drial DNA refers to the radioactivity of mitochon-drial DNA made in uninfected HeLa cells. The

amount of radioactivity in HeLa cell chromosomal DNA between4and 8 hp.i.wasthe 100%reference

point for both chromosomal and Ad2 DNA synthe-sized in theinfected cells.

of these DNAs. Recently it has been demon-strated thatsynthesis of the linear adenovirus DNA begins at or near the left end for one

strand andtheright end for thecomplementary strand. Replication proceeds in the 5' to 3' di-rectiononeach ofthese strands (12). This mode

ofreplication generates large regions of single-stranded DNA inadisplacement reactionvery

similar to the D-loop displacement shown for mitochondrial DNA (27). In addition, both of thesemammalian DNAsarepeculiarly

resist-ant to the effect of protein inhibitors, which rapidly shut down the synthesis ofmostother eucaryoticaswellasprocaryoticDNAs (13, 30).

The inhibition ofchromosomal DNA

synthe-sis inadenovirus-infected cells isnot well

un-derstood (23); however,it maybesecondaryto

theprofound shutoff of host protein synthesis.

Similar controls do not affect mitochondrial

DNAsynthesisinadenovirus-infected cells,

al-though mitochondrial DNA synthesis is

pre-sumablydependentonenzymessynthesizedon

cytoplasmic polyribosomes. Perhaps the

mito-chondrial DNA is resistant to shutoff because

each round ofreplication does not require the

synthesis of proteins de novo, or the effect of

adenovirus infection on chromosomal DNA

mayinvolvethe inhibition ofselectedproteins

andleaves theproduction of factors needed for

mitochondrial DNA synthesis intact. Although

adenovirus DNA issynthesized in the nucleus

and mitochondrial DNAinthecytoplasm, itis

possible that these two processes share some

replicativeenzymes. There is noevidence that

adenovirus induces the synthesis ofa newDNA

polymerase (2, 15). However, the virion does

code for its own DNA-binding protein (32).

Since adenovirus infection inhibitsnotonly the

synthesis ofchromosomal DNA but also that of

SV40 (13) or vaccinia DNA (6) in cells coin-fected with either of these viruses, the mainte-nance ofmitochondrial DNAsynthesisat

nor-mal levels in adenovirus-infected cells is also

unique inthisregard.

Inall of these experiments, the incorporation

of[3H]thymidineinto an appropriately

charac-terized chromosomal, mitochondrial, or viral

DNA isassumed to correlate with therate of

synthesis of that macromolecule. Since adeno-virus genes do not code for a viral thymidine

kinase (20) and thechange in levels of

thymi-dine kinase in exponentially growing

unin-fectedcells varies less than30%afterinfection

(7), there is probably a good correlation

be-tween [3H]thymidine incorporation and DNA

synthesis. Since chromosomal,

mitochondrial,

andviral DNAweredeterminedunder identical

conditions inthecell, anomalies oflabelingof

mitochondrial DNA could only arise ifit

ob-tained [3H]TTP fromapooluniquetothe

syn-thesis of that macromolecule. Aseparate

thy-midine kinase has beenreported for

mitochon-drial DNA, but its enzymatic activitydid not

change after adenovirus infection (20). Since

adenovirus-infected cells are notorious for the

artifactual translocation of macromolecules

uponcell fractionation (e.g., virion is isolated

inthe cytoplasm asmentioned above), we did

not attempt to measure intramitochondrial

pools of TTP after infection. It is furthermore

unlikely thatcompensatingartifacts oflabeling

inourexperimentswouldcombineto yield con-stantratesof[3H]thymidineincorporation into

mitochondrial formIDNA fora 24-hperiod.

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LITERATURE CITED

1. Berk, A. J., and D.A. Clayton. 1974. Mechanism of mitochondrial DNA replication in mouse L-cells: asynchronousreplication of strands, segregation of circulardaughtermolecules, aspects of topology and turnover of an initiation sequence. J. Mol. Biol. 86:801-824.

2. Bolden,A., J.Aucker, and A.Weissbach.1975. Synthe-sisofherpes simplexvirus, vaccinia virus,and adeno-virus DNA inisolatedHeLacell nuclei.I. Effect of viral-specificantisera and phosphonoacetic acid. J. Virol. 16:1584-1592.

3. Branton,P. E., W.P. Cheevers, and R. Sheinin. 1970. The effectofcyloheximideon DNA synthesis in cells productively infected with polyoma virus. Virology 42:979-922.

4. Edvardsson, B.,E.Everitt,H.Jornvall,L. Prage,and L.Philipson. 1976. Intermediatesin adenovirus as-sembly.J.Virol. 19:533-547.

5. Ellens,D.J., J. S.Sussenbach,and H.S. Janz. 1974. Studiesonthe mechanismofreplicationof adenovi-rus DNA. III. Electron microscopy of replicating DNA.Virology61:427-442.

6. Giorno, R., andJ. R.Kates.1971.Mechanismof inhibi-tion of vaccinia virusreplication in adenovirus-in-fectedHeLacells.J.Virol.7:208-213.

7. Green, M. 1962. Studies onthebiosynthesis ofviral DNA. Cold Spring HarborSymp. Quant. Biol. 27: 219-235.

8. Hodge, L. D., and M. D. Scharff.1969.Effect of adeno-virus on host cell DNA synthesis in synchronized cells.Virology37:554-564.

9. Horak,I.,H.G. Coon,and I.B.Dawid.1974. Interspe-cific recombinationofmitochondrialDNAmolecules inhybridsomaticcells.Proc.Natl.Acad.Sci. U.S.A. 71:1828-1832.

10. Horwitz,M.S. 1971.Intermediatesinthesynthesis of type 2 adenovirus deoxyribonucleic acid. J. Virol. 8:675-683.

11. Horwitz, M. S. 1974. Location of the origin ofDNA replicationin adenovirustype 2. J. Virol. 13:1046-1054.

12. Horwitz,M.S. 1976.Bidirectionalreplicationof adeno-virustype 2 DNA. J. Virol. 18:307-315.

13. Horwitz, M. S., C. Brayton, and S. G. Baum. 1973. Synthesis oftype 2adenovirusDNA inthepresence ofcycloheximide. J. Virol. 11:544-551.

14. Hudson, B., and J. Vinograd. 1969. Sedimentation velocity properties ofcomplex mitochondrial DNA. Nature(London)221:332-337.

15. Ito, K., M. Arens, andM.Green.1975.Isolationof DNA Polymerase-yfromanadenovirus2DNAreplication complex.J. Virol. 15:1507-1510.

16. Kaplan,A.S., andT. Ben-Porat.1966.Thereplication

of thedouble-stranded DNA of ananimalvirus dur-ing intracellularmultiplication. Int. Congr. Micro-biol.Symp.9:463-482.

17. Kasamatsu, H., D. L. Robberson, and J. Vinograd. 1971. A novel closed-circular mitochondrial DNA

ceptible cells. J. Virol. 3:25-32.

20. Kit,S., W. C. Leung, G. Jorgensen, D. Trkula, and D. R. Dubbs. 1974. Subcellular localization and proper-ties of thymidine kinase from adenovirus-infected cells. J. Gen. Virol. 24:281-292.

21. Levine, A. J. 1971. Induction of mitochondrial DNA synthesisin monkey cells infectedby simian virus 40 and (or)treatedwith calf serum. Proc. Natl. Acad. Sci.U.S.A.68:717-720.

22. Maizel, J. V., Jr., D. 0. White, and M. D. Scharff. 1968. The polypeptidesofadenovirus. I. Evidence for multi-pleprotein components in the virion and a compari-son oftypes 2, 7a, and 12. Virology 36:115-125. 23. Philipson, L., U. Pettersson, and U. Lindberg. 1975.

Molecular biologyofadenoviruses.In Virology mono-graphs, vol. 14.Springer-Verlag, New York. 24. Radloff,R., W. Bauer, and J. Vinograd. 1967. A

dye-buoyant-densitymethod forthedetection and isola-tion ofclosed circular duplexDNA: theclosedcircular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521.

25. Radsak,K., and M. Albring. 1974. Herpessimplex vi-rus-inducedenhancement of mitochondrial DNA syn-thesis inthe absence ofvirus replication. J. Gen. Virol.25:457-463.

26. Radsak,K.D.,and H. W. Freise. 1972.Stimulationof mitochondrial DNAsynthesisin HeLa cellsby her-pexsimplexvirus(1). LifeSci.11:717-724. 27. Robberson, D. L., andD. A.Clayton.1972.Replication

ofmitochondrial DNA in mouse L cells and their thymidine kinase-derivatives: displacement replica-tion on a covalently-closed circular template. Proc. Natl.Acad.Sci.U.S.A.69-.3810-3814.

28. Schildkraut, C.L.,and J. J. Maio.1969. Fractionsof HeLaDNAdifferingintheir content ofguanine and cytosine. J.Mol. Biol.46:305-312.

29. Schilling,R., B. Weingirtner, and E.-L.Winnacker. 1975.Adenovirustype2DNAreplication.II.Termini ofDNAreplication.J.Virol. 16:767-774.

30. Storrie, B., andG. Attardi. 1972. Expression ofthe mitochondrialgenome in HeLacells. Xm.Effectof selectiveinhibition ofcytoplasmicormitochondrial proteinsynthesisonmitochondrial nucleic acid syn-thesis. J.Mol. Biol. 71:177-199.

31. Tolun, A., andU. Pettersson. 1975.Terminationsites for adenovirus type 2 DNA replication. J. Virol. 16:759-766.

32. vanderVliet,P.C., A. J. Levine, M. J. Ensinger,and H.S.Ginberg.1975.ThermolabileDNAbinding pro-teinsfromcellsinfectedwith atemperature-sensitive mutantofadenovirus defectiveinviral DNA synthe-sis.J.Virol.15:348-354.

33. Vesco, C., and C. Basilico.1971.Inductionof mitochon-drialDNAsynthesisby polyomavirus.Nature (Lon-don)229:336-338.

34. Wong-Staal, F.,J.Mendelsohn,and M. Goulian.1973.

Ribonucleotides in closed circular mitochondrial DNAfrom HeLa cells. Biochem.Biophys.Res. Com-mun. 53:140-148.

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Figure

FIG.1.fractionDNAgradientsleftandinfectedlabeledmlwere Neutral sucrose gradient analysis of mitochondrial DNA synthesis in mock-infected and Ad2- HeLa cells
FIG. 3.20[3H]thymidinedrialcells.tureaswith described h. CsCl-EtBr gradient analysis of mitochon- DNA synthesis in HeLa cells infected for 16 to Mitochondrial DNA was isolated from a mix- of 108 Ad2-infected HeLa cells labeled with between 16 and 20 h p.i
FIG. 4.pelleted,pointDNAcells[3H]thymidinesisdineaftersomaldrialMitochondrialanddrialsizedbothsucroseamountthe Mitochondrial, Ad2, and host chromo- DNA synthesis in Ad2-infected HeLa cells

References

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Parameters with high values for S i are respon- sible for most of the output variance, and by knowing their true values it is possible to reduce output uncertainty at

development and poverty eradication are central to the SDGs, and also that sustainable development provides the context in which the Paris Agreement operates, it is critical that

The narrowing of the gender gap on corporate management boards has been addressed in debates on quotas as important for company performance (to ensure that companies access the

nome, any inhibition of the net synthesis of viral DNA in the productive cells should de- crease the amount of EBV DNA per cell. If we assume that the VCA-negative cells in the

In this paper, we provide a focused mapping review and synthesis of the extent to which vicarious trauma is considered within the published nursing research literature.. In