Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

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0022-538X/80/00-0041/09$02.00/0

Revertants of

Adenovirus Type

12-Transformed Hamster

Cell

Line T637 as Tools in the Analysis of Integration

Patterns

DIRK EICK, SILVIA STABEL,ANDWALTER DOERFLER*

Institute of Genetics, University of Cologne, Cologne, Germany

Spontaneously arising morphological revertants of the adenovirus type 12

(Adl2)-transformed hamstercell line T637 had been previously isolated, and it

had been demonstrated that in theserevertants varying amounts of the integrated

Ad12genome wereeliminated fromthe host genome. In this report, the patterns

of persistence of the viral genome in the revertants were analyzed in

detail.

In some of the revertant cell lines, F10, TR3, and TR7, all copies ofAdl2 DNA

integrated inline T637werelost.InlinesTR1, -2, -4 to -6, -8 to -10, and -13 to -16,

only the right-hand portionofoneAd12 genome was preserved; it consisted of theintactrightsegmentofAdl2DNA and was integrated at the same site as in

line T637. In revertantlinesG12, TR1l,and TR12, one Ad12 DNA and varying

parts of a second viral DNA molecule persisted in the host genome. These

patterns of persistenceofAdl2DNAmoleculesin different revertants supported amodel foramode of integration of Adl2DNA inT637 hamstercellsinwhich

multiple (20 to 22) copies of the entire Ad12 DNA were

serially

arranged,

separated from each other by stretches of cellular DNA. Theoccurrenceof such

revertantsdemonstrated that foreignDNA sequencescouldnotonly be acquired

but could also be lost from eucaryotic genomes. There was very little, ifany,

expressionof Adl2-specific DNAsequencesintherevertantlines TR7 and TR12. Moreover, Adl2 DNAsequenceswhichwerefoundtobeundermethylated in line T637were completely methylated in therevertant cell lines G12,TR1l, TR12, and TR2. Thesefindingswereconsistent with the absence ofTantigen from the

revertantlines reportedearlier. Henceitwasconceivable that the expression of

integrated viral DNA sequences was somehow dependentontheir positions in

the cellulargenome. In celllineTR637, the earlysegments ofAdl2DNA were

expressed and undermethylated; conversely, in the revertant lines G12, TRll, TR12, and TR2, thesamesegmentsappearedtobeexpressedto alimitedextent

andwerestrongly methylated.

Mammaliancells haveamechanismthat

en-ables them to

incorporate foreign

DNA into their genomes. The

uptake

and

integration

of

viral DNA have sofar been studied most

thor-oughly,

in

particular

in virus-transformed cells (for review,seereference3). The

integration

of specific genes and of

bacteriophage

DNA into

the genomesof mammalian cellshas also been

reported (9, 19). On the other

hand,

different clonesof

morphological

revertantsof adenovirus

type 12

(Adl2)-transformed

hamster cellshave

been shown tohave losta

portion

or allof the

integratedviral DNA sequences(7,8).

Similarly,

revertants of simian virus 40-transforned (14)

and polyoma-transformed cells (2) have also

been described. Thus, foreign DNA sequences

can also be eliminated from the genomes of

mammalian cells. The

question

arises whether

in theprocessofexcision of

foreign (viral) DNA,

cellular sequences

adjacent

to viral sequences

are removedatthesametime. Since these

cel-lularDNA sequences arepossibly of the repeti-tive type (5, 13), their loss may not affect cell survival.

The T637line ofAdl2-transfonned hamster cells has been studied

extensively

in our

labo-ratory (5, 10, 13, 16). Approximately 20 to 22

copies of Adl2DNA percellareintegrated into

thegenomeof thesecellsat two orthree

differ-entsites.Most,

perhaps all,

of the Adl2genomes areseparated by cellularDNA sequences. A true

tandemarrangementofAdl2 DNA hasnotbeen Qbserved (5, 13).

The fourearly regionsof Adl2 DNA are

ex-pressed asmRNAin T637cells (10).T637cells

synthesize the

Adl2-specific

Tantigens. Spon-taneously arising morphological revertants of

T637cells have been isolated and characterized

(7,8).Therevertantsexhibita

morphology

strik-ingly different from that of T637 cells and are

T-antigennegative

according

tothe

immunoflu-orescence test (7). Among 18 revertant clones

41

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42 EICK, STABEL, AND DOERFLER

analyzed, 3 seem to have lost most, if not all,

Adl2 DNA sequences. Fourteenclones have lost

considerable amounts of viral DNA. Several of

the 18 clones have not been independent

iso-lates. The revertants are not useful in the

anal-ysis of Adl2 genesessentialforthe maintenance

of the transformed state, because all revertant

clones are aboutas oncogenic as the T637line

orthe BHK-21 parent line from which the T637

cellsoriginated (7).

In the present report, we have used the

re-vertants as tools in the analysis ofintegration

patternsof Adl2 DNA in the T637line.

Accord-ing to the patterns of persisting Adl2 DNA

sequences,the18clones fall into five classes. We

haveanalyzedindetail which parts of the viral

genomehave beeneliminated from each of the

revertant lines. The data obtained further

sup-port themodel presented in theaccompanying

paper (13) for the integration of Adl2 DNA in

T637 cells, i.e., multiple viral genomes being integratedpossiblyinto repetitiveDNA. Intact

viral DNA moleculesareseparated bystretches

ofcellular DNA, and multiple cell-virus DNA

combinationsareserially arranged. There is

lit-tle, if any, expression of the remaining Adl2

genomesin therevertantlines, andthepattern

ofmethylationhas becomestrikinglyaltered in therevertantsincomparisonwith the T637line.

MATERIALS AND METHODS

Cells and viruses.Theoriginandthemethods of

propagation oftheT637 cells (6)and ofthe morpho-logical revertants (7) were previously described.

Among the 18 revertant clones, G12 and F10 were

obtained first and independently from lines TRI through TR16.The clones ofthe seriesTRIthrough TR16wereisolated fromasingle culture of T637 cells which had been derivedfrom asingle independently reisolated clone of T637 cells. It was demonstrated that manyofthe TRclones (TRI,-2, -4, -5, -6, -8, -9, -10, -13, -14, -15, and -16) exhibitedidentical

integra-tionpatternsofAdl2DNAand, therefore,were likely torepresent reisolates of the sameoriginalrevertant

clone.

Adl2was propagatedon human KB cellsgrowing insuspensioncultureormonolayerculture.The meth-odsofAdl2propagation, virus purification, and isola-tion ofAdl2DNA weredescribed elsewhere (10).

IsolationoftheEcoRIfragmentsofAdl2DNA.

Isolationproceduresareoutlined in detail elsewhere (5, 13). The same methods were used to purifythe

BamHI orHindIIIfragments ofAdl2 DNA. In later

experiments, EcoRI fragments of Adl2 DNA were usedwhich had been cloned in the bacterialplasmid

pBR325 (S. Vogel, M. Brotz, U. Winterhoff, and W.

Doerfler, manuscriptinpreparation).

Other methods. The methods used in the isolation

ofcellularDNA(16),in nick translation ofAdl2DNA or viral DNA fragments (11), and in the analysisof

viral DNAsequences in cellular DNAby blotting(12), by DNA-DNA hybridization (18), and autoradiogra-phyarealldescribedin detail elsewhere (5, 13,16).

Methylation of integrated Adl2 DNA se-quences. Themethodsofcleavageof DNA fromT637 cellsand from therevertantswiththeMspIandHpaII restrictionendonucleasesaredescribedelsewhere(15). Intact,32P-labeledAdl2DNAwasusedas hybridiza-tionprobe.

RESULTS

Survey of integration patterns of Adl2

DNAinallrevertantlinesand inT637 cells.

The DNAs from all revertant lines (FIO, G12,

and TRi through TR16) and from the parent

line,T637,wereisolated andanalyzed by South-ern blotting upon cleavage with theEcoRI

re-striction endonuclease. The DNA on the blots

was subsequently hybridized with Adl2 DNA

which was32p labeledby nick translation. The

datapresented in Fig. la demonstrate five

dis-tinct patterns of integration in revertant lines

F10, G12, TR12, TR1l, and TR2. Moreover, it wasestablishedthat the revertant linesTRl,-2,

-4, -5, -6,-8, -9, -10, -13, -14, -15,and-16exhibited

identical patterns (Fig. 1). Thus, these latter

isolates probably represented reisolates of the

samerevertantclone.Aspreviously reported (7), inrevertantlines F10, TR3, and TR7, viral DNA

sequencescouldnotbedetected (Fig. la,b), even

thoughamuchmoresensitive methodfor DNA-DNA hybridization (18) was applied in this

studythan in earlier studies (7, 8). In Fig. la,

the five principal integration patterns of Adl2

DNA inrevertantlines FIO, G12, TR12, TRI1,

and TR2 are presented. In the DNA from line

F10,viral DNAsequenceswere notapparent. In

the DNAs from linesG12,TR1l,andTR12, the

FIG. 1. Analysis of the DNA from 18 morphological revertants and from line T637 for the patterns of integration ofAdl2DNA. DNAs from revertant celllines or fromAdl2as indicated werecleavedwith the restriction endonuclease EcoRI, and the fragments were separated by electrophoresis in horizontal 0.5% agarose slabgels. Each gel slot contained 10 LgofDNA. The DNA fragments were then transferred to nitrocellulose filters by the Southern technique and hybridized withAdl2DNA which was32Plabeled bynick

translation. Adl2-specificDNA fragments werevisualized byautoradiography. In the gel slots designated 5 x Adl2, five genome equivalents ofAdl2 DNA mixed with 10 ,ug of B3 DNA used as substrate were

electrophoresed after EcoRI digestion. The calculation of genome equivalents was based on a molecular weight of 20x 1i;forAdl2DNA (4) and a DNA content of 1.3 x 10V jigper T637 cell (6). All methods used areoutlinedindetail elsewhere (5, 13, 16). In (a), theEcoRI map ofAdl2DNA and the sizes of the EcoRI

fragmentsA to Earegivenin kilobasepairs(kbp).

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REVERTANTS OF Adl2-TRANSFORMED CELLS

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intemal

EcoRI

fragments

B,

D,

and E of Adl2 DNA

comigrated

with the corresponding

frag-mentsinthe virion marker DNA. The intensities

of these bands in the lines mentioned were,

however,

considerably

reduced in comparison with the T637 parent line. The DNA from all threerevertantlines also contained

off-size

band

3of T637 DNA(13). In

addition,

the DNAfrom line TR12carriedaweak off-sizefragment (band 2), and the DNA fromlineTRll containedan

Adl2-specific fragment intermediate in size

be-tween the EcoRI fragments A and B of Adl2

DNA (Fig. la). The DNAs from line TR2 and all its subclones contained off-size band3, aband

comigrating

with the EcoRI

fragment E,

anda

band of

slightly higher

molecular weight than the EcoRI

fragment

C of virion marker DNA. The identities ofmostof these bandswere de-termined

by using specific

viral DNAfragments

as

hybridization

probes.

The amountsof Adl2 DNA persisting in the

revertant

lines

were quantitated by

photomet-rically

comparing

intensities of Adl2-specific bands in the

autoradiograms

of Fig. 1. This method is described in theaccompanyingreport

(13). Theresults of this quantitation indicated that the DNA from therevertantlines TR1,-2,

-4 to-6, -8 to-10, and-13to -16containedabout

one copy ofafragment of Adl2 DNA percell (seebelow). The DNAs from therevertantlines G12,

TR12, TR1l,

and TR2 contained

consid-erably

less than five

copies

ofAdl2 DNA and

morethanonecopy,probably abouttwo copies,

atleast ofsomepartsofthe Adl2genome.

Analysis

of

integration

patterns in

re-vertants,

using specific

Adl2 DNA

frag-mentsas

hybridization

probes.

Werepeated

the Southem blotting experiments with the DNAsfrom line T637 and fromthe revertants

F10,

G12,

TR12,

TR1l,

andTR2, using

32P-la-beled EcoRI

fragment

A, B,C,orDas

hybridi-zation probe. In some experiments, the

32P-la-beled BamHI

fragments

orsome of the cloned EcoRIorBamHI

fragments

of Adl2 DNAwere

usedasprobes also. The data presentedin Fig.

2 canbe

summarized

asfollows.

(i) Off-size band 3 (13; Fig.3) in revertant

lines

G12, TR12,

TR1l,

and TR2contained sequences

that were homologoustoEcoRI fragmentA of

Adl2 DNA. Homology to fragment A was also

found in the DNA from line G12 in a band

comigrating with EcoRI fragment B, and in

DNAfrom line TRll in the

Adl2-specific

band

electrophoresing

in a position between EcoRI

fragments

Aand B (Fig. 2a). In the DNA from

lineTR12, off-size band 2hybridized to EcoRI

fragments

AandC.

(ii) In the DNA from lines G12, TR12, and

J. VIROL.

TR1l, the

Adl2-specific

bandcomigrating with the authentic EcoRIfragment B

represented

in factfragment B. In the DNA from lineTR2, the

Adl2-specific

band

electrophoresing

in a

posi-tion slightly above EcoRI fragment C

repre-sented

part of the EcoRI

fragment

B ofAdl2 DNA (Fig. 2b). This fragment showed weak homology to fragment A also, suggesting that the truncated B

fragment

might

still be linked

tothesamecellular DNA site viaashort

frag-ment from the

original

left terminus of Adl2 DNA

(Fig. 2a).

The weak

homology

to EcoRI fragment B in off-size band 3

(Fig.

2b and

3)

might have been due to a contaminant in the probe.

(iii)EcoRI fragment Cwasmissing altogether fromthe DNA of line TR2.

Homology

toEcoRI fragmentC of Adl2 DNAwasfound in the DNA from line TR12 in off-size band 2 and in the DNA from line TRll in the

position

between

E&oRI

fragments A and B

(Fig. 2c).

In the DNA fromline

G12,

aband

comigrating

withEcoRI fragment B also

displayed

homology

toEcoRI

fragment

C. The weak apparent

homology

of off-size band 3 in the revertant cell lines to

EcoRI

fragment

C could

possibly

be

explained

by hybridization of the C

fragment

due to the

inverted

terminal repeat.

(iv) The DNA from lines

G12,

TR12, and TRll contained the authenticD

fragment.

This fragmentwasabsent from theDNAofline TR2 (Fig.2d).

(v) None of the EcoRIfragmentswas detect-able in theDNAof lineF10.

All of the data presented are summarized schematically in Fig. 3. All DNA bands with

homologies

toEcoRI

fragments

A,B,

C,

andD wereidentifiedby hybridization tothe isolated EcoRI

fragments

of Adl2 DNA as indicated (Fig. 3). The identities of the EcoRIfragments E and F were deduced from the fact thatthey comigrated with the authentic virion marker

DNAfragments.

Right-hand

end of Adl2 DNA is

pre-served intact in revertant cell lines. The data presented in Fig. 4 demonstrate that the right terminal 2.9 to 3 kilobase pairs (kbp) of

Adl2

DNA were persisting intact in the T637

cell line andinthe revertantcell lineTR2. The

rightterminalHindIII fragment E ofAdl2DNA

(2.9 to 3kbp)was

32p

labeled bynicktranslation

and was used inhybridization experimentswith

the DNAs from cell line T637 and from the

revertantlineTR2. TheDNAs of these

cell

lines

were cleavedwith the restriction endonuclease

MboI or PstI; the fragments were electropho-resed, blottedonto

nitrocellulose

filters,and

hy-bridized to theprobe (Fig. 4).The 1.97- and

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FIG. 2. Analysisofintegrationpatternsof fiverevertantlines and ofT637cells,usingEcoRIfragmentA, B, C,orDofAdl2DNAasthe hybridizationprobe. Experimentalconditionsweresimilartothose described

inthelegendtoFig. 1.The DNAs ofrevertantlinesFIO, G12, TR12, TR11, and TR2wereanalyzed, using EcoRIfragment A(a),B(b),C (c),orD (d)ashybridizationprobe. Thesefragmentswere32plabeled bynick

translation.

kbp (not visible) MboI fragmentsaswellasthe

1.52- and 0.83-kbp PstI fragments comigrated

with the corresponding Adl2 virion marker

DNAfragments, whereas the terminal 1.16-kbp

MboIandtheterminal1.15-kbpPstIfragments

were displaced to off-size positions due to the

linkage of viral to cellular DNA (Fig. 4). We

conclude that the right terminal 2.9- to 3-kbp

45

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Ad12 T637 TR1 2 3 4 5 6 7 8 9 10 11 12 13 14 i-AC

2-AC

3-

--AC

-A - - -A -

--AC

-

-B(A)--A

15 16 G12 F10 T637 Adl2

1-AC 2-AC

---A 3-A

-AC

-B I E IF A I

FIG. 3. Schematic representation ofAdl2-specific DNA bands in the DNAs of line T637 and of 18

morphologicalrevertants. Theidentitiesof all bandswereindicatedby the letters Athrough F, which refer

tothe EcoRIfragments ofAdl2 DNA. TheEcoRImapof Adl2 DNAisalsodepicted.

segment ofAdl2 DNA waspreserved intact in the DNAs of the T637 line and of the revertant cellline TR2. Therewas averylimited number ofoff-size bands in line T637 and only one off-size band in line TR2. The latter off-off-size band wasidentical to oneoff-size band inline T637,

suggesting that the sites ofintegration in line

T637 and in the revertantwerethesame.

Expressionand methylation oftheearly

regions in the revertant cell lines. It has

beenpreviously reportedthatthe revertantcells

do not express the Adl2-specific T antigen as determinedby immunofluorescence (7, 8).

Pre-liminarydata(S.Schirm and W.Doerfler,

man-uscript in preparation) indicated that in the

revertantlines TR7 andTR12,verylittle,ifany,

Adl2-specificRNA couldbedetectedby

hybrid-ization of32P-labeled Adl2 DNA or ofthe left

terminal BamHI fragment A (see Fig. lb in

reference 13) to total cytoplasmic RNA

ex-tracted fromrevertantcelllines TR7andTR12

andcovalently linked to diazobenzyloxymethyl

paperbythemethod of Alwineetal.(1).InT637

cells,onthe otherhand,Adl2-specificsequences

werereadilyfoundbythesamemethods. Itwas

concludedthat Adl2 DNAdidnotappeartobe

expressed extensively as mRNA in revertant

lines TR7andTR12. Forline TR7,thisfinding

was expected since Adl2 DNA sequences did

notpersistinthis line(Fig. lb).

Wehavereportedearlierthat incellline T637

theearly regionsof Adl2DNA correspondingto

EcoRI fragments A and Careundermethylated,

whereas the late regions in EcoRI fragment B

are strongly methylated (15). Similar results

havebeen obtained with Ad2 DNAsequencesin Ad2-transformed hamster cells (17). It was,

therefore, of interesttoinvestigatetheextentof

methylationof theAdl2 DNAsequencesinthe

revertantcell linesG12, TR12, TR1l,andTR2. TheDNAsofcelllinesT637, G12,TR12, TR11, and TR2 were cleaved with the restriction

en-donuclease MspI or HpaII, and the fragments

were electrophoresed and transferred to

nitro-cellulose filtersasdescribed (15, 17). The DNA

on the filters was then hybridized with 32P-la-beled Adl2 DNA. The results of these experi-ments (Fig. 5) demonstrate that theAdl2 DNA sequences were almost completely methylated in the revertants and undermethylated in the T637 line aspreviously found (15). The restric-tion endonuclease MspI cleaved at all of the

CCGG sites in Adl2 DNA in the revertants, whereas the enzyme HpaII did not (Fig. 5).

Thus,the Adl2 DNAsequencespersistinginthe

revertants mighthave come under completely

different control as a consequence of their

re-positioning inthe reversionprocess. Itwasalso

conceivable, though unlikely,thatunmethylated

sequences ofAdl2 DNA might have been lost preferentially.

DISCUSSION

The analysisofthepatterns ofintegration of

Kbp

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FIG. 4. Presenceoftherightterminalsegmentof

Ad12 DNA in celllines T637 andTR2. TheDNAs

from Adl2 orfrom cell lines T637 and TR2were

cleaved with restrictionendonuclease MboIorPstI

asindicated. Thefragmentswereseparated by

elec-trophoresison2.0%agaroseslabgelsandtransferred to nitrocellulose filters bythe Southernprocedure.

The DNA was then hybridized to the 32P-labeled HindIII fragmentEofAdl2 DNA whichcontained

the rightterminal 2.9to 3kbp ofAdl2DNA. The

numbers on theleft margin ofeach autoradiogram designate the sizes in kilobasepairs ofindividual

DNAfragments. Themapson the bottoms ofeach

graph indicate the relative positions of the right

terminal MboI and PstIfragmentsandtheir sizes in kilobasepairs. Mapsweretakenfromreference5.

Adl2 DNAinmorphologicalrevertants of T637

cellshas revealed five different modesof

persist-ing Adl2 genomes (Fig. 3 and 6). In lines F10,

TR3, and TR7, apparently all viral DNA

se-quences have been lost. In line TR2 and its

identicalsubclonesTR1, -4 to-6, -8 to-10,and

-13to-16,allviralgenomeshave been lostexcept

aportionofoneviralDNA molecule. This

inter-pretation is consistent with and based upon

model (a) in the accompanyingpaper (13), i.e.,

a serial arrangement ofmultiple copies of the

entire Adl2 genome integrated into cellular

DNAseparated fromoneanotherby repetitive

cellular DNA sequences. An episomal location

of the integrated Adl2 DNA copies on avery

FIG. 5. Extentof methylation ofAdl2DNAin line

T637and in revertant lines G12, TR12, TR11, and TR2. TheDNAsofAdl2orof thecelllinesas

indi-cated were cleaved with restriction endonuclease MspIorHpaII. Fragmentswereseparated by

electro-phoresison1.3%agaroseslabgels.AU other

condi-tions were described elsewhere (5, 13, 15, 16);

32p-labeled,intact Adl2 DNAwasusedashybridization

probe.

large extrachromosomal element would also

ex-plain the occurrence ofrevertants. At present,

there isnoevidenceforsuch episomalstructures.

Inthe process of deletion of viral and cellular DNA, the entire block of DNA comprising all

the viral and cellular DNAsequencesis excised,

leaving only one viral DNA equivalent which

hasbeen truncated intheprocessinsideEcoRI

fragment B (Fig. 3 and 6). This model is in

agreementwiththepersistence of off-size band

3 homologous to EcoRI fragment A, of

frag-mentsE andF, and ofatruncated Bfragment

(Fig. 3).It hasalso beendemonstrated that the

right terminalsegmentofAdl2 DNAcomprising

2.9 to 3kbp persists intact incelllines T637 and

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FIG. 6. Proposed models for the integrcltion of Adl2DNA infourrevertantlines. The letters AthroughF refertothe EcoRI fragments of Adl2 DNA. The shadedareasrepresentcellularDNAsequences;thevertical

arrowsindicatethe known viralortheunmappedcellular EcoRI sites.TR2, G12, TR11, andTR12referto individualrevertantlines(see text).

TR2 (Fig. 4). The sites of linkage of Adl2 to

cellular DNA in lines T637 and TR2seemtobe identical.

In lines G12, TR1l, and TR12, one entire Adl2genomehasbeen conservedintheprocess ofexcision. In addition, the adjacent viral ge-nome,which isseparated bycellularsequences, has been truncated at different sites inside the

right terminal EcoRI fragment A. Truncation

might also have occurred inside the cellular

DNA sequences separating the two persisting viral DNA molecules. The schemespresentedin

Fig. 6 (G12, TR11, and TR12) indicate the

hy-pothetical sites of truncationwithin theEcoRI

fragmentAof the secondpersistingAdl2 DNA

molecule. The nucleotide block consisting ofa

truncatedfragment A,ofcellularsequences,and of the left terminalfragmentC of theviral DNA molecule persisting intact varies in length in each of the threelines,dependingonwhere the break insidefragmentAhasoccurred(Fig.3 and

6).InthehypotheticalschemespresentedinFig.

6, the exactlocations of theEcoRI sites in the

cellular DNA sequences andthelengthsof the cellular DNA separating viral DNA sequences arenotknown.

The patterns ofpersistenceof theviral DNA sequencesremaining in the genomes of the

re-vertantsanalyzedsupportthegeneralmodel(a)

proposed for the mode of integration of Adl2

DNA in T637 cells (13). The mechanism of excision of DNA sequences from transformed cellsisnotknown. Ifthe modelselaborated here are correct, viral and cellular sequences have been eliminatedjointly. Thefact thatasmany asabout 20 viralgenomescanbeexcisedjointly

strengthens the notion that many Adl2 DNA

copies areintegratedinto cellularsequences en

blocasdepictedinmodel(a) inthe

accompany-ingreport(13).We havenotyetdeterminedhow and at whatsite(s)theremaining truncatedviral genomes arereconnectedtocellular DNAupon

completion ofthe excision event. It cannot be

ruledoutatthepresenttime that theremaining

viral sequences in revertants have been

com-pletely excised and reintegrated at a different

site. However, we consider this possibility

un-likely,because the sizesof off-sizeband 3 in the

T637 line and the corresponding band in the revertantlinesareapparently identical (Fig. 3).

Moreover, the sites ofintegration at the right

andperhapsthe leftends ofAdl2 DNA inlines

T637 andTR2arethesameor verysimilar (Fig.

4).

Itcannot be stated how frequently DNA

se-quences areexcised fromvirus-transformed cells orfrom mammalian cells in general. Investiga-tions on this fascinating subject may be

facili-tatedbytheuseofvirus-transformed cells. It is quite interestingto notethat the

revert-antsanalyzed donotseemtocontain the

Adl2-specificTantigen that line T637 synthesizes (7,

8). By DNA-RNA hybridization techniques

us-ingaverysensitivemethod(1),wehave detected

minute amounts, ifany, of Adl2-specific RNA

in the cytoplasm of the revertant line TR12.

Apparently, the Adl2 genome isnot expressed

in these revertants. In line T637, on the other

hand, all four early regions of Adl2 DNA are

expressedasmRNA (10),and these transcripts

canreadilybedetected by themethods used in

the presentstudy. Wehave alsoshown that the

extent ofDNA methylation ofAdl2 DNA

se-quencesinalloftherevertantcell lineshas been

TR 2

G12

TR11

TR12

J. VIROL.

on November 10, 2019 by guest

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[image:8.510.66.461.58.237.2]

(9)

markedly enhanced incomparison with that in the parent line T637 (Fig. 5). Thisfinding would alsoimply that the Adl2 genome copies persist-ing in the revertants have become genetically inactivated, perhaps as a consequence of their repositioning in the cellular genome. We have reported previously (15, 17) on an inverse cor-relation between thelevels of DNA methylation and the extents ofexpression of the same seg-ments of viral genomes in adenovirus-trans-formed cells. It is rather lesslikely that some of the Adl2sequences in line T637 had been un-dernethylated to begin with and that these Adl2 molecules had been lostpreferentially.

The findings presented on the altered levels ofexpression and methylation of Adl2 genes in revertantssuggest thatexpression of viral genes intransformedcells may be somehow dependent ontheir

position

in thecellular genome.

ACKNOWVLEDGMEN1TS

We thankJiirgen Groneberg for a gift ofcellular DNA preparations, Hanna Mansi-Wothke for the preparation of media,BirgitKierspelfortypingthemanuscript,andSabine SchirmforagiftofpurifiedBamHIfragmentsofAdl2 DNA. Thisresearchwassupported bythe Deutsche Forschungs-gemeinschaftthroughgrantSFB74.

ADDENDUM INPROOF

(i) Theresults ofrecentexperiments have shown that inthe revertantlinesG12 and TRll sequencesat

the right terminal 2,900 to 3,000 base pairs ofthe persistingAdl2 DNA have beenrearranged andthus

are nolongercolinear with thevirionDNA sequences,

ashas beenfound for the parent line T637.

(ii) In the cytoplasmicRNAsof therevertantlines, very few, ifany, sequenceshavebeendetectablethat hybridized withAdl2 DNA.

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