JOURNAL OFVIROLOGY, Nov. 1991, p. 5944-5951 Vol.65,No. 11 0022-538X/91/115944-08$02.00/0
Copyright © 1991, AmericanSocietyforMicrobiology
Regulated
Replication
of
an
Episomal
Simian Virus 40 Origin
Plasmid
in
COS7 Cells
THOMAS CHITTENDEN,t ALANFREY,:AND ARNOLD J. LEVINE*
Departmentof MolecularBiology, Princeton University, Princeton, New Jersey 08544-1014
Received 8April 1991/Accepted30July1991
Thereplicationofasimian virus40(SV40)origin-containing plasmid, pSLneo,instablytransfected COS7cells has beenstudied. pSLneo containstheSV40originofreplicationand encodes thepositiveselectable marker for
G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, itwas anticipatedthatextensiveplasmid replicationwouldprecludeisolation ofpermanentcell linescontaining pSLneo. However,significantnumbersof G418-resistantcolonies arose after transfection of COS7 cells withpSLneo.Cell lines establishedfromthesedrug-resistant colonies containedbetween 100 and1,000extrachromosomalpSLneo copies per cell.EpisomalplasmidDNA inpSLneo/COS7lineswasstablymaintained after 2 months ofcontinuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that
replicationof pSLneo closelyparalleledthatof cellularDNA. Onaverage,plasmidDNA did notreplicatemore than once during asingle cellgeneration period. Regulation ofpSLneoreplication appeared to benegatively
controlled by a cis-acting mechanism. Endogenouscopies ofepisomal pSLneoremained at a stable low copy numberduring thesimultaneous, high-levelreplication ofanewly transfected plasmid encodingSV40 largeT
antigen in the same cells. These results indicate that regulated replicationof an SV40 origin plasmidcan be
acquiredin a cell anddoesnotrequirethepresenceof additionalgeneticelements. The molecular mechanismby whichcellsenforce thisregulationonextrachromosomalSV40plasmidsremains to be defined.
Mammalian chromosomal DNA replication involves the coordinate functionofalargenumber ofindependent origins ofDNAsynthesis(8, 15, 16). Faithfulduplication ofthecell genomerequiresacellular control mechanismto ensurethat eachreplication origininitiates replication onlyonce during a single cellcycle. In the absence of such control, repeated initiations by a single replication origin would result in amplification ofadjacent DNA sequences. The problemof regulating multiple replication origins is compounded by the observationthatchromosomereplication generally proceeds by a distinct temporal and spatial program (3, 11, 26; reviewedinreference15). Areplication origin that functions early in the S phase must be prevented from reinitiating replicationduring the remainder of the S phase, despite the
continued presenceof all factorsnecessaryforinitiation. It has been suggested that reinitiation is prevented by a cis-acting negative control mechanism that distinguishes newly
replicated DNA from unreplicated DNA. However, at presentthere is only limited experimental evidence to sup-porttheexistence of suchamechanism (21, 23).
A majorobstacle to the study of replication control has been thedifficulty inisolating chromosomal origins of DNA replication from mammalian cells. The bovine
papillomavi-rus(BPV) and the Epstein-Barr virus (EBV) both replicate in
a fashion that closely parallels duplication ofcell chromo-somes. In most cases, BPV and EBV persistas multicopy
extrachromosomal plasmids in infectedcells,witheach viral
episome replicating onlyoncepercellgeneration period (1,
2). BPV and EBV rely upon the cellular DNA replication apparatus for their duplication.It is likely thattheseviruses
*Corresponding author.
tPresent address: Division ofNeoplastic Disease Mechanisms, Dana-Farber CancerInstitute, Boston, MA 02115.
tPresent address: Department of CellBiology, NewYork Uni-versityMedicalCenter, New York, NY 10016.
also exploit the cellular mechanisms that regulate DNA replication to ensure long-term, stable maintenance of their
owngenomes in infected cells. These features of BPV and EBV genome replication make these viruses attractive model systems for the study of replicationcontrol.
In contrast to BPV and EBV, simian virus 40 (SV40) bypasses cellularcontrols onreplication andexhibits uncon-trolled, high-level replication in permissive cells (27). Plas-mids containing the SV40 origin of replication are rapidly amplified when transfected into COS7 cells (5). These cells constitutivelyexpressSV40large T antigen (T-Ag), the only viral protein required for SV40 replication. SV40 plasmids undergo multiple rounds of duplication within one cell generation, usually accumulating to over 10,000 plasmid copies per cell just 48 h after transfection. High-level,
runawayreplication by SV40 plasmids is thoughttobe lethal
to cells (22, 24). Thus, very few COS7colonies are report-edly obtained if one selects for the presence of an SV40 origin plasmid ina stabletransfection assay (22, 28).
In amodel system described by Roberts and Weintraub, BPVgenetic elementsimposed aregulated mode of replica-tionuponSV40inacis-dominant fashion (22,23). Chimeric plasmidscontaining both the SV40 origin of replication and theBPV genomereplicated onlytolowlevels in COS7 cells when testedin transient replication assays. In these
experi-ments, suppression of SV40 replication wasdependentupon the presence ofspecific cis- and trans-acting BPV genetic elements. Furthermore, it was possible to establish stable COS7 linescontainingrelatively lowcopynumbers of extra-chromosomal BPV-SV40 hybrid plasmids. The BPV-SV40 plasmids in the stable COS7 lines replicated in acontrolled fashion,with each plasmid undergoing onlyoneduplication
percellcycleperiod (23). Introduction ofa secondplasmid, which contained an SV40 origin and expressed additional T-Ag, resulted in runaway replication of the newly trans-fected plasmid, while in the same cells the endogenous 5944
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BPV-SV40 hybrid plasmids remained at a constant, low copy number. This result indicated that all trans-acting factors necessary for runaway SV40 replication were present in these cells; thus, replication of BPV-SV40 composite plasmids must be negatively controlled by a cis-acting mech-anism. Replication control was suggested to be mediated through BPV genetic elements because SV40 plasmids lack-ing BPV sequences could not establish permanent COS lines.
The experiments presented here were originally under-taken todetermine whether EBV sequences could similarly control SV40 replication in EBV-SV40 hybrid plasmids transfectedinto COS cells. In these experiments, substantial numbers ofCOScolonies were obtained after stable trans-fection ofa control plasmid containing no EBV DNA se-quences,pSLneo. Thisplasmidharbors only an SV40 origin and a selectable marker gene (for G418 resistance). Trans-fection by pSLneo was predicted to be lethal, since it replicates in a runaway fashion in short-term assays. Sur-prisingly, G418-resistantCOS colonies arising after transfec-tion with pSLneocould be readily cloned to derive perma-nent cell lines. The pSLneo/COS lines stably maintained between 100 and 1,000 extrachromosomal pSLneo copies per cell, and the replication ofpSLneo DNA in these cells occurred in a regulated fashion (on average, once per cell generation period). Furthermore, pSLneo replication ap-peared to be negatively regulated by a cis-acting mechanism. These resultsindicate that controlled episomal replication of SV40-basedplasmids can be imposed bya cellular mecha-nismthatdoes not requirethe presence ofexogenous viral DNA sequences. Itremainspossiblethat processes control-ling pSLneo replication inthis system arerelatedtocellular mechanismsthatregulate chromosomal DNA replication.
MATERIALSANDMETHODS
Plasmid constructions. The construction of pSLneo was described previously (18). pSLneo, a 4.5-kb derivative of pSV2neo (25), containstheHindlll (nucleotide [nt] 5172)-to-PvuII (nt 272) SV40 region encompassing the origin of replication(nucleotide numbering accordingtothe systemof Tooze[27]).The SV40earlypromoterin thisplasmiddirects theexpression ofthebacterial
neomycin
phosphotransferase
gene (neo), which provides G418 resistance in mammalian cells (25). A control plasmid that is defective for SV40 replication, pSLori-, was constructed from pSLneo by introducingasmalldeletionwithin theSV40origin. pSLneo was digested with
Sfil,
whichcuts within the originpalin-drome(nt5234). The
resulting
3-bp overhangwas removed bysubsequenttreatmentwithT4DNApolymerase, and the plasmid was recircularized to create pSLori-.pSLori-doesnotdetectablyreplicateinCOS7 cells whentested ina transient assay. pSVLwas constructed
by
cloning
a7.4-kb EcoRI fragment ofbacteriophage
lambda DNA into the uniqueEcoRIsite withinpSLneo.
Theplasmid pSVTAg
(gift
fromT. Shenk's
laboratory)
containstheHpaII
(nt
346)-to-BamHI (nt 2533) SV40earlyregion,
including
theorigin
of replicationandT-Aggene,cloned intopML2
(aderivative of pBR322) (19).Transfection assays. COS7 cells
(5)
were grown in Dul-becco modifiedEagle
mediumsupplemented
with 10%fetal calfserum. Confluent 100-mm-diameterplates
of cells weretrypsinized,
and thecellswerereplated (1:10
dilution)
1day
before transfection.
Typically,
1,ugofplasmid DNA,
with 10jxg
ofsalmon sperm carrier DNA, was transfectedby
the calciumphosphate
coprecipitation
method(6).
Thecells
were
subjected
to aglycerol
shock3to4haftertransfection and refedwith fresh medium. For someexperiments,
plas-mid DNA
(without
carrier DNA) was transfectedby
the DEAE-dextran method (20). For transientreplication
as-says,cellswereharvested forpreparation
ofDNAfrom16to 68 h after transfection. Forisolation ofpermanentcelllines,
cellswere
split (1:10)
3days
aftercalciumphosphate
trans-fection into mediumcontaining
500 ,ug of G418 per ml.Drug-resistant
coloniesgenerally
aroseafter 10to 12days
in selective media. Individual G418-resistant COS7 colonies were isolatedapproximately
3 weeks after transfectionby
trypsinization
inglass cloning rings
and thenreplated
into 30-mm-diameter dishes. Cell cultures wereexpanded
into 100-mm-diameterplates
andsplit
1:5 or 1:10during
subse-quentpassaging.
Throughoutexpansion
andgrowth of G418-resistant celllines,
selection was maintained at 500 ,ug of G418 per ml.TotalDNAwasprepared
from transfectedcellsby
the method of Roberts and Weintraub(22).
In some cases, thelow-molecular-weight
DNAfraction wasisolatedby
themethodofHirt(12).
ForSouthern blotanalysis,
DNAwas fractionated on agarose gels, denatured, and electro-blotted onto
nylon
membranes.32P-labeled probes
wereprepared by
nick translation ofplasmid
DNA,resulting
inspecific
activities ofabout108 dpm/,lg
or greater.Hybrid-izationsandwasheswerecarriedoutasdescribed
by
Church andGilbert(4).
Density transfer analysis. Subconfluent 100-mm-diameter
plates
ofcells were incubated in mediumcontaining
50,iM
bromodeoxyuridine (BUdR)
for 24 or 88 h. Total cellular DNA was harvested and fractionatedby
CsCldensity
gra-dientcentrifugation
asdescribedby
Roberts andWeintraub(23).
Fractions(0.4
ml)
were collectedby
dripping
from the tube bottom. The refractive index of every other fraction was determined to monitor fraction densities. Fractions were desaltedby
passage over G-50spin
columns and transferredto anylon
membranewithaMinifoldII slot blot apparatus(Schleicher
&Schuell).
Theblotwasprobed
first with32P-labeled
pBR322
todetectplasmid
DNA. A controlsample
ofCOS7 DNA was included on the same blot to ensure that theprobe
was notbinding
nonspecifically
to chromosomal DNA sequences.Following
autoradiography,
the
pBR322
probe
wasstripped
from the blot withboiling
water.
Complete
removal of the firstprobe
wasconfirmedby
autoradiography.
Cellular DNA in the fractions wassubse-quently
detectedby
rehybridizing
the blot with32P-nick-translatedDNAfromCV-1 cells
(which
donotcontainSV40
DNA
sequences).
Therelativeamountsofplasmid
DNAand cellular DNA within eachfractionweremeasuredby
analy-sisof
appropriate
autoradiographs
withaBio-Rad model 620densitometer.
Flow
cytofluorimetry.
Cellswereprepared
forflow cytom-etryessentially
asdescribedby
Khochbinetal.(13). Fixed,
permeabilized
cells were incubated with aT-Ag-specific
monoclonal
antibody, pAb423 (10)
(undiluted
culturesuper-natants).
Foreachcell lineanalyzed,
ablank in which fixedcellswereincubatedinmedium
lacking
theprimary antibody
wasalso
prepared. Samples
werethenincubated ina1:1,000
dilution ofgoat
anti-immunoglobulin
conjugated
with fluo-resceinisothiocyanate
(Cappel Laboratory).
Fluoresceinisothiocyanate
fluorescence of treated cellswasquantitated
by
analysis
with a fluorescence-activated cell sorter. The meanfluorescence(log
meanchannelnumber)
of10,000
cells was measured for eachsample.
Specific
fluorescence(SF)
was defined as mean fluorescence ofthe cell line - mean
fluorescence ofthe
corresponding
blank. The fluorescenceofon November 10, 2019 by guest
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5946 CHITTENDEN ET AL.
14days 25days
pSLneo
pSLorI-pRSVneo
FIG. 1. Production of G418-resistant COS7 cell colonies by
transfectionwithSV40-basedplasmids.COS7cellsweretransfected
with 1,ug of the indicated plasmid DNA and grown in medium
containing500 p.gof G418perml. Plates werestained with Giemsa
stainafter14 days(left) or25 days(right)in selective medium.
acell line relative to COS7 cellswas calculated as follows:
relative fluorescence =1o(SFof cellline/SF of COS7cells) - 1
RESULTS
Establishmentofstable COS7 lines containing
extrachromo-somal pSLneoDNA. The original goal of these experiments
was to determine whether DNA sequences from the EBV
origin of replication could act to negatively controlSV40 replication in EBV-SV40 chimeric plasmids. By analogy to
BPV, the presence of certain EBV sequences on an SV40-neo plasmid might permit isolation of permanently
G418-resistant COS7 colonies dueto the suppression ofrunaway
SV40 replication. As a control for these experiments, an
SV40-based plasmid lacking any EBV sequences, pSLneo
(18),wasalso tested.pSLneocontainsaportionof theSV40
early region encompassing the origin of replication, a gene
encoding G418 resistance (neo) expressed from the SV40 early promoter, and bacterial plasmid sequences derived
frompML2(19).ItwasanticipatedthatpSLneowould yield
few, if any, G418-resistant COS7 colonies, because its
overreplication would be lethal to cells. However, pSLneo
consistently produced unexpectedly high numbers of
G418-resistantCOS7colonies(Fig. 1),withanestimated
transfec-tion efficiencyof 0.001%. These pSLneo colonieswere not
abortive in the sense that they continued to grow after 3
weeks ofG418 selection (Fig. 1) and could be established
intocelllines withhigh efficiency(>50%). Furthermore, the
numbers ofG418-resistant colonies obtained with pSLneo
were only slightlyreduced(two-tothreefold lower) relative
to those obtained with plasmids that lacked a functional
SV40 origin (pSLori- and pRSVneo [Fig. 1]) and require
integration of their DNA for long-term maintenance. pSLori- is identical in structure topSLneo, except that the functional
SV40
origin has been eliminated by a 3-bp dele-tion within the origin palindrome. pRSVneo completely lacksSV40origin sequences and expresses G418 resistance from the Rous sarcoma virus long terminal repeat. The capacity of pSLneo to produce colonynumbers comparable to those produced bypSLori- suggestedthat thereplication of pSLneo was notsubstantially toxic to theCOS7
cells that produced these colonies.Multiple independent G418-resistant colonies werecloned and expanded in culture to establish cell lines. To examine the state of thetransfected pSLneoDNA inthese cell
lines,
low-molecular-weight DNA was prepared by the method of Hirt (12) and analyzed by
Southern
blotting (Fig. 2). Inde-pendent pSLneo transfectedCOS7
lines (termedSLi,
SL3, and SL10, etc.) contained between 100 and 1,000 extrachro-mosomal copies of pSLneo per cell. Extrachromosomal DNA was not detected in four G418-resistant lines estab-lished after transfection with a plasmid lacking a functional SV40 origin (i.e., pRSVneo [Fig. 2]). Much of the pSLneo DNA persisted as form I closed circular DNA. Higher-molecular-weight species were also present in most clones and appeared to be multimers or concatemers of pSLneo. Digestion of the Hirt extracts withBamHI,
which cuts once within pSLneo, reduced the higher-molecular-weight mate-rial to a single species of unit length (Fig. 2).Southern
blot analysis of high-molecular-weight DNA confirmed that the pSLneo DNA was predominantly episomal in these cell lines (data not shown), although the possibility of a small number of integrated pSLneo copies could not beexcluded. Similar copy numbers (500 to 1,000 per cell) and higher-molecular-weight species were observed in cell lines containing BPV-SV40 chimeric plasmids (23).To assess the stability of pSLneo episomes, one pSLneo/ COS7 cell line,
SLi
(containing approximately 400 pSLneo copies per cell), was passaged extensively either in the presence or in the absence of G418. The copy number of the pSLneo plasmid DNA in the cultures was monitored by Southern blot analysis of Hirt extracts (Fig. 3). In the presence of G418, very little of the pSLneo DNA was lost from the culture. After 2 months of continuous culture in G418 medium, approximately 200 copies of pSLneo per cell remained (P17 [Fig.3]).
These results indicate that pSLneo was capable of stable extrachromosomal maintenance in COS7cells. When the cells were passaged in the absence of G418 selection, the plasmid DNA was eventually (4 to 6 weeks) lost from the culture (Fig. 3). The instability of pSLneo in the absence of selection is similar to the observed rate of loss ofBPV-SV40 plasmids under equivalent condi-tions (23).Regulated replication of pSLneo. It was possible that pSLneo/COS7 cell lines were heterogeneous cultures wherein runaway plasmid replication within a fraction of the cells provided resistance to the remaining population (which
lacks the plasmid). It has been reported that this situation
accounts for cell lines that maintain extrachromosomalSV40 plasmids harboring a
GPIT-based
selectable marker (23, 28). Alternatively, pSLneo replication might proceed in a regu-lated fashion analogous to BPV-SV40 plasmids in stable COS7lines, wherein the timing of plasmid replication closely paralleled cellular DNA replication. Density transfer exper-iments were performed to examine the replication of pSLneo DNA in stably transfectedCOS7 cells. G418-resistantCOS7 colonies arising after transfection with pSLneo were pooledand expanded in culture. Subconfluent plates of
cells
were J. VIROL.on November 10, 2019 by guest
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[image:3.612.80.285.74.351.2]BamHI Uncut
Copies/cell
-., I
0
a) 8
c 0
°o 0 0 > ° CO
o $ J J j 4J J4 CEn
n uC co to o co Co a :Co
o) - M It Ul)
C.I)
-i -J -j -J -j -J
Co co co Co co co
5a
U,
W
40* ,- do 4m b 4tqw
4.A -0. I
FIG. 2. Southernblot analysis of low-molecular-weight
DNA
fromG418-resistant pSLneo/COS7 cell lines. DNA was prepared from seven independent G418-resistant clones (SL1, SL3, SL10, SL11, SL12, SL14,and SL15)isolated after stable transfection ofCOS7 cells with pSLneo. OnepRSVneo/COS7clone(RSVneo)was alsoanalyzed. DNA prepared from106cells was fractionated on an agarose gel either uncut(right)orafter digestion withBamHI(middle). DNAwas then blotted and probed with32P-labeledpBR322 to detect plasmidsequences. Copy number controls (left)were madeby mixingappropriate quantities ofBamHI-digestedpSLneo with the Hirt extract from106COS7 cells. Thearrowindicatesthe position ofform I circular plasmid DNA.then incubated in media containing BUdR for either 24 h (less than the estimatedcell generation time of 36 h) or 88 h. TotalDNAwaspreparedfromthecells andfractionated on CsCl density gradients, and the amount of pSLneo or cellular DNA in eachfraction was determined by slot blot analysis (Fig. 4). Thisanalysis revealed that the replication ofpSLneo DNA closely paralleled the replication of chro-mosomal DNA sequences. Twenty-four hours after the addition ofBUdR,significantamountsofpSLneo DNA were present only in the heavy-light hybrid DNA fractions and were not detected in the heavy-heavy (both DNA strands substituted with BUdR) portion ofthegradient (Fig. 4,top), indicatingthatpSLneo plasmidsdid not on averagereplicate more than once during this period. Only after cells were incubated inBUdR for times greaterthan onecell division (88
h)
didwedetect substantialamountsofpSLneo
DNAin the heavy-heavy density fractions (Fig. 4, bottom). Similar results were obtained after analysis of a cloned pSLneo/ COS7 cell line (SL1) (datanotshown). While thepossibility of low levelsof uncontrolled pSLneo replication cannotbe totally excluded, these results indicate thatthemajority
of pSLneoplasmids in stable COS7 linesreplicated in a regu-latedfashionandclosely followedcellular DNAduplication. Furthermore, these results suggest that there are very few"jackpot"
cells within the culture thatspontaneously
un-dergouncontrolled pSLneo replication.cis-acting negative control ofpSLneo replication.
pSLneo
replicatestohighlevels(10,000copiespercell)inatransient replication assay of COS7 cells (see
Fig.
6). However, in stablepSLneo/COS7
celllines, pSLneo
is maintained at a lowercopy number (100 to1,000percell)
andreplicates
inacontrolled fashion. To examine the nature ofthis apparent suppression ofpSLneo
replication
in stablepSLneo/COS7
lines, we performed transfection assays with two different
test
plasmids.
The firstplasmid,
pSVL,
is identical to+G418 -G418
---_
0
M co P3 P8 P13 P17 P4 P7 P10 P12
[image:4.612.157.469.75.297.2];
tFIG. 3. Stability of extrachromosomal pSLneo DNA in C057 cells.ApSLneo/C0S7cellline,SLI-, waspassagedin thepresence (+G418)orabsence(-G418)ofdrugselection.Typically,thecells
werepassagedatconfluency(trypsinizedandsplit1:10or1:5)every 4 days. Seventeen passages (P17) corresponds to 2 months in continuous culture. Cellswere harvested at theindicated passage number for isolation of low-molecular-weight DNA. DNA
corre-sponding to 106 cells was analyzed by Southern blotting. Plasmid DNAwasdetectedbyhybridizationwith12P-labeledpBR322. Lane Mcontains 50 pg ofuncutpSLneoDNAtoserve as amarker. The
arrowindicates thepositionof form I pSLneoDNA.
lf:-w
i-4ow
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[image:4.612.339.537.393.626.2]5948 CHITTENDEN ET AL.
COS7
C)c cL
pSVL pSVTAg pSVL
SL1
pSVTAg
16 36 68 16 36 68 16 36 68 16 36 68
4 4 .~4.t.,. ;
_
h.
A
C
88hrspostBUDR
25
20
I.-0
I-5' 15
-10'
5'-0 5 1 0 15 20 25 30 FRACTION NO.
FIG. 4. Comparison ofpSLneoDNAandcellularDNA
replica-tioninpSLneo/COS7 cells by density transferanalysis.Acultureof pooled G418-resistant COS7 colonies derived by transfection of pSLneo was incubated in BUdR medium for 24 h (top) or 88 h (bottom). Total cellular DNA was harvested and fractionated on
CsClgradients.Aportion of each fractionwasanalyzed byslotfilter hybridization. The filterwasfirstprobedwith32P-labeledpBR322, andtherelativeamount(percentageof totalsignal forallfractions) ofpSLneoDNAin each fractionwasquantitated by densitometry of the resulting autoradiograph. The filter was stripped of the first probe andrehybridized with32P-labeledCV-1DNAto measurethe relative amount of cellular DNA in each fraction by the same procedure. Also shownarethe fraction densitiesand theregions of the CsCl gradient corresponding to unsubstituted (LL), single-strandedBUdR-substituted (HL), anddouble-stranded BUdR-sub-stituted (HH)DNA.
pSLneo except that it contains an additional fragment of lambda DNA to make it distinguishable in size from pSLneo. The second plasmid, pSVTAg, contains the SV40 origin of replication as well as the T-Ag-coding sequences. Replica-tion ofthese plasmids in COS7 cells and SL1 cells, which containapproximately 400 copies of episomal pSLneo, was tested. TotalDNA was isolated from the cells 16, 36, or 68 h after transfection of plasmid DNA, digested with DpnI to eliminate unreplicated DNA, and analyzed by Southern blotting (Fig. 5). pSVL replicated to high levels in COS7 cells(Fig.5,arrowA); however, very little pSVL replication
[image:5.612.57.303.72.422.2]wasdetected in SL1 cells. This suggested that a trans-acting factor necessary for SV40 replication was limiting in SLI cells. Thelimiting factorwasT-Ag because pSVTAg, which
FIG. 5. Replication of pSVL and pSVTAg in pSLneo/COS7 cells.pSVL andpSVTAgwere transfected into eitherCOS7 cells
(left)orSL1cells (right), apSLneo/COS7cell line. Bothplasmids contain the SV40origin ofreplication; pSVTAgalso encodes the
T-Ag gene.Total DNA washarvested16, 36,or68hafter
transfec-tion. In eachcase, 2p.gofrecoveredDNA wasdigestedwithDpnI
to eliminateunreplicated DNA,Southernblotted,andhybridizedto 32P-labeled pBR322.Arrows indicate thepositionsof formIplasmid DNA forpSVL (A),pSVTAg (B),andtheendogenouscopies(about 400per cell) ofpSLneowithinSL1cells (C).To ensurethatDpnI
eliminated allunreplicatedplasmid DNA,1ngofpSLneowasmixed with 2,ugofCOS7DNA(DpnICTL)and treated inparallelwiththe othersamples.
encodes additional T-Ag, replicated to high levels in SLi cells(Fig. 5,arrowB).
Significantly,
the copy number of the endogenous pSLneo plasmids in SLi cells (arrow C) was unaffected by the transfection andoverreplication
of the T-Ag-encoding pSVTAg plasmid. This result suggests that insufficient T-Aglevels were notsolelyresponsible
for limit-ing the replication ofpSLneo in SLi cells. Since pSVTAg overreplicated in SL1 cells, all trans-acting factors neces-saryforrunawayreplication
of SV40originplasmids
mustbe present. Therefore, maintenance of the resident pSLneo plasmids at a low copy number under these conditions indicates that pSLneo replicationin SLi cells is negatively controlled by acis-acting
mechanism. Similar results werereported for COS7 lines containing BPV-SV40 composite plasmids (23). Newly transfected plasmids that expressed additional T-Ag overreplicated, while the copy number of the endogenous BPV-SV40 plasmids remained unaffected. The resultspresented here indicatethat cis-actingnegative controlofSV40replicationcan alsooccurin the absenceof BPV orEBVgeneticelements.
ThissuppressionofpSLneo replicationcould beexplained by mutation of the pSLneo DNA sequences (for example, within the SV40 origin of replication) to make up a less active substrateforthereplicationmachinery.This explana-tionpredictsthatplasmidDNAisolatedfrompSLneo/COS7 cell lines will be defective for high-level replication when retransfected into COS7 cells. To examinethispossibility,a
24hrs
density g/mi
HH HL LL [1.9
,1.8
II F
IR:1.
~
Q.
J. VIROL.
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[image:5.612.318.555.74.316.2]pSLneo pSLRi pSLR2
-E
I-10 1 0.5 24 48 2448 24480
FIG. 6. Transient replication analysis of pSLneo plasmids res-cuedfrompSLneo/COS7 cells. Two plasmids, pSLR1andpSLR2, wererescued inE. colifromtheHirtextract of apSLneo/COS7cell line (SL1) and subsequently transfected into COS7 cells. For comparison, the parentalplasmid, pSLneo, was tested in parallel. One microgram ofplasmid DNA wastransfectedin eachcase. Total DNAwasharvested24 or48 h aftertransfection,digested withDpnI
andBamHI, Southernblotted,andprobedwith32P-labeledpBR322 todetectplasmidsequences. Copy number controls (left) contained the indicated amounts of BamHI-digested pSLneo DNA. One nanogram corresponds to approximately 5,000 copies per cell, assuming a 10% efficiency of transfection. One nanogram of pSLneo, mixedwith 2 ,ugofCOS7 DNA, was treated in parallel with theothersamples to ensure thatDpnIcompletely digested unrepli-catedDNA(DpnICTL).
number of plasmids present in SLi cells were isolated by transformingcompetentEscherichiacoli(7) with Hirt DNA preparations. Plasmids rescued from SLi cells were identi-cal to pSLneo by a numberofcriteria. First, the plasmids producedthe same pattern ofrestrictionenzyme-generated fragments as pSLneo (data not shown). Second, the SV40 origin DNA sequences from two rescued plasmids were determinedandfoundtobeidenticaltopSLneo. Inaddition, all sequences wereidenticaltothepublishedsequenceofthe wild-type PvuII (nt 272)-to-HindIlI (nt 5172) SV40 origin region. Third,the rescuedplasmids (pSLR1andpSLR2)had the same capacity for overreplication in COS7 cells as pSLneowhentested inatransientreplicationassay
(Fig.
6). From these results, it maybe concluded that thecis-acting
negative control of pSLneo replication does not involve genetic changesin the plasmidstructure.
Modifications suchas DNA methylation could be
impor-tantfor suppression ofpSLneo replication. However, such modifications ofplasmid DNA would not be maintained in theexperiment describedabove, inwhichpSLneo plasmids were first rescued and then replicated in E. coli. This possibility has been tested by
monitoring
thereplication
capacity of extrachromosomal plasmid DNA
purified
di-rectly frompSLneo/COS7cells(withoutpriorisolation inE.CTL SLl SL23
18 48 18 48 18 48 hours
*
w
FIG. 7. Transientreplication analysis of pSLneo DNApurified from pSLneo/COS7cell lines.COS7 cells were transfected bythe DEAE-dextran method with low-molecular-weight DNA prepara-tions from two pSLneo/COS7 cell lines (SL1 and SL23) or with pSLneo DNA purified fromE. coli(CTL). DNAcorrespondingto approximately 5 ng of pSLneo was transfected in each case. The low-molecular-weightDNA extract fromabout 5 x 106COS7cells was included as carrier DNA in the CTL transfection. Low-molecular-weightDNA was thenisolated 18 or 48h after transfec-tion, digested with BamHI (single site within pSLneo),andSouthern blotted. Plasmid sequences were detected by hybridization with
32P-labeledpBR322.
coli). Low-molecular-weight DNA was prepared from two
independent pSLneo/COS7 cell lines (SL1 and SL23). An initialSouthernblot wasperformed to quantitate the amount of episomal pSLneo presentin each Hirt DNApreparation (data not shown). Portions of the low-molecular-weight
DNA samples corresponding to approximately 5 ng of
pSLneoDNA were thentransfected intoCOS7 cellsby the
DEAE-dextran method (this transfection procedure
ap-peared to be more efficient than the calcium phosphate
method withsmallamounts ofDNA). Forcomparison, 5 ng ofpSLneo purified fromE. coliwasalso transfected.
Low-molecular-weight DNA was isolated 18 and 48 h after transfection, and replication of pSLneo was monitored by Southern blot analysis (Fig. 7). pSLneo DNA from two
pSLneo/COS7 celllines (Fig. 7, SL1 andSL23) showedthe samecapacityforreplicationaspSLneoDNApurifiedfrom E. coli(Fig. 7, CTL).Thisresultsuggestedthatsuppression
ofpSLneo replicationin stable pSLneo/COS7 lines did not
involve DNA modifications (such as methylation) that are
maintained uponpurificationoflow-molecular-weightDNA.
Analysis ofT-AginpSLneo/COS7cell lines. The
transfec-tion data described abovestrongly suggestthatsuppression
ofrunaway pSLneoreplicationoccursbyacis-acting mech-anism. Ifnegativecontrol ofpSLneo replicationismediated
in cis, then all trans-acting factors required for high-level SV40 replication should remain intact in pSLneo/COS7 cells. The inability ofpSVL to replicate in SL1 cells (see above)suggestedthatT-Aginthese cells wasnotavailable forreplicationofnewlytransfectedplasmids.In order to test thecapacityofT-Agproduced by SLicells tosupportSV40 replication,SLicellswerepassagedfor several weeks in the
absence of G418. During this time, pSLneo plasmids were
lost from the culture (Fig. 3), permitting isolation of
SL1-derived cell lines that had been cured ofplasmid DNA. The ng
on November 10, 2019 by guest
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[image:6.612.79.271.72.333.2] [image:6.612.349.516.74.252.2]5950 CHITTENDEN ET AL.
TABLE 1. Quantitation of T-Ag levels inpSLneo/COS7 celllines
by flowcytofluorimetrya
Cell lineb fluorescencecSpecific fluorescencedRelative
COS7 92.0 1.00
SL1 65.1 0.51
SL3 67.7 0.54
SL21 68.1 0.55
SL22 69.0 0.56
SL1-p, clone 1 87.6 0.89
SL1-p, clone2 92.8 1.02
a Fixed, permeabilized cells were incubated sequentially in anti-T-Ag
specific monoclonalantibodyand fluoresceinisothiocyanate-conjugatedgoat anti-mouse immunoglobulin and analyzed as described in Materials and Methods.
b SL1, SL3, SL21, and SL22 are COS7 celllinescontaining100 to1,000 episomal copies of pSLneo per cell.SL1-p clone 1and SL1-p clone2 are
independent clones isolated afterSL1cellswerecured ofpSLneoDNAby
passageinthe absenceof G418.
cSpecific fluorescence represents themeanfluorescence(logmeanchannel number)of the sample after adjustment fornonspecificfluorescence.
dRelative fluorescence referstothespecificfluorescencecomparedwith
thatofCOS7 cells(defined as 1.00) (see Materials and Methods formethodof
calculation).
cured SLi cells (SL1-p) were
equivalent
to COS7 cells in their capacity to support high-level replication (5,000 to10,000 copies per cell) of transfected
SV40
plasmids in a transient assay (data not shown). Since SL1-p cells were derived fromSL1 cells,this
resultindicatesthat T-Ag in the parental SL1 cell line was not genetically defective for high-level SV40plasmid replication. Had there been a heri-tabledefect inT-Ag orothertrans-actingcomponentsofthe replication machinery in SL1 cells, SL1-p cells would nothave supported overreplication ofSV40plasmids.
T-Ag in pSLneo/COS7 cell lines was also examined by biochemical methods. Immunofluorescenceexperiments us-ing the T-Ag monoclonal antibody pAb423 demonstrated
nuclear
localizationofT-Agin pSLneo/COS7cell lines (not shown). T-Ag levels inanumber of cell lineswere quanti-tatedby flowcytometry(results summarizedin Table1). The fourpSLneo/COS7 linestested(SL1, SL3, SL21, andSL22) contained40 to50%less T-Ag than theparentalCOS7cells. There is some heterogeneity of T-Ag levels within COS7 cells; thus, it is possible that COS7 cells expressing less T-Ag were selected for during isolation of pSLneo/COS7 lines. However, when plasmid DNA was eliminated from SL1 cellsby passaging in the absenceofG418 (as in Fig. 3), theamountofT-Ag returned toCOS7 levels (SL1-p [Table 1]). This finding suggests that SL1 cells do not have a heritablereduction in T-Ag expression. Instead, diminished T-Aglevels in SL1 cells mightreflect altered T-Ag metabo-lism(i.e.,reduced half-life) due to the involvement of T-Ag in replication of plasmid DNA within these cells. In either case,whilelower T-Ag levels might be important for limiting theextentofpSLneo replication, such reductions do not by themselves provide a mechanism for the observed cis-acting suppressionof pSLneo replication.DISCUSSION
The experiments presented here demonstrated that an SV40-based plasmid, pSLneo, can replicate in a regulated fashion in stablytransfected COS7 cells. The properties of pSLneo replication in these cells were similar to those reported for BPV-SV40 composite plasmids in several re-spects. First, pSLneoDNAwas stably maintained at 100 to
1,000 extrachromosomal
copies
per cell under continuous G418 selection but was lost from the culture after 4 to 6 weeks in the absence ofdrug
selection.Second,
pSLneo
replication
paralleled
cellularDNAreplication.
Onaverage, pSLneo DNA did notundergo
more than one round ofduplication
in asingle
cellcycle.
Third, suppression
ofhigh-level pSLneo
replication appeared
to be enforcedthrough
acis-acting
mechanism. SincepSLneo
lacks any BPV or EBV sequences, additional viralgenetic
elementsare
apparently
not necessary to establishnegative
control overreplication
ofanSV40-based
plasmid
in atleast someportion
ofthe COS7 cells inculture.These
findings
suggest thatsuppression
ofSV40plasmid
replication
ismediatedby
acellularmechanism whichisnotabsolutely
dependent
on the presence ofspecific
negative
controlDNAsequences incis.
Indeed,
the SV40genome is predicted to lack such sequences, since the virus has evolved toreplicate
predominantly
inanuncontrolled fash-ion. It is also unlikelythat pSLneofortuitously
contains aDNA sequence that limits
replication,
sincepSLneo
over-replicates
when tested in atransient assay. There is prece-dence forreplication
controlexhibiting
anindependence
of DNA sequence. Avariety
ofplasmid
DNAs,
regardless
of theirstructures,replicated
in acellcycle-controlled
fashion(only
oncepercellcycle)
when microinjected intoXenopus
laevis eggs
(9).
In some cases, initiation and control ofplasmid
DNAreplication
in human cells occurs with little apparentrequirement
forspecific
DNA sequence elements (14).Suppression
ofoverreplication
ofpSLneo
plasmids
in stableCOS7linesappearsto actin cis.Supertransfection
ofpSLneo/COS7
cells withaSV40plasmid encoding
additionalT-Ag
resulted in uncheckedreplication
ofthenewly
trans-fectedplasmid,
while the copy number of the residentpSLneo
episomes
remained constant.Experiments
wereperformed
to assess the nature ofthesuppression
mecha-nism.cis-acting
control didnotinvolve mutation ofpSLneo
DNA.PlasmidDNAthat
persisted
inCOS7cellswaslargely
identical in structure towild-type pSLneo
anddisplayed
equivalent
potential
forhigh-level
replication
when rescued in E. coliand reintroduced into COS7 cells. Plasmid DNApurified
directly
from Hirt extracts ofpSLneo/COS7
cells exhibitedthecapacity
for runawayreplication
upon retrans-fection into COS7 cells.Thesedatasuggestthatsuppression
does not involve
simple
covalent modification ofplasmid
DNA,
such asmethylation.
Theseexperiments
do notruleout
possible
alterationsimparted by
the cell which are notretained
during
the isolation ofplasmid
DNA, such ascompartmentalization
ofplasmid
DNA or association with cellularproteins. Conceivably, replication
may berepressed
by
anorigin-binding
cellularprotein
orby
anepigenetic
mechanism
involving
the stable association of chromatinproteins
with pSLneo DNA.The mechanism that dictatesregulatedreplicationappears
to be heritable to
daughter
plasmid
molecules. Our results suggestthatoncereplication
controlhas beenestablished,
it is maintainedthroughfuture cellgenerations. Density trans-ferexperiments
indicated that very few cells in a culture escapenegative
control andoverreplicate pSLneo. Further-more,relatively
lowpSLneo
copy numbers weremaintained overmany celldivisionsandwereunaffectedby introduction ofaT-Ag-expressing plasmid.
Heritability appears to reside inpSLneo episomes
andnotthecellularenvironment,sincepSLneo/COS7
cells that had been cured ofpSLneo DNAsupported
runaway SV40plasmid
replication.An apparent self-contradiction posed by these findings is J. VIROL.
on November 10, 2019 by guest
http://jvi.asm.org/
that one plasmid, pSLneo, replicated in two different modes: runaway (in transient assays) and controlled (in stable cell lines). One explanation for this might be the operation ofan inefficient (or slow) process for the imposition ofcontrolled replication upon pSLneo. In a transient replicationassay, a fraction of transfected pSLneo DNA may, in fact, replicate in acontrolledfashion. This phenomenon wouldbemasked in a short-term assay by high-level replication of other
plasmid molecules that have escaped replication control. In
contrast, in a stable transfection experiment, cellsthathave managed to control pSLneo replication may be selectedfor because their growth is not impaired by overreplication of
plasmid DNA. The presenceof BPVgeneticelementsincis, while not required, would then function to substantially enhance the efficiency (or rapidity) with whichSV40 plasmid replication is brought under negativecontrol.This is consis-tent with the observations thatBPV-SV40 plasmids exhibit detectable reductions in replication in atransientassayand give rise to G418-resistant COS7 colonies with increased frequency (22).
There are no clear reasons at this timewhypSLneois able to establish stable G418-resistant cell lines, while others have reported that SV40-neo-based plasmids produce only abortive drug-resistant COS7 colonies (22). In the studies reported here, about five times more plasmid DNA (1 ,ug versus 200 ng) was employed in stable transfection assays than in other studies (22), which may also havecontributed to the ability to isolate stable pSLneo/COS7 lines. Conceiv-ably, differences in the plasmidconstructions used may be important. However, the capacity to produce stable G418-resistant COS7 cells is not unique to pSLneo, since trans-fection of pSV2neoalsoyieldedstable COS7 linescontaining episomal plasmid DNA(data not shown).
SV40 DNA replication provides a particularly attractive model system for study ofeukaryotic DNA replication in that T-Ag is the only viral proteinrequired forthis process and cell-free replication systems are available (17). A sub-stantial limitation of SV40 as a model for chromosome replication is its failure to exhibit analogous regulation of DNA replication. These studies may have removed this limitation, and further study of negative control of SV40 plasmid replicationinstableCOS7 lines may well extend the utility ofSV40as amodeltoincluderegulation of replication origins. It is possible that the mechanisms that control pSLneo replication in COS7 cells are directly related to those thatgovern chromosomal DNA replication.
ACKNOWLEDGMENTS
Wethank James Sherley and Mark Labow for helpfuldiscussions andcritical reading of themanuscriptand KateJames for herhelpin its preparation.
This work was supported by a grant CA49271 from NIH. T. Chittendenwas supported by apredoctoralfellowshipfromtheNew Jersey Commissionon CancerResearch.
REFERENCES
1. Adams, A. 1987. Replication of latent Epstein-Barr virus ge-nomes in Raji cells. J. Virol.61:1743-1746.
2. Botchan, M., L. Berg, J. Reynolds, and M. Lusky. 1986. The bovine papilloma virus replicon, p. 53-67. InP. Howley(ed.),
Papillomaviruses. John Wiley&SonsLtd.,Chichester, United Kingdom.
3. Brown, E. H., M. A.Iqbal,S.Stuart,K. S.Hatton, J.Valinsky, and C. L. Schildkraut. 1987. Rate ofreplication ofthe murine immunoglobulin heavy-chain locus: evidencethat the region is
partofasinglereplicon.Mol. Cell. Biol. 7:450-457.
4. Church, G. M., and W. Gilbert. 1984. Genomic
sequencing.
Proc. Natl.Acad. Sci. USA 81:1991-1995.
5. Gluzman, Y. 1981. SV40-transformed simian cellssupportthe replicationofearly SV40mutants. Cell 23:175-182.
6. Graham,F.L.,and A.J.vander Eb. 1973. Anew
technique
for the assayofinfectivityofhumanadenovirus 5 DNA.Virology
52:456-467.
7. Hanahan,D.1983.Studiesontransformation of Escherichiacoli
withplasmids. J. Mol.Biol. 166:557-580.
8. Hand, R. 1978. EucaryoticDNA: organizationofthegenome forreplication. Cell15:317-325.
9. Harland, R.M., and R. A.Laskey. 1980. Regulated
replication
ofDNAmicroinjectedinto eggs ofXenopuslaevis. Cell 21:761-771.
10. Harlow,E.,L. V. Crawford,D.C.Pim,and N. M. Williamson. 1981. Monoclonal antibodiesspecificfor simian virus40tumor
antigen. J. Virol. 39:861-869.
11. Hatton, K. S., V. Dhar, E. H. Brown,M. A. Iqbal,S. Stuart, V. J.Didamo,andC.L.Schildkraut.1988.
Replication
program of active and inactive multigene families in mammalian cells. Mol. Cell. Biol. 8:2149-2158.12. Hirt, B. 1967. Selective extraction of polyoma DNA from infectedmousecell cultures.J. Mol. Biol. 26:365-369. 13. Khochbin, S.,A. Chabanas,P. Albert,andJ. Lawrence. 1989.
Flow cytofluorimetric determination of
protein
distributionthroughoutthecellcycle. Cytometry10:484-489.
14. Krysan, P. J., and M. P. Calos. 1991.
Replication
initiates atmultiple locations on an
autonomously
replicating
plasmid
in human cells. Mol.Cell. Biol. 11:1464-1472.15. Laskey,R.A.,M.P.Fairman,andJ. J.Blow.1989. S
phase
of the cellcycle.Science 246:609-614.16. Laskey,R.A., and R. M. Harland.1981.
Replication
origins
in theeukaryoticchromosome. Cell 24:283-284.17. Li,J., and T. Kelly. 1984. Simian virus 40DNA
replication
in vitro. Proc. Natl. Acad.Sci. USA81:6973-6977.18. Lupton,S.,and A.J.Levine.1985.
Mapping
genetic
elements ofEpstein-Barrvirusthatfacilitateextrachromosomal
persistence
of Epstein-Barr virus-derived
plasmids
in human cells. Mol. Cell. Biol.5:2533-2542.19. Lusky,M.,and M. Botchan.1981. InhibitionofSV40
replication
insimian cellsbyspecific pBR322sequences. Nature
(London)
293:79-91.
20. McCutchan,J.H.,andJ.S.Pagano. 1968. Enhancementof the
infectivityofSV40DNAwithDEAE-dextran. J.Natl. Cancer Inst. 41:351-357.
21. Rao, P.,andR.Johnson. 1970. Mammalian cell fusion: studies
onthe regulationof DNA
synthesis
andmitosis. Nature (Lon-don)225:159-164.22. Roberts, J. M., and H. Weintraub. 1986.
Negative
control of DNA replication incomposite
SV40-bovinepapilloma
virusplasmids. Cell 46:741-752.
23. Roberts, J. M., and H. Weintraub. 1988.
Cis-acting
negative
control ofDNA
replication
ineukaryotic
cells. Cell 51:397-404. 24. Roman,A.,and R.Dulbecco. 1975. Fateofpolyoma
formIDNAduringreplication. J. Virol. 16:70-74.
25. Southern, P.,and P.Berg. 1982. Transformation ofmammalian cellstoantibiotic resistancewithabacterialgeneundercontrol of the SV40 early
region
promoter. J. Mol.Appl.
Genet. 1:327-341.26. Stubblefield, E. 1975.
Analysis
of thereplication
pattern of Chinesehamster chromosomesusing
5-bromodeoxyuridine
sup-pression of 33258 Hoechstfluorescence. Chromosoma 53:209-221.
27. Tooze, J. (ed.). 1981. Molecular
biology
ofthe tumorviruses,part 2. DNA tumor viruses, 2nd ed. Cold
Spring
HarborLaboratory,Cold
Spring
Harbor, N.Y.28. Tsui,L.-C.,M.L. Breitman,L.Siminovitch,andM.Buchwald. 1982. Persistence of