Selective sites of adenovirus (foreign) DNA integration into the hamster genome: changes in integration patterns.

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JOURNALOFVIROLOGY,Jan. 1994. p. 187-194 Vol.68,No. 1 0022-538X/94/$04.00()+

Copyright

Selective Sites of Adenovirus

(Foreign)

DNA

Integration

into

the

Hamster

Genome: Changes

in

Integration

Patterns

GERTRAUD OREND, ANDREAS LINKWITZ,t ANDWALTER DOERFLER*

Institiute

for Genetics, University of

Colognze,

D-50931

Colognze,

Germany Received28 June 1993/Accepted 29September 1993

Weinvestigatedwhether, upon the integration of multiplecopies of adenovirus type 12 (Adl2)DNA into an established mammalian (hamster) genome, the pattern of foreign DNA insertion would remain stable or change withconsecutive passages of cells in culture. By the injection of purified Adl2 into newborn hamsters, tumors were induced, cells from these tumors were cultivated, and five independent cell lines, HT5, H201/2, H201/3, H271, and H281, wereestablished. These cell lines carried different copy numbers ofAdl2 DNA per cell in an integrated form anddiffered inmorphology. Cell line HT5 had been passed twice through hamsters astumor cells and wassubsequently passaged in culture. Patterns ofAdl2 DNAintegration were determined by restrictioncleavage of the nuclear DNA withBamHI, EcoRI,HindIII,MspI,orPstI followed by Southern blot hybridization using

32P-labeled

Adl2DNA or its cloned terminal DNA fragments as hybridization probes. In this way, theoff-sizefragments, which represented the sites of linkage betweenAdl2 andcellular DNAs, were determined. At early passage levels in culture, the integration sites of Adl2 DNA in the hamster genome, as characterized by the positions of off-size fragments in agarose or polyacrylamide gel electrophoresis, were different in the fivedifferenttumor cell lines. Upon repeated passage, however, theoff-sizefragment patterns generated by the five restriction endonucleases became very similar in the five tumor cell lines. This surprising result indicates that under cell culture conditions,Adl2-transformed tumor cell lines that carry the foreign

(Adl2)genome in selective, probably very similar sites of the cellular genome evolve.

Studies onthe integration offoreign (viral) DNA intothe preexisting genomes ofmammalian cells can serve an

exem-plary

function in the analysis of the mechanisms and the outcome ofinsertional recombination between foreign DNA and an established genome. These interactionsare of interest not only in tumor virology and gene therapy but also for

models about the evolutionary role of foreign DNA integra-tion. A largenumber ofadenovirus-transformed cell lines or adenovirus type 12 (Adl2)-induced tumor cells or cell lines

havebeeninvestigatedforthesites ofviral DNAinsertion (for

a review, see reference 1). For a number of junction sites

between adenovirus and cellular DNAs, the nucleotide se-quences have been determined (2,6, 12). Inthese cell lines,a

specific cellular nucleotide sequence at the insertion sites for

adenovirusDNAhas not beenfound. However,allintegration sites of adenovirus DNA investigated in that respect have

proved tobe transcriptionally active both in the transformed cells and in the original cells prior to adenovirus infection, integration, and transformation (3, 18). Short patch homolo-gies betweenviral and cellularjunction sequenceshaveoften,

but not always, been found.

Recently,we have initiated work oncell-free systems, using

nuclear extracts from hamster or insect cells to elucidate details of the mechanism of insertional recombination (5, 17, 24, 25). The recombinants generated in the cell-free in vitro system arealsocharacterized bypatchhomologiesbetween the

recombinationpartnersatthesites of

junction (24,

25). Several times,the hamster cell sequence CCTCTCCG has been found tobe therecombination target, or to belocated closeto

it,

with adenovirus DNA in several independent cell-free

recombina-tionexperimentsin which extracts

purified

todifferentextents

Corresponding author. Mailing address: Institute for Genetics, UniversityofCologne,Weyertal 121,D-50)931K6ln, Germany.Phone: 49-221-470-2386. Fax:49-221-470-5163.

t Present address: BayerAG, Leverkusen, Germany.

have been used (24, 25). The same hamster cell nucleotide

sequence has been identified at the site of linkage to Adl2

DNA in the Adl2-induced hamstertumor CLACI (21).

In a series of

Ad12-induced

hamster tumor cell lines (11,

14), we have now analyzed the dynamicsof the sites of viral DNA insertion.

MATERIALS ANDMETHODS

Most of the techniques used in this study have been de-scribed previously (14).

Adl2-induced hamster tumor cell lines. Hamster tumors were induced by the injection of Adl2, and the cell lines H201/2, H201/3, H271, and H281 were established from tu-mors T201/2, T201/3 (two different tumors in the same ani-mal), T271, and T281 and passaged in Dulbecco medium enriched with 10% fetal calfserum(14). Conditioned medium

was obtained by growing BHK-21 hamster cells in regular

Dulbecco medium with 10% fetal calfserum for24h, sterile filtering this medium, and then using itin a 1:1 mixture with fresh Dulbecco medium

supplemented

with 5% fetal calf

serum for the cultivation of Adl2-induced tumor cells. The hamster tumorT313 was induced, and the cell line H313was established morethan 10 years ago

(11).

The H313 cellswere passagedtwicethrough hamsters thatdevelopedtumorsupon

injection of H313 cells. From the second tumor passage,cell

line HT5 was derived. The tumors and cell lines T-H201/2, T-H201/3a, T-H271, T-H281, H313, and HT5 contained

ap-proximately 10, 3, 2 to 3, 5, 30, and 10 Adl2 genome

equivalents percell,

respectively

(14).

The cell lines in culture werepassagedatdifferent

split

ratios

ranging

from 1:2to 1:10. Analyses by Southern blot (19) and DNA-DNA

hybridiza-tion. In different

experiments,

DNAwas cut with one of five different restriction

endonucleases,

BamHI,

EcoRI,

HindlII,

MspI,

and PstI. The

generated

fragments

were transferred in

alkaline medium(0.25M

NaOH,

1.5 M

NaCl,

or0.4 M

NaOH)

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TABLE 1. Characteristics ofAdl2-induced tumorsandof hamster tumorcell linesestablished from them

Tumororcell line No.of

Adl2

genome Similar

Adl2

Denovomethylationof Cellmorphology

equivalents/cell' integration

patternsb

integratedAdl2

DNAC

Tumor

T313c 30 + +

T5d 10 +

T191 3-5 + +

T211 3 + +

T201/2 10 - +

T201/3 3 - +

T271 2-3 - +

T281 5 - +

Celllinee

H313 30 + + Trapezoid

HT5 10 + - Trapezoid

H191 3-5 + Fibroblastic

H211 3 + Fibroblastic

H201/2 10 + + Trapezoid

H201/3

a 3 + + Trapezoid

b NDf ND ND Fibroblastictotrapezoid&

H271

Grown in normal medium 2-3 + + Fibroblastictotrapezoid

Grownin conditioned ND ND ND Fibroblastictotrapezoid

medium

H281 5 + + Fibroblastic totrapezoid

Data takenfrom reference 14.

bFor tumors,symbols represent similarities among the tumors and corresponding celllineslisted. For cell lines, symbols represent similarities observed in HT5 at p6,H201/2 atplO, H201/3aatp12,andapproachinglate stagesof cell lines H271 atp13and H281 at pll.

cDatataken in part from reference 10.

dAgift of Ingrid Kuhlmann, Universityof Konstanz. The integrated adenoviral DNA is already heavily de novo methylated in tumor T5. The methylation state does notfurther increaseduringcultivation of cell line HT5.

e In cell lines H313, HT5,H191, H211, and H201/2, the described parameters applied to several passages. Cell lines H201/3a, H271, and H281 were analyzedin p3, p5,and pl,respectively.

fND,CompleteAdl2genomes are not detectable. Themajority ofpreviously integrated Adl2 sequences is deleted. gUponcultivation,cellmorphologychangedfrom fibroblastic totrapezoid.

toHybond N+ nylon membranes. As a32P-labeled hybridiza-tion probe, Adl2 DNA or one of the terminal fragments of Adl2 DNA, the EcoRI C (left end) or the BamHI E (right

end)

fragment, was used. Hybridization and wash conditions

were described previously (7, 14). These probes could be repeatedly removed by boilingin 2 x SSC(1x SSCconsists of 0.15 M NaCl and 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate.

In some

experiments,

DNA fragments were separated by electrophoresison5% denaturing polyacrylamide gels contain-ing 7 M urea, electrotransferred to HybondN+ nylon

mem-branes, and then hybridizedto

32P-labeled

probesasdescribed previously (14).

Restriction maps ofAdl2 DNA. The complete nucleotide

sequenceof Adl2DNA wasdetermined inourlaboratory

(20).

The restriction maps presented in Fig. 2fwere derived from thissequence.

RESULTS AND DISCUSSION

Alterations in cell morphology and integration patterns of

Adl2 DNA in Adl2-induced hamster tumor cell lines. The

Adl2-inducedhamster tumor cell lines H201/2, H201/3, H271,

H281, and HT5 were described previously (14), and their

characteristics are summarized inTable 1. Both the morphol-ogy ofthe tumorcells in culture (Fig. 1) and the patterns of

viralDNAintegration underwent distinct changesas cells were

explanted

from the tumor-bearing animals and repeatedly

passaged in culture. Changes in integration patterns were

manifestedby alterations in the positions ofthe off-sizeAdl2

DNAfragments upon agarose orpolyacrylamide gel

electro-phoresis. These fragments constituted the junctions between

the termini of Adl2 DNA and the abutting cellular DNA sequences.

Patterns ofAdl2DNAintegration inAdl2-inducedhamster tumor cells. The DNA extracted from the original tumors, T201/2, T201/3, T271, and T281, was analyzed by using one of

the five restriction endonucleases BamHI, EcoRI, HindlIl,

PstI, and MspI and by Southern blotting. The integration

patterns of Adl2 DNA in tumors T201/2 and T271 are

reproduced inFig. 4a and 3, respectively (seebelow). Some of

the integration patterns ofAdl2 DNA in these tumors were

described in detail previously(14). As a probe for

hybridiza-tion, the

32P-labeled

Adl2 DNA orthe terminal Adl2DNA fragment EcoRI C (left) or BamHI E (right) was used in

successive hybridization experiments. The autoradiograms in Fig. 2a through d present the cleavage patterns of the

inte-grated Adl2genomes generated byEcoRI (Fig. 2a),BamHI (Fig. 2b), HindIII (Fig. 2c), PstI (Fig. 2d), andMspI (Fig. 2e)

with the DNA of cell lines HT5, H201/2, H201/3a, H271, and H281. The restriction maps for AdI2DNA foreach of these

restriction endonucleases are reproduced in Fig. 2f. These maps were drawn according to the complete nucleotide se-quence of Adl2 DNA that was recently determined in this

laboratory (4a, 20). As expected, the internal viral DNA

fragments comigratedwith the Adl2DNAmarker fragments and were identical in the DNAs from the different tumors,

indicating that the Adl2 genomes were integrated in an orientation colinear with that inAdl2virion DNA. Theoff-size

fragmentswith sequence homologies to the left- or the

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ADENOVIRUS DNA INTEGRATION AT SELECTIVE SITES 189

FIG. 1. Changes in cell morphology upon continued passage in culture. (a) Cell line H271 at an early (A) or intermediate (B)stage; (b) cell lineH271 at alate stage.(c)cell line H281 atintermediate stages, early (C) and late (D) cell morphology; (d) cell line HT5; (e) cell line H201/3b (inthis cell line, completeAdl2genomes are no longerdetectable[Table 1]); (f) cell line H201/3a. Photographs were taken under a Leitz inverted microscope at x100magnification.

terminal Adl2DNAfragment or toboth terminiaremarked by arrowheads inthe autoradiograms.

It was apparentfrom the datapresented in Fig. 2ato ethat the off-sizepatternsdifferedamongtheDNAsamples from the different tumorsand from those of cell lines H313 and HT5 (Table 1). The cleavage patternsof theDNAsfrom the latter

two lines are presented for comparison. However, their pat-terns wereidentical tothose approached by cell linesH201/2, H201/3a, H271, and H281 aftercontinuous cell culture

(Fig.

2ca). After cleavage withoneof the five restriction

endonucle-ases EcoRI, BamHI,

HindIll,

PstI, and MspI

(Fig. 2),

the integration patterns of Adl2 DNA in the five different cell linesinvestigatedwereverysimilar; in particular, the distribu-tions of the off-size bands

(arrowheads

inFig. 2)were practi-cally identical. This

finding

suggested that the sites ofviral DNA integration were highly selective at this stage. As

ex-plained above, cell line H313 was generated about adecade ago

(11),

and cell line HT5 was derived from it after two hamster tumor cell passages. Cleavageandoff-sizepatternsof these celllineswereidentical for theDNAsfromH313andT5.

However, thecopynumber of30Adl2genome equivalents in cellline H313 wasdecreased to 10incell line HT5(Fig. 2ca).

For each of the panels in Fig. 2, the hybridization probe is indicated foreasyappraisal ofthe results.

Intermediate stages ofintegrationpatterns in the transition from tumor cells to tumor cell lines.Upon continuouspassage

of theAdl2-induced hamster tumor cellsin

culture,

the size distribution of the off-sizefragments of the

integrated

Adl2

genomes began to change. These alterations first became

apparentin passage 1(pl), p3, p5, andplforcell lines H201/2, H201/3a, H271, and H281, respectively. We have not

illus-trated these

changes

for all four cell lines but ratherpresenta typical example. Theautoradiograms in Fig. 3 demonstrate the HindlIl cleavage patterns of integrated Adl2 DNA in the

DNAfrom thetumorT271and from celllineH271in different

passages. It was apparent that starting with p1 1, the distribu-tion of the off-sizefragments

(designated

by arrows)

began

to

change and, after passage

p15,

finally

attainedapatternsimilar

to the patterns in the other Adl2-induced tumor cell lines H201/2, H201/3a, and HT5. Similar resultswereobtained for theDNAfrom cell line H281 after

cleavage

of theDNAwith HindIll. Some of the Adl2 off-size bands in the HindlIl cleavage patterns ofDNAfrom cell lines H271 andH281 in

higher passage numberswere identical in their size distribu-tions. Similar

findings

were made with the restriction

endo-nucleasesBamHI,EcoRI, and MspI

(data

not

shown).

Apparentsimilarities in the patterns of off-sizefragmentsof Adl2 DNA in five different Adl2-induced hamster cell linesas documented by cleavage with five different restriction endo-nucleases. As demonstrated

by

the data in

Fig.

2 and

3,

the

cleavage

patternsofDNAfrom the five

independently

estab-lished cell lines

generated

with fivedifferentrestriction

endo-nucleases showedverysimilar distributionsofmanyof theleft

and

right

terminal off-size bands. It was

particularly striking

that these off-size bands had thesamesizes in cell lines

H201/2,

H201/3a, H271, and H281 in

comparison

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190 OREND ET AL.

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FIG. 2. Patternsofintegrationof Adl2 DNA inthegenomesfrom five different Ad12-inducedhamstertumorcelllines. The DNAs fromcell lines HT5 atp6,H201/2 atplO,H201/3aatp12, H271 atpl1 (aand b) and p13 (c and d), and H281 atpIl (aandb) and p14 (c andd)were

extracted and cleaved withrestriction endonucleasesasdescribed in the text.Thefragmentswereseparatedby electrophoresison0.8% agarose

gels (ato d)andhybridizedto32P-labeled Adl2 DNA(panelsegmentsa),to the left-terminal EcoRI Cfragment (panel segmentsb),ortothe right-terminal BamHI-E fragment of Adl2 DNA (panel segments c). Amounts of authentic, virion-extracted Adl2 DNA cleaved with the corresponding restriction endonuclease andrepresentingapproximately 10(leftmarkerlane)or20(rightmarkerlane)genomeequivalentsper

cellwerecoelectrophoresed asfragmentsize references. Genomeequivalentswerecalculatedonthe basis oftheamountof cellular DNA used

perslot: aandb,10,ug;candd,40fig; ca, 10 jigforcell lines H313 andHT5;e,50 jLg.(a) CleavagewithEcoRI, usingAdl2 DNA(aa)orthe EcoRl C(ab)orBamHI E(ac) fragment of Adl2 DNAasthe hybridization probe. (b) CleavagewithBamHI. (c) Cleavage with HindIll.(d)

xEcoRI

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H271

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Psti EcoRI BamHI Hindlll CleavagewithPstI. (e)Cleavage withHpaIIorMspI,using theEcoRI C(ea) orBamHI E(eb)fragment as the hybridization probe. Fragments 5 represent the very terminal 143-bpMspIfragments at both ends of theAdl2genome. Off-size bands are designated byarrowheads. In panelca, HindIll-cuthamster liverDNAwascoelectrophoresed as a negative control. In panels a and b, DNA from cell line H271 was examined before theintegrationpatternofAdl2DNAhad reached the final stage. In panel ca, equal amounts of DNA were used in all lanes. The autoradiograms for T5 and HT5wereexposedfour timeslongerthan that for H313. (f) Restriction maps of theAdl2genomefor the restriction endonucleases PstI, EcoRI,BamHI, andHindIll.

line HT5. Since thecell lines investigatedwere derived from tumorsinducedby Adl2inhamsters, itwas notsurprisingthat mostof the cell lines contained multiple copiesof

Adl2

DNA

(Table 1).Inaddition,someofthecell linescarried additional, submolar fragments of Adl2 DNA which gave rise to weak bands inpositions not identical with anyof the Adl2 marker

DNAbands(Fig. 2). The nature of these weak bands was not

investigatedin detail.

Apartfromthe fact thatthe five cell lines harbored different copy numbers of Adl2 genomes,the increase in the de novo methylation ofthe integrated Adl2 genome in the different cell lines followed different kinetics in the transition from VOL.68, 1994

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H271

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FIG. 3. Changes in the distribution of off-size bandsintheDNAfrom the Adl2-inducedhamster tumor T271 and the hamster tumor cell line H271. For details, seethe textand legendtoFig. 2. As the hybridization probe, Adl2 DNA (a)orthe left-terminal EcoRI Cfragment (b) or

right-terminal BamHI Efragment (c) of Adl2 DNAwasused.Inpanels b andc,lanesp15containedmorethan 10 p.gofDNA,anamountthat

wasloaded in all of the other gel slots. Thesignals in this lanewerethereforemoreintense. The data showninpanelawerederivedfrom two differentelectrophoresis experiments and aligned accordingtothepositions of markerDNAfragments.

tumor tocell line.Moreover,the patterns ofmethylationinthe integrated Adl2 genomes in later passages ofthe cell lines

were not identical (Fig. 4) (14, 14a) for the cell lines that exhibitedsimilarintegrationpatternsandsites,asdocumented

by the size distributions of the off-size fragments. An example ispresented inacomparison between theHpaII (H) and MspI

(M) cleavage patterns of DNA from cell lines HT5 and H201/2. The HpaII cleavage patterns of Adl2 DNA were

distinctly different in thetwocell lines(Fig. 4b and c). Similar results were obtained after HhaI cleavage (data not shown). Thus, thepatternsofmethylation in the integrated Adl2 DNA

weredifferent, attesting tothe independent origins of thetwo cell lines.

Intwoother cell lines also derived from the sameseriesof

Adl2-induced hamster tumors, H191 and H211, the integra-tion patterns of Adl2 DNA clearly differed from those just described,evenafter extensive subcultivationof these celllines (datanotshown). We also analyzed thestateofmethylationin several subclones oftheAdl2hamster tumorcell lines H191 andH281, which differedintheir Adl2integrationpatterns.By usingsubfragments of Adl2DNAashybridization probes, cell

line-specific methylation patterns of integrated Ad12 DNA

were found (datanot shown).

It is concluded thatpossiblyas aconsequenceof thetransfer from cells from afreshly induced hamster tumor to thevery

different conditions of cellculture, patternsofforeign (Adl2) DNAintegration can change, leading either to striking simi-laritiesof Adl2DNAintegrationpatternsortoapartial loss of Adl2genomes(Table 1). The datasuggestthat viral DNAcan

be rearranged to sites of linkage to cellular DNA that are

similar or identical after fiveindependent integration events. Apparently, under certain, so far unidentified conditions,

rearrangementscanbehighly selective, perhapsevendirected

tothesamecellular DNAsequences.The latterpossibilitywill have to be further investigated by cloning some of these secondary junction sites and bycomparing the cellular nucle-otide sequences. The data argue for the chromosomal

insta-bilityof the Adl2genomes,which have becomeintegrated into

the cellulargenomeduringtumorinduction,upontransfer and propagation oftumorcells under cell culture conditions.

Evaluation of results. There is much evidence thatarguesin favorofnonspecific insertion of adenoviralDNAorretroviral

genomesintocellulargenomes. It islikely thatmanydifferent cellular DNAsequences can serve asintegrationtargets,with

apossible preference for transcriptionally activesequences or

possibly for origins of DNA replication. On the other hand, it has been observed that Adl2 genomes frequently become

associated with human chromosome 1 (13, 15) or that the genome of adeno-associated virus can become linked to

hu-man chromosome 19 (8, 9, 16). In the present report, the evidence suggests that Adl2 DNA can become integrated at

verysimilar sites ofhamster cell DNA intumorcells that have been explanted from the tumor and have subsequently been carried in culture for longer periods of time. Apparently, initially the Adl2 integration siteswere different in the

differ-ent tumors analyzed. However, upon cultivation of cells, integrated viral DNAsequencesmusthave beenrearrangedor

transferred to cellular sites such that the junction fragments appearedverysimilarwhenanalyzed with five different restric-tion endonucleases. Thereare numerous previouslydescribed examplesinwhich thisremarkablereorganization didnotseem

to occur (1, 22, 23), perhaps because the cell culture and

passage conditions used in earlier workas well as the devel-opmentalstagesof the animalsmayhave been different.

Thepossibility existed that inthecourse ofourstudies,we

would have selected for a subpopulation of Adl2-induced

tumor cells from each of the tumors in which Adl2 had originally been insertedataspecific site which then would have

been thesame orverysimilarin alltumors investigated here. The following observations argue against this possibility and demonstratetheindividuality of the cell lines. (i) The cell lines analyzed exhibit different morphologies (Fig. 1). (ii) They contain different amounts ofintegrated Adl2 DNA per cell (Table 1). (iii) The five different cell lineswere derived from

fourtumorinductionexperiments, anddifferent passage

pro-tocols have been followed. (iv) One of the cell lines was

H 271

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FIG. 4. (a) HindIII restriction patterns of integrated Adl2 ge-nomes in the Adl2-induced hamster tumor T201/2 and the corre-sponding cell line H201/2. As the hybridization probe, 32P-labeled Adl2 DNA was used.The Adl2integration patterns differed between tumor andcelllines,asindicatedby arrowheads and the rectangle. In lane pl, less than 10 ,.g of DNA was loaded on the gel. Lane SS contained 10 p.g of sonified salmon sperm DNA. (b) Methylation patterns of integrated Adl2 genomes in the Adl2-induced hamster tumor lines HT5 and H201/2 were different. The high degree of 5'-CCGG-3' methylation in the integrated genomes of T5 remained stable, whereas upon cultivation of cell lineH201/2,anincrease in the extent of 5'-CCGG-3', approaching a comparably high degree of methylation in Adl2 DNAof the tumor T5, was observed. The DNA from the two celllines or from different passages of cell line H201/2 or from Adl2 were cleaved with MspI (M) orHpaII (H), as indicated. Experimental procedures for the generation of Southern blots and theseautoradiograms aredescribedin the text andlegendtoFig.2. As the hybridization probe,32P-labeledAdl2 DNA was used. The HpaII fragments ofauthenticAdl2marker DNA are designated A to X. (c) Tabulationof the increase in 5'-CCGG-3' methylation of Adl2 DNA in tumorT201/2and in cell line H201/2 in comparison with the high degree of Adl2 DNAmethylationin tumor T5.Values indicate no (0), partial (1), or complete (2) methylation of 5'-CCGG-3' sequences.

A-

B-C,D

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E/F 0 2 2 2 2

G 0 1 1

established completely independentlymorethan 10years ago and has been subjected to two successive hamster tumor passages. (v) The rates of de novomethylation as well as the final methylation patterns of theintegrated Adl2 genomes are similar butnotidentical

(Fig.

4b).

(vi)

The transition patterns of Adl2DNAintegration for the cell lines have been different, and in different cell

lines,

some of the off-size bands are in nonidentical positions.

The experimental conditions that can lead to changes in the Adl2 integration patterns, as evidenced by the approach to

similar off-size band patterns

generated

with five different restriction endonucleases and thenot infrequent loss of Adl2 genomesfrom Adl2-transformed cells orfrom Adl2-induced

tumor cells

passaged

in culture

(4, 10),

are not understood. The presence of

apparently

similar patterns of Adl2 DNA

integration in five independently isolated hamster tumorcell linesmight reflect

genomic instability

of theforeign DNA in these cell lines. We have initiated work to clone the sites of junction between Adl2 DNA and the

adjacent

hamster cell

DNA from different Adl2-induced tumor cell lines. In this way, wehope to assess thepossibility of selective sites ofAdl2

DNA

integration

and document itat thenucleotide level. ACKNOWLEDGMENTS

We thank Ingrid Kuhlmann, University of Konstanz, Konstanz, Germany,formakingcell line HT5 available. WeareindebtedtoPetra Bohmfor excellent editorial work.

This research was supported by the Deutsche Forschungsgemein-schaft through SFB274-TP1 and bythe Bundesministerium fur For-schungundTechnologie through GenzentrumKoln,TP2.03.

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