Vol. 39, No. 1JOURNAL OF VIROLOGY, July 1981, p. 1-100022-538X/81/070001-10$02.00/0

Characterization of AKR Murine Leukemia Virus Sequencesin AKR Mouse Substrains and Structure of Integrated

Recombinant Genomes in Tumor TissuesWIM QUINT, WIM QUAX, HERMAN VAN DER PUTTEN, AND ANTON BERNS*

Laboratory of Biochemistry, University of Nijmegen, 6525 EZ Nijmegen, The Netherlands

Received 8 January 1981/Accepted 2 April 1981

A specific cDNA probe of AKR murine leukemia virus (AKR-MLV) wasprepared to detect AKR-MLV sequences in normal and tumor tissues in a varietyof AKR mouse substrains. AKR strains contained up to six endogenous AKR-MLV genomes. All substrains tested had one AKR-MLV locus in common, andclosely related substrains had several proviruses integrated in an identical site.Virus-induced tumors in the AKR/FuRdA and AKR/JS strains showed a rein-tegration pattern of AKR-MLV sequences unique for the individual animal,suggesting a monoclonal origin for the outgrown tumors. An analysis of tumorDNAs from the AKR/FuRdA and AKR/JS substrains with restriction enzymescleaving within the proviral genome revealed a new EcoRI restriction site andBamHI restriction site not present in normal tissues. The positions of these sitescorresponded both with cleavage sites of EcoRI and BamHI in integrated Molo-ney recombinants and with the structure of isolated AKR mink cell focus-formingviruses. All tumors analyzed to data contain nearly identical integrated recombi-nant genomes, suggesting a causal relationship between the forination of recom-binants and the leukemogenic process.

It has been established that the endogenousAKR murine leukemia viruses (AKR-MLVs) aretransmitted from parent to offspring within avariety of inbred strains of mice (1). The segre-gation ofAKR-MLV proviral loci has been iden-tified by the backcross of mice of the AKR strainand the virus-negative NIH/Swiss strain; thesestudies determined the presence of at least threeAKR-MLV proviral loci (15). Two high-induc-ibility loci (Akv-1 and Akv-2) have been mappedon chromosomes 7 and 16, respectively (24, 25)and have been characterized by restriction en-donuclease mapping (27). AKR mice expresshigh levels of endogenous AKR-MLV through-out their life-span and show a high incidence ofleukemia (70 to 95%) after a latency of 7 to 9months (25). Leukemogenesis in AKR/J mice isaccompanied by an amplification ofAKR-MLVsequences in tumor tissues caused by reintegra-tions of endogenous MLV sequences (2, 4, 27).Recombinants between ecotropic AKR virus

and endogenous xenotropic MLVs have beendetected in the thymuses of leukemic and pre-leukemic AKR mice and in leukemic tissues ofNIH/Swiss mice that inherit either the Akv-1 orthe Akv-2 virus-inducing locus (13). RNase T,fingerprinting and heteroduplex analysis showedthat these mink cell focus-forming (MCF)-typeviruses are env gene recombinants with consid-

erable heterogeneity among the various isolates(9, 14, 19, 23). In contrast, Moloney MLV (M-MLV)-induced tumors in mice all contain simi-lar recombinant genomes, indicating the neces-sity for a specific type of recombination (H. vander Putten et al., Cell, in press). Therefore, wesearched for common characteristics of recom-binant virus genomes integrated in the DNA ofAKR tumor tissues.

In the study presented here, a cDNA probethat was specific for AKR-MLV and that didnot cross-hybridize with other endogenous viralsequences was used. We applied the Southernblotting technique (26) to identify and charac-terize the different endogenous AKR-MLV se-quences in a number of AKR substrains and ina variety of nornal and malignant tissues ofthese strains.

MATERIALS AND METHODSVirus. AKR virus, isolated from Akv-1 congenic

mice, was propagated in monolayers of NIH/3T3 cellsin roller bottles on a Smith Kozoman autoharvester(Bellco). The AKR virus was isolated as describedpreviously (32).

Mice. BALB/c and 129 mice of inbred strains andthe outbred strain S.E.(Swiss) were obtained from theCentral Animal Laboratory of the University ofNijmegen, Nijmegen, Holland. AKR/FuRdA micewere obtained from Netherlands Cancer Institute,

2 QUINT ET AL.

Amsterdam, Holland. AKR/J mice were from theWeizmann Institute of Science, Rehovot, Israel. Sub-strain AKR/Cnb was from Centre d'Etude de l'EnergieNucleaire, Mol, Belgium. Organs from AKR/JS micewere obtained from R. Jaenisch, Heinrich Pette-Insti-tut, Hamburg, Federal Republic of Germany. AKR/Abom Fib mice were obtained from the Breeding andResearch Center, GI. Bomholtgaard Ltd., Ry, Den-mark. Organs from the Akv-2 strain of mice weregenerously supplied by A. Colombatti, Istituto di An-atomia, University of Padova, Padova, Italy. AKRclone 623 was kindly provided by D. Lowy, NationalCancer Institute, Bethesda, Md.

Preparation and selection of the cDNA probe.AKR viral cDNA was prepared in the endogenouspolymerase reaction as described previously with mi-nor modifications (32). For the removal of sequencesfrom the AKR-MLV cDNA which cross-react withother endogenous viruses, AKR-MLV cDNA was se-

lected against DNA from 129 mice exactly as describedby Berns and Jaenisch (2).

Extraction of DNA, electrophoresis, and elu-tion. DNA was isolated, purified, and cleaved withrestriction endonucleases as described previously (32).Restriction endonuclease fragments were separated onthe basis of size by electrophoresis in 0.5 or 0.8%neutral agarose gels as reported previously (32). DNAwas eluted and concentrated from 2-mm gel slices byelectrophoretic elution; preparative elution of DNAfragments from agarose gel slices was carried out for14 h at 70 V in a buffer containing 12.5 mM Tris baseand 1.5 mM EDTA, adjusted to pH 8.0 with boricacid. The eluted DNA (400-,ld samples) was adjustedto 0.3 M NaCl and 0.5% sodium dodecyl sulfate, ex-

tracted twice with 1 volume ofTE buffer (10mM Tris-hydrochloride [pH 7.4], 1 mM EDTA)-saturatedphenol (pH 8) and twice with 1 volume of CHCI-isoamyl alcohol (25:1), and precipitated twice withethanol.

Blotting and hybridization. After electrophore-sis, the DNA was transferred with 1Ox SSC (lx SSC:0.15 M NaCl, 0.015 M trisodium citrate) to nitrocel-lulose filters as described previously (30). Before hy-bridization, the filters were incubated for 2 h at 42°Cin a volume of 200 ,ul of pre-hybridization mix (50%formamide, 5x SSC, 5x Denhardt solution [lx Den-hardt solution: 0.02% bovine serum albumin, 0.02%Ficoll, 0.02% polyvinylpyrrolidone], 250 ug of dena-tured salmon spenn DNA per ml, 0.05 M sodiumphosphate [pH 7.0]) per cm2. Hybridization was per-formed at 42°C for 16 h in a volume of 50 PI/cm2 witha solution containing 50% formamide, 5x SSC, lxDenhardt solution, 0.02 M sodium phosphate, 10%dextran sulfate, 100 ug of denatured salmon spermDNA per ml, and 0.5 x 106 to 1 x 106 cpm of cDNAper ml (33). After hybridization, the filter was washedwith hybridization mix from which the probe anddextran sulfate were omitted for 2 h at 42°C, once for15 min with 2x SSC-0.1% sodium dodecyl sulfate at50°C, once with 0.lx SSC-0.1% sodium dodecyl sulfateat 45°C, and once with 0.1x SSC-0.1% sodium dodecylsulfate at 67°C. The filters were dried and autoradi-ographed for 2 to 6 days with Kodak Royal X-Omat Rfilm and intensifying screens (30).

J. VIROL.

RESULTS

Characterization of the AKR cDNAprobe. For the detection of AKR-MLV-specificsequences in the genomes of different mousestrains, an AKR-MLV-specific 32P-labeledcDNA probe was prepared and selected as de-scribed by Berns and Jaenisch (2). The unfrac-tionated AKR-MLV cDNA contains sequenceswhich cross-hybridize with other endogenousmouse viruses. These sequences were removedby hybridizing the unfractionated AKR cDNAto DNA from mice of the 129 strain, which lacksthe AKR-type virus (2). For a determination ofthe specificity of AKR-MLV cDNA, the probewas hybridized to EcoRI restriction endonucle-ase DNA fragments from liver DNA of micewhich lack AKR-MLV-type proviruses (129 andSwiss) (17) and mice which contain one copy ofAKR-MLV per haploid genome (BALB/c andNIH/Swiss Akv-2 congenic) (8, 22, 32). No hy-bridization was detected with 129 and SwissDNAs, whereas the AKR-MLV cDNA probehybridized to a single EcoRI restriction frag-ment of 20 kilobase pairs (kbp) in BALB/c DNAand a fragment of 26 kbp in NIH/Swiss Akv-2congenic DNA (Fig. 1A). It has already beensuggested that this specific probe could repre-sent 30 to 35% of the genome (2), allowing rec-ognition over the entire genome. Indeed, theAKR-MLV-specific cDNA displayed hybridiza-tion with all four BamHI restriction endonucle-ase fragments (27) of a cloned AKR-MLV inte-grated provirus (clone 623) at a concentrationidentical to the relative number of AKR se-quences in cellular DNA samples (18). However,not all four BamHI restriction endonucleasefragments of clone 623 were recognized equally(Fig. 1B). The fragment corresponding to the 5'end of the viral RNA showed less hybridizationas compared with the fragment assigned to the3' end of the genome.Endogenous AKR-type sequences of

AKR mice. To characterize the endogenousAKR-MLV sequences in the AKR strains, weused the Southern blotting technique (26). Re-striction endonuclease EcoRI does not cleave invitro-synthesized full-lengthAKR proviral DNA(28) and cloned AKR-MLV DNA (18). Thisimplies that the sizes of cellular EcoRI DNAfragments containing AKR proviral sequenceswill depend on EcoRI recognition sites presentin cellularDNA flanking the integrated provirus.Restriction endonuclease BamHI cleaves AKRproviral DNA at three sites, resulting in twointernal DNA fragments of 1.9 and 3.0 kbp andtwo viral DNA fragments linked to the adjacentcellular sequences (27).

AKR-MLV SEQUENCES IN AKR MOUSE SUBSTRAINS

AKbp

3 7 -

11 -

5-

a b c d

-26-2 0

FIG. 1. Characterization of the selected cDNAprobe. (A) EcoRI-digested DNAs were analyzed bythe Southern procedure (26) and hybridized with thespecific cDNA probe. EcoRI does not cleave the viralDNA (28). Lane: a, liver DNA of a 129 mouse; b,BALB/c liver DNA; c, S.E.(Swiss) liver DNA; d,NIH/Swiss Akv-2 congenic liver DNA. The molecularsizes ofthe AKR-MLV-specific restriction endonucle-ase fragments are indicated at the right. Adenovirustype 2 viral DNA and phage A DNA, digested withEcoRI, HindIII, andBamHI, were used as molecularweight markers. (B) BamHI digestion of 50 pg ofAKR-MLV clone 623 (18), analyzed by the Southernprocedure and hybridized with a specific cDNAprobe. The positions of the internal proviral frag-ments (3.0 and 1.9 kbp) and the fragments containingthe 3' (21.6-kbp) and 5' (2.3-kbp) junctions with cellu-lar andphage X DNAs are indicated at the right.

Liver DNA from AKR/A, AKR/JS, andAKR/FuRdA mice showed two, four, and sixEcoRI restriction endonucleaseDNA fragments,respectively, which hybridized to the AKR vi-rus-specific cDNA probe (Fig. 2, lanes b, d, ande). All fragments were larger than 8.8 kbp andmight therefore contain a complete AKR-MLVgenome. This was also suggested by nearly equalamounts of radioactivity detected in each band.The six EcoRI restriction endonuclease DNAfragments of the AKR/FuRdA strain weretreated with restriction endonuclease BamHIand further analyzed for the presence of AKR-MLV-specific sequences (27). EcoRI restrictionendonuclease fragments from liver DNA (300lsg) ofAKR/FuRdA mice were separated on the

basis of size by electrophoresis on a 0.4% neutralagarose gel. DNA from 2-mm gel slices waselectroeluted and digested with restriction en-donuclease BamHI, and DNA fragments wereseparated on 0.8% agarose gels. The six endoge-nous AKR-MLV-containing fragments appearedto contain the 3.0- and 1.9-kbp internal BamHIDNA fragments, as indicated by the increasedintensity in the autoradiograph in the corre-sponding positions. Fragments of identical sizecould be obtained from a cloned AKR proviralgenome (clone 623). In addition to the internalproviral DNA fragments, a clearly unique distri-bution of additional fragments was found in allsix AKR-MLV-containing EcoRI fractions (Fig.3A). These additional fragments represented theoverlap of the 3' and 5' parts of AKR-MLVgenomes with the flanking cellular sequences.Each of the AKR-MLV-containing EcoRI frag-ments was characterized further with restrictionendonucleases BamHI plus BglI and PstI. BglIreduced the 3.0-kbp BamHI DNA fragment to2.0 kbp, whereas the 1.9-kbp BamHI fragmentwas trimmed to 1.8 kbp (data not shown), in

Kbp a b c d e f g3 7 - 5t-r;0;55

11

63-

4.9-

3.5

2.8>0:X -3~-.02.8- 3

2.3

1.8-

FIG. 2. Identification of endogenous AKR-MLVsequences in substrains ofAKR mice. EcoRI-digestedDNAs (lanes a to e) and BamHI-digested DNAs(lanes f and g) were analyzed by the Southern pro-cedure. The lanes represent liver DNAs from theAKR/Cnb (lane a), AKR/A (lane b), AKR/J (lane c),AKR/JS (lanes d and g), and AKR/FuRdA (lanes eand f) strains. The molecular sizes ofsome individualAKR-MLV-containing fragments are indicated at theright. The molecular weight markers at the left areas described in the legend to Fig. 1.

VOL. 39, 1981 3

4 QUINT ET AL.

55 21 1 8I i

14 13 l 1I

6.3 -

4.9 -

3.5 -;

2.8-

2.3-

1.4 -

55 21 18 14 13B IKbp

_1 1

-8.2

_6.3

-4.9

FIG. 3. Characterization of the six endogenous AKR-MLV genomes present in the AKR/FuRdA strain.EcoRI restriction fragments from liver DNA (300 Ig) ofAKR/FuRdA mice were separated on the basis ofsizeon a 0.4% agarose gel. DNA from 2-mm gel slices was electroeluted. (A) Digestion ofDNA from the 2-mm gelslices with BamHI after separation of the fragments on 0.8% agarose gels and Southern analysis. Lane a

represents 10 Aig of AKR/FuRdA liver DNA digested with EcoRI plus BamHI. The positions of the twointernal BamHI fragments (3.0 and 1.9 kbp) are indicated at the right. The molecular weight markers at theleft are as described in the legend to Fig. 1. (B) Digestion ofDNAs from the six AKR-ML V-containing EcoRIfragments with PstI after separation of the fragments on 0.8% agarose gels and Southern analysis. Lane a

represents 10 jig of AKR/FuRdA liver DNA digested with EcoRI plus PstI. The position of an 8.2-kbpfragment is indicated at the right. The molecular weight markers at the right are as described in the legendto Fig. 1. The molecular sizes given at the top correspond to the positions of the six endogenous AKR-MLVgenomes shown in Fig. 2, lane e.

agreement with the location of the BglI sites inthe molecularly cloned provirus (clone 623). Re-striction endonuclease PstI cleaves the uninte-grated AKR-MLV genome only within the ter-minal redundancy, generating an 8.2-kbp frag-ment (28). Digestion with PstI of the six AKR-MLV-containing EcoRI restriction endonucle-

ase fractions yielded in all instances the 8.2-kbpintemal fragment (Fig. 3B). Therefore, theAKR/FuRdA strain contains six endogenousAKR-MLV proviral genomes, integrated in dif-ferent sites of the mouse genome.

Digestion of DNA from the AKR/A strainwith EcoRI gave rise to two fragments of 55 and

AKbp

37

1 1

3.0

1.9

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AKR-MLV SEQUENCES IN AKR MOUSE SUBSTRAINS

55 164 1

kbp

15..11..llo.Cd

&3,

49-3

3.7..

28..

I&

_ a5

- Lo

_ tS

FIG. 4. Characterization of the two endogenousAKR-MLV genomes present in the AKR/A strain.The 55- and 16-kbp EcoRI restriction fragments fromliver DNA (300ug) ofAKR/A mice were isolated as

described for AKR/FuRdA mice. The isolated frag-ments were cleaved with BamHI and analyzed by theSouthern procedure. The molecular weight markersat the left are as described in the legend to Fig. 1.

16 kbp, respectively. More detailed analyses ofeach fragment by digestion with BamHI re-vealed the 3.0- and 1.9-kbp intemal BamHI frag-ments and two additional fragments represent-ing the 3' and 5' overlaps with cellular sequences(Fig. 4). The 55-kbp EcoRI DNA fragment ofthe AKR/A strain contained the same flankingfragment of 3.5 and 4.0 kbp as found after diges-tion of the 55-kbp EcoRI fragment present inthe AKR/FuRdA strain of DNA (compare Fig.3A and 4). BamHI digestion ofthe 16-kbp EcoRIfragment from the AKR/A strain gave rise tofragments not seen in the AKR/FuRdA strain.Therefore, one of the two AKR-MLV proviralgenomes within the AKR/A strain is integratedin the same chromosome site as occupied in theAKR/FuRdA strain, whereas the other is uniquefor the AKR/A strain.

Similarly, one of the four endogenous proviralgenomes present in a 55-kbp EcoRI DNA frag-ment in the AKR/JS strain has the same chro-mosomal location as found in the AKR/FuRdAand AKR/A strains. Double digestion with re-

striction endonucleases BamHI and EcoRI ofliver DNA of the AKR/FuRdA and AKR/JSstrains showed, besides two internal fragments,

clearly one other common fragment of 3.5 kbp(Fig. 2, lanes f and g). This fragment was alsofound after digestion with BamHI of the isolated55-kbp EcoRI DNA fragment from the AKR/FuRdA and AKR/A strains (Fig. 3A and 4).Double digestion with restriction endonucleasesEcoRI and HindIII showed two fragments of 30and 25 kbp within the AKR/FuRdA and AKR/JS strains (data not shown). HindIII cleaves thein vitro-synthesized full-length AKR proviralDNA once (28). These fragments can only be-long to the AKV locus contained in the 55-kbpEcoRI DNA fragment. In all substrains of AKRmice, the 55-kbp EcoRI restriction endonucleasefragment could be detected, whereas the num-bers and integration sites of the other endoge-nous AKR-MLVs varied (Fig. 2).The same three AKR-MLV-containing EcoRI

fragments present in the AKR/J genome werealso detected within the AKR/JS strain ofDNA(Fig. 2, lanes c and d). The AKR/JS strains had,in addition, an AKR-MLV-containing EcoRIfragment of 17 kbp. This indicates that germ linereinsertions of AKR-MLV occur rather fre-quently and that substrains which have beenseparated recently have more AKR-MLV-con-taining EcoRI fragments in common.We conclude that the different AKR sub-

strains contain various numbers of endogenousAKR-MLV genomes and that only a single AKRprovirus is integrated in an identical site in allAKR strains. Closely related substrains have, ingeneral, several proviruses in common.Integration of AKR-MLV sequences in

tumor DNA. Previously, it has been shown byliquid hybridization studies that leukemogenesisin AKR mice is accompanied by an amplificationof AKR-MLV sequences in tumor tissues (2).These experiments did not provide informationabout specific integration sites and the structureof reintegrated viral genomes; therefore, AKRsequences were analyzed in tumor and nontumortissues ofAKR/FuRdA mice. DNA was isolatedfrom these tissues and analyzed by restrictionendonuclease digestion, blotting, and hybridiza-tion to a specific cDNA probe. Figure 5 (lanes bto i) shows the distribution pattern of AKR-MLV provirus-containing fragments from tumorDNAs of different animals, as well as from tumorDNAs obtained from different anatomical sites(thymus, spleen, and lymph nodes) of one ani-mal.

Additional fragments were observed in tumorDNAs that were absent in normal liver DNAs.These reintegrated AKR-MLV sequences, rang-ing in size from 2 to 26 kbp, displayed a uniquepattem for each individual animal. However,tumor DNAs from different tissues within oneanimal showed the same reintegration pattern.

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6 QUINT ET AL.

Kbp a b c d e

37 -

22 -

1 5 -

6.3-

4.4-

2.8-

1.8-

'IG. 5. Detection of new AK]ing fragments in the DNAs of ItAKR/FuRdA mice. Ten microgfrom several tumors was digestEalyzed as described in the text. IDNA; b, c, and d, DNAs from thlymph nodes of animal 1; e ar

spleen and lymph nodes of aninfrom the spleen and lymph nodefrom the thymus of animal 4. Tmarkers at the left are as descrFig. 1.

These observations indicatemors are composed largely ofdants of one or a few transfiare metastasized at differen'Furthermore, the sizes of t1restriction endonuclease DNcontain the endogenous AKremain unchanged in tumorsults have been obtained formitted M-MLV (16). The r

MLV sequences in tumor tissilevel of hybridization as comnetically transmitted AKR-Apected for integrations probaonce per diploid genome equ

Individual EcoRI DNA:from a lymphoma of an Alwere analyzed for the presen(

istic intermal BamHI DNA I

the characteristic fragmentsdiagnostic for the authentic A

f g h i a new fragment of 2.2 kbp was detected afterBamHI digestion of some EcoRI DNA fractions

w__wcomprising newly acquired AKR sequences.BamHI digestion of DNA from tumor tissues of

i<:~w~ different animals gave rise to a 2.2-kpb BamHINIfbIIJ fragment, indicating that this fragment is de-

rived from modified AKR genomes (Fig. 6, lanes2 to 5). This fragment was localized on thephysical map of the AKR genome: the 2.2-kbpBamHI fragment was trimmed 0.5 kbp in size byXhoI digestion, generating a 1.7-kbp fragmentnot seen in DNAs from normal tissues (Fig. 6,lanes 6 to 8) and not present in XhoI-digestedDNAs of tumor tissues (data not shown). Thispositioned the new 2.2-kbp BamHI fragmentfrom 4.1 to 6.3 kbp on the physical map (Fig. 7).Probably due to methylation, the 3-kbp internalBamHI fragment was only partly digested byXhoI, resulting in the presence of both a 2.5-kbpfragment and a 3-kbp fragment in double digestsof BamHI plus XhoI. Similarly, digestion ofDNAs from all tumor tissues isolated so far bya combination of EcoRI and XbaI revealed afragment of 1.0 kbp (Fig. 6, lanes 9 and 10) notdetectable in DNAs from normal tissues (Fig. 6,

R-MLV-type-contain- lane 11).eukemic tumors from Figure 7 shows the localization of this new,rams of tissue DNA fragment on the viral genome. The position ofed by EcoRI and an- the EcoRI cleavage site (position 6.8) was fur-Lane: a, normal liver ther confirmed by double digestion with EcoRIe thymus, spleen, and and PstI. PstI cleaves AKR-MLV proviral DNAid f. DNAs from the in the long terminal redundancy at map posi-sal 2; g and h, DNAs tions 0.1 and 8.3 kbp. Digestion with PstI and'he molecular weight EcoRI should generate two fragments of 1.5 and-ibed in the legend to 6.7 kbp. Figure 6 (lanes 12 to 16) shows that the

1.5-kbp fragment could only be found withinDNAs from tumor tissues. Hybridization to a6.7-kbp fragment was also seen in DNAs from

that outgrown tu- normal tissues and in DNAs from mice lackingf the clonal descen- the endogenousAKR genomes (data not shown).ormed cells, which Apparently, a large number of other endogenoust anatomical sites. viruses can give rise to a 6.7-kbp fragment afterie different EcoRI digestion with EcoRI and PstI. The large molar{A fragments that excess of a fragment of this size might result inR-MLV sequences the recognition of this fragment by a smalltissues. Similar re- amount of nonspecific cDNA still present in ourr genetically trans- selected cDNA preparation.reintegrated AKR- The presence of an EcoRI cleavage site and aues show a reduced BamHI cleavage site within reintegrated viralkpared with the ge- genomes indicates the acquisition of non-AKR-4LV copies, as ex- MLV sequences. In all of the tumors analyzed,Lbly occurring only the same new fragments were found, indicatingivalent. a rather conserved organization of recombinantfractions obtained proviral DNAs in tumors. Similar results wereKR/FuRdA mouse obtained in M-MLV-induced tumors of BALB/ce of the character- Mo and BALB/c mice, in which EcoRI andfragments. Besides BamHI sites have been located at nearly iden-of 3.0 and 1.9 kbp tical positions in recombinant genomes (van deriKR MLV genome, Putten et al., in press).

WAR* ON

."WA

J. VIROL.

AKR-MLV SEQUENCES IN AKR MOUSE SUBSTRAINS

kbpi 2 3 4 5

15- t _ _ii

6 7 8 12 13 14 15 16

.67-

4.4

3.0

- 2.2 -

1..7_

FIG. 6. Recombinant AKR-MLV sequences i.* AKR/FuRdA and AKR/JS mice. Ten micrograms ofDNAfrom tumors of different animals was cleaved with the indicated restriction endonucleases and analyzed bythe Southern procedure. Lane: 1, BamHI digestion of normal AKR/FuRdA liver DNA; 2 to 5, BamHIdigestionpatterns oftumorDNAsfromfourAKR/FuRdA animals, showing the new 2.2-kbp BamHIfragment;8, digestion with XhoIplus BamHI ofnormal AKR/FuRdA liver DNA; 6 and 7, AKR/FuRdA tumor DNAs,revealing the additional 1.7-kbp XhoI-plus-BamHI fragment; 11, XbaI-plus-EcoRI digestion ofnormal AKR/JS liver DNA; 9 and 10, XbaI-plus-EcoRI cleavage patterns oftumor DNAs from a spleen ofan AKR/FuRdAanimal (lane 9) and a thymus of an AKR/JS animal (lane 10) containing a 1.0-kbp fragment; 12, PstI-plus-EcoRI digestion of nornal AKR/FuRdA liver DNA; 13 to 16, AKR/FuRdA tumor DNAs from four animals,digested with PstIplus EcoRL The molecular weight markers at the left are as described in the legend to Fig.1.

Unintegrated AKR MuIV DNA(8.8 Kbp)

BamHI Xbal Pstl

j j BamHl EcoRi

AKR/FuRdA,AKR/JS tumors 4

FIG. 7. Structure of integrated recombinant proviral genomes. The upper line represents the map ofrestriction endonuclease cleavage sites on the unintegratedAKR-MLVgenome (partly obtainedfrom reference28). The boxes at the ends indicate the long terminal redundancies. The scale of the map is shown in themiddle. The line at the bottom represents the recombinant structure, with the positions of the EcoRI andBamI sites. The difference between the broken-line frame (representing recombinants as found in M-MLV-induced tumors) and the solid frame (minimum substituted sequences as indicated by BamHI and EcoRIdigestions in this paper) reflects the most probable positions of the recombinant sites.

DISCUSSION these viral sequences can be detected by DNAprobes complementary to most leukemia vi-

Many type C viruses are part of the genome ruses. To detect more specifically the AKR en-

complement of inbred mice (7). The majority of dogenous virus, Steffen et al. removed most of

VOL. 39, 1981 7

Bar HI Bam HI Xhol

Kbp

o 1 2 3 41 1 5 6 7 ! 8 91~~ ~ ,, , , ,,-II I I fI I I I

8 QUINT ET AL.

the AKR viral sequences which this virus has incommon with M-MLV (27). Cross-hybridizationcould still be observed with other endogenousviruses. Also, subgenomic segments from molec-ularly cloned AKR-MuLV DNA (clone 623)have been used as probes to analyze and identifyecotropic sequences integrated in the genomesof several mouse strains. These probes, however,do not show hybridization with the endogenousecotropic MLVs present in some of the mousestrains from which this virus can be induced,such as C57BL (9), or still seem to cross-hybrid-ize with non-AKR endogenous type C viruses,such as in 129 mice (5). Furthermore, the detec-tion of endogenous ecotropic sequences by theseprobes is restricted to defined regions of theendogenous ecotropic genomes. Therefore, weselected the AKR cDNA probe by hybridizationto 129 mouse DNA, which lacks the AKR en-dogenous virus, and isolated the single-strandedDNA fraction by hydroxyapatite chromatogra-phy. This single-stranded DNA fraction did notshow any hybridization to chromosomal DNAfrom strains which have been reported to lackthe endogenous AKR virus (129 and Swiss); onthe other hand, DNA from strains which havebeen shown to contain a single AKR endogenousvirus exhibit hybridization with a single uniquerestriction fragment (BALB/c and NIH/SwissAkv-2 congenic).The selected probe represents 30 to 35% of the

viral genome (2) and recognized the four AKR-MLV-specific BamHI restriction endonucleasefragments at a dilution similar to the amount ofAKR proviral sequences present in cellularDNAsamples used in our agarose gels. This indicatesthat the sequences present in the cDNA-specificprobe are distributed over the entire AKR ge-nome, although not all fragments are recognizedequally; DNA fragments corresponding to theenvelope region, where most dissimilarities withother mouse type C viruses are found by heter-oduplex analysis (9), seem to be recognized pref-erentially.

Ihle and Joseph presented evidence for threeAKR-MLV proviruses in AKR mice (15). Weobserved six endogenous AKR-MLV sequencesin the AKR/FuRdA strain and two genomes inthe AKR/A strain, whereas four genomes weredetected in the AKR/JS strain; other substrains,such as AKR/Cnb and AKR/J, appear to con-tain various numbers of integrated AKR ge-nomes (Fig. 2). The characterization of the sixAKR-MLV-containing EcoRI fragments withinthe AKR/FuRdA strain by digestion withBamHI, BglI, and PstI suggests that they rep-resent six complete authentic AKR-MLV ge-nomes. This was substantiated further by back-

J. VIROL.

cross studies, which revealed the independentsegregation of all six proviral loci, each with anidentical intemal restriction pattem (data notshown). Similarly, BamHI digestion of the twoAKR-MLV-containing EcoRI fragments de-tected in the AKR/A strain indicated that inthis strain two complete AKR-MLV genomesare present. Digestion of DNAs from all AKRsubstrains (14 different strains have been testedso far) with EcoRI revealed an AKR-specificfragment of 55 kbp. The other AKR sequencesare detected in differently sized fragments. A 55-kbp fragment has also been found in the AKR/N, AKR/J, and AKR/Cu mouse strains by oth-ers (9, 27).The differences in number and size of endog-

enous AKR-MLV-containing fragments amongseveral substrains demonstrated considerablegenetic variability. Since most AKR substrainshave segregated for long periods of time (up to50 years), independent germ line reintegrationsmay have taken place, giving rise to the highvariability in integrated AKR genomes in thesestrains. The AKR endogenous viral genome,contained in the 55-kbp EcoRI fragment (prob-ably corresponding to the Akv-1 locus [6, 27])and present in all substrains, may have acted asa progenitor ofother endogenousAKR genomes.The AKR/J and AKR/JS substrains have threeproviruses integrated in the same chromosomalsite, whereas the AKR/JS strain possesses oneadditional provirus not present in the AKR/Jstrain. This indicates that three proviral lociwere present before separation and that theintegrated structure is stable for many genera-tions. The additional provirus within the AKR/JS strain may have arisen from a new integrationevent after separation, or the difference mayhave been due to the loss of one provirus by theAKR/J substrain. Another explanation for thehigh variability of the endogenous AKR ge-nomes can be given by assuming a high excisionor mutation rate for some of these integratedgenomes, possibly by virtue of their transposon-like feature (29, 31). However, our recent anal-ysis of 14 different AKR substrains, all withunique integration sites, did not give any indi-cation of the occurrence of excision as an expla-nation for the variability in number and integra-tion site of endogenous AKR genomes in thisstrain. Therefore, we believe that most provi-ruses became stably integrated in the chromo-somal genome by accidental germ line integra-tion.The hybridization pattern of amplified AKR-

MLV sequences observed in tumor tissues wasidentical for tumors obtained from different an-atomical sites ofa single animal but was different

AKR-MLV SEQUENCES IN AKR MOUSE SUBSTRAINS

among individual animals. This suggests a mon-oclonal origin of outgrown tumors which metas-tasized at different anatomical sites of a singleanimal. The number of somatically acquiredAKR-MLV sequences in tumors of AKR micediffers considerably. Similar results have beenobtained for tumors from BALB/Mo mice (16),for M-MLV-infected BALB/c mice, and for M-MTV-type sequences in mice with mammarycarcinomas (10, 20). Within the tumors of differ-ent animals, no common reintegration site wasdetected (Fig. 4, lanes b to i). This suggests thata specific reintegration site of these viruses isnot an etiological cause of leukemia in mice.Further characterization of viral integrationsites are being performed to clarify this point.Furthermore, the genetically transmitted AKRgenomes seem to be unaffected in these tumors;the AKR-MLV-containing restriction endonu-clease fragments observed in nontumor tissuesare present in similar amounts in tumor DNAs.An analysis with restriction enzymes cleaving

within proviral genomes revealed internal frag-ments in tumor DNAs not seen in normal tis-sues. A 2.2-kbp BamHI fragment could be as-signed exclusively to the newly acquired se-quences in tumor DNAs. This fragment waslocalized on the restriction endonuclease map ofthe AKR genome between map positions 4.1 and6.3 kbp. In all tumor DNAs, AKR proviruseswere detected with an EcoRI restriction site atposition 6.8 kbp. The presence of this EcoRI siteand a BamHI site within these reintegrated viralgenomes, which were detected in all tumorsanalyzed, suggests that a recombination hastaken place under strong selection pressure. Theobserved restriction pattern in the recombinantregion is in agreement with the restriction mapof integrated Moloney recombinants detected inM-MLV-induced lymphomas (van der Putten etal., in press). An EcoRI site at position 6.9 kbpfrom the 5' end was also found within proviralDNA of the AKR MCF-247 recombinant virus(21). Therefore, it is likely that the newly ac-quired cellular sequences are derived from en-dogenous xenotropic viral sequences and repre-sent AKR MCF proviruses. The recombinant-specific sequences (solid-line box between mappositions 6.3 and 6.8 on the restriction endonu-clease map of Fig. 7) represent the minimum(observed) length of the substitution. The posi-tions are in full agreement with the MCF-spe-cific regions found by heteroduplex analysis ofAKR MCF and Moloney MCF isolates (6.05 to6.95 kbp from the 5' end of the RNAs) (9). Themajor envelope glycoprotein of M-MLV codedfor by a 2.7- to 3.0-kbp-long fragment located atthe 3' end of the genome starts at position 6.4

kbp from the 5' end of the genome (11, 12; C.Van Beveren, personal communication). There-fore, a heteroduplex analysis (3), our data, andthe data obtained by an analysis of recombinantstructures within DNAs of M-MLV-induced tu-mors (van der Putten et al., in press) stronglysuggest that the amino terminus of the majorglycoprotein of MCF is contributed by xeno-tropic viruses, whereas the carboxy terminusoriginates from the parental ecotropic virus. Thepresence of the same conserved recombinantproviral structure in DNAs of all AKR-MLV-and M-MLV-induced leukemias (we have ana-lyzed 30 different tumors to date) strongly sug-gests that the formation of these recombinantsis a prerequisite for leukemic transformationrather than the accidental consequence of neo-plastic outgrowth.

ACKNOWLEDGMENTSWe are grateful to H. Bloemendal, in whose laboratory this

investigation was carried out. We also thank Els Hulsebos fortechnical assistance, A. Groeneveld for growing the viruses,and F. Janssen for collecting the different mouse strains.

This study was performed as part of the Ph.D. Study of W.Quint and was supported by the Foundation for MedicalResearch, which is subsidized by the Netherlands Organiza-tion for the Advancement of Pure Research.

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