long-range mapping of mis-2, a common provirus integration site

Vol. 67, No. 10JOURNAL OF VIROLOGY, OCt. 1993, P. 5733-57390022-538X/93/105733-07$02.00/0Copyright X 1993, American Society for Microbiology

Long-Range Mapping of Mis-2, a Common ProvirusIntegration Site Identified in Murine LeukemiaVirus-Induced Thymomas and Located 160

Kilobase Pairs Downstream of MybLUC VILLENEUVE,1 XIAOYAN JIANG,1 CHANTAL TURMEL,2

CHRISTINE A. KOZAK,3 AND PAUL JOLICOEURl 4*

Laboratory ofMolecular Biology, Institut de Recherches Cliniques de Montreal, Montreal, Que'bec,Canada H2W lR71*; Xerox Research Centre of Canada, Mississauga, Ontario, Canada LSK 2L12;

Laboratory ofMolecular Microbiology, National Institute ofAllergy and Infectious Diseases,Bethesda, Maryland 20892'; and Departement de Microbiologie et d'Immunologie,

Universite de Montreal, Montreal, Quebec, Canada H3C 3J7, andDepartment of Experimental Medicine, McGill University,

Montreal, Canada H3G IA44

Received 29 March 1993/Accepted 21 June 1993

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in miceor rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens mostfrequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirusinsertions, recognized as common provirus integration sites, have been instrumental in identifying novelcellular genes involved in tumor formation. We have searched for new common provirus integration sites inMoloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of thesetumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%)percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstreamof myb. The Mis-2 region may contain a novel gene involved in tumor development.

Moloney murine leukemia virus (Mo-MuLV) is a highlyleukemogemic retrovirus which induces T-cell leukemia insusceptible mice and rats (9, 11). In general, the tumorsinduced by this virus are clonal or oligoclonal (18). Mo-MuLV is a nondefective virus which contains only threegenes, gag, pol, and env (27). Its genome does not encode acell-derived oncogene. The determinants of its leukemoge-nicity appear to be multiple and have been mapped withinthe U3 long terminal repeat (LTR) and in other regions of thegenome (1, 5).The mechanism by which Mo-MuLV induces T-cell trans-

formation is not yet completely understood, but importantobservations have been made. In the early phase of thedisease, the virus seems to be able to stimulate hematopoi-etic cell proliferation in the spleen, which is thought to be acritical event in initiation of the disease (4, 33). The ability ofits provirus to act as an insertion mutagen is also thought tobe one of the genetic events which is necessary for theinitiation and/or development of MuLV-induced T-cell lym-phoma (3, 18; for reviews, see references 15, 22, and 34).The myc proto-oncogene has frequently been found to beactivated by the integration of proviruses in Mo-MuLV-induced tumors (2, 29). In addition, several other novelregions have been identified as targets of Moloney provirusintegration (15, 22, 34). In some of these common integrationsites, genes corresponding to putative oncogenes have beenidentified, and the mutation by provirus insertion was foundto be associated with the development of T-cell leukemia.The development of T-cell leukemia following Mo-MuLV

infection is very likely a multistep process. Identification of

* Corresponding author.

the genes involved in this process remains an important taskfor an understanding of this disease. To identify some ofthese genes, we have searched for new targets of provirusinsertion in rat Mo-MuLV-induced T-cell leukemia. Wereport here the identification of a novel common provirusintegration site (Mis-2), located on chromosome 10 at about160 kbp downstream of myb.

MATERIALS AND METHODS

Animals and viruses. Tumors induced in Fisher 344 andLewis rats by Mo-MuLV have been described previously(18, 36).DNA extraction and restriction endonuclease digestion.

High-molecular-weight tumor DNAs were prepared fromprimary thymomas as previously described (23). They weredigested with various restriction endonucleases (New En-gland Biolabs, Inc., Beverly, Mass.; Boehringer MannheimBiochemicals, Montreal, Qu6bec, Canada; and P-L Pharma-cia, Montreal, Quebec, Canada), under conditions recom-mended by the manufacturers, as previously described (18).DNAs for pulsed-field gel electrophoresis were prepared asdescribed previously (28). Plugs of DNA were digested in aminimal volume, under conditions recommended by themanufacturer, and directly loaded onto the gel. The Saccha-romyces cerevisiae VP-148 and A-364, Candida shehatae,and Schizosaccharomyces pombe yeast chromosomes wereprepared as described previously (28). Bacteriophage andplasmid DNAs were prepared essentially as described else-where (19, 25).Agarose gel electrophoresis and hybridization procedure.

Digested DNA samples were electrophoresed on 1% agarosegels and transferred onto nitrocellulose or nylon membranes

5733

5734 VILLENEUVE ET AL.

(Amersham Co., Montreal, Quebec, Canada) by the South-ern technique (32) as described previously (18, 23). SpecificDNA fragments were detected by hybridization with 32p_labeled cloned DNA fragments. These cloned DNA frag-ments were excised from vectors and separated by agarosegel electrophoresis. DNA fragments were isolated by elec-troelution onto NA45 affinity paper (Schleicher & Schull),phenol-chloroform extracted, and precipitated with ethanolas described previously (23). Probes were labeled by therandom-priming method with the Klenow fragment of DNApolymerase I in the presence of hexamers (6). Nylon mem-branes were hybridized in 4 x SET (1x SET is 0.15 M NaCl,30 mM Tris, and 2 mM EDTA [pH 7.4]) with 0.1% sodiumpyrophosphate, 0.2% sodium dodecyl sulfate (SDS), 10%dextran sulfate, and 0.5 mg of heparin per ml at 65°C for 16h. After hybridization, all membranes were washed in 2xSSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)for 15 min at room temperature, in 0.1 x SSC-0.5% SDS for1 h at 65°C, and three times for 2 min each time in 0.1 x SSCat room temperature. Membranes were air dried and ex-posed on RP-Royal X-Omat film (Eastman Kodak Co.,Rochester, N.Y.) with a Cronex Lightning-Plus intensifyingscreen (DuPont Co., Wilmington, Del.) at -70°C. Prepara-tion of agarose gels and pulsed-field gel electrophoresis wereperformed at room temperature with 0.8% agarose gels in 1 xTris-borate-EDTA buffer. Filters were prehybridized for 4 hin 10% dextran-1% SDS-1 M NaCl. Fresh buffer containing100 ,ug of sheared DNA per ml was added for hybridization.Molecular cloning procedure. DNA from the Mo-MuLV-

induced tumor 1213-2 (80 ,ug) was digested with EcoRI andcentrifuged on a sucrose density gradient (10 to 40%) aspreviously described (18, 24, 35). Fractions of 0.5 ml werecollected and ethanol precipitated. Aliquots of DNA fromeach fraction were separated by agarose gel electrophoresis,transferred onto a nitrocellulose membrane, and hybridizedwith an Mo-MuLV U3 LTR-specific probe (26). Fractionscontaining viral fragments from 10 to 15 kbp were pooled andligated with EcoRI-cleaved Charon 4A arms (1 ,g). Theligated DNAs were packaged as described elsewhere (18).Recombinant phages harboring viral sequences were identi-fied with the 32P-labeled Mo-MuLV U3 LTR-specific probe.

Phages were eluted, and their DNA was extracted on adouble 5 and 40% glycerol cushion. Inserts were subclonedinto plasmid vectors as described elsewhere (24, 35).

Probes. The Mis-2-specific probes fragments 1 to 5 weresubcloned from pMo-2A, a proviral insertion cloned fromtumor 1213-2. The c-myb probe was MM49 DNA (7). TheMo-MuLV LTR probe consists of a 0.3-kbp PstI-SacI frag-ment from a mutated Mo-MuLV LTR (23, 24, 26).

Somatic cell hybrids. Isolation of the mouse-hamster cellhybrids has been described previously (12, 14). Mousechromosomes were identified directly by Giemsa-trypsinbanding followed by staining with Hoechst 33258 (Hoechst-Roussel Pharmaceuticals, Inc., Somerville, N.J.). Hybridcells were typed for the presence of specific mouse markersas described previously. For analysis in this study, 26hybrids were selected from a larger panel of 76 hybrids.

Genetic crosses. C58/J mice were obtained from The Jack-son Laboratory, Bar Harbor, Maine. NFS/N mice wereobtained from the Division of Natural Resources, NationalInstitutes of Health, Bethesda, Md. Wild-derived mice [Musmusculus musculus (Skive)] were provided by M. Potter(National Cancer Institute) from his colony at HazeltonLaboratories, Rockville, Md. M. musculus musculus (Skive)males were mated with C58/J or NFS/N females, and the F1

A1 2 3 4 5

Kbp .0t;: 'I KbpKbp20 -

14 -

13

9.2.-#,

3.0--

ENZYME: R H X X XT T T T N

PROBE: U3 FR 2,6 FR5B

S X R HS S S H RX SH

BB' B BNB B

R HS S S H ,.* RCLONE MO-2A

PROBES 5 B5 X S 2.6

x 4 x xL 13 _ _J 3

L2Kb, r-------------- 9.2 ----- --- H2Kb ~~H H

FIG. 1. Restriction enzyme analysis of Moloney provirus withinthymoma 1213-2 DNA. (A) Southern blot analysis of provirusespresent in Mo-MuLV-induced thymoma. DNAs (20 pg) were di-gested with various restriction endonucleases, electrophoresed on1% agarose gels, transferred to nitrocellulose membranes, andhybridized with a 32P-labeled viral U3 LTR probe or with Mis-2probe fragment 2.6 (FR2.6) or 5 (FR5). Normal (N) (lane 5) or tumor(T) (lanes 1 to 4) rat DNAs were digested with EcoRI (R) (lane 1),HindIII (H) (lane 2), or XbaI (X) (lanes 3 to 5). 3 P-labeledHindIII-digested A DNA was used as markers. G, germ line frag-ment. (B) Schematic representation of the provirus inserted withinMis-2 and cloned from tumor DNA 1213-2 (clone Mo-2A). Openbox, LTR; close bar, unique-copy cellular probes; line, cellularsequences. At the bottom are shown lengths (in kilobase pairs) ofrearranged fragments generated with XbaI or HindIII. Restrictionendonucleases: B, BamHI; H, HindIII; R, EcoRI; S, SacI; X, XbaI.

females were mated with M. musculus musculus males toproduce backcross progeny (10, 13).DNA was extracted from the livers of inbred mice, M.

musculus musculus, and the backcross progeny. DNAs weredigested with restriction enzymes, electrophoresed on 0.4%agarose gels, and transferred to nylon membranes (HybondN+; Amersham) as described above. Hybridization andwash conditions were as described above.

RESULTS

Cloning of virus-cell junction fragments of Mo-MuLV-induced thymoma 1213-2 and identification of Mis-2. Toidentify novel common proviral integration sites in Mo-MuLV-induced thymomas, the experimental approach usedto identify other provirus integration sites was used (18).Among a series of EcoRI-digested thymoma DNAs, whichcontain several newly acquired Moloney proviruses hybrid-izing with a Mo-MuLV U3 LTR-specific probe (data notshown), one (tumor 1213-2) was selected for its lowernumber of proviruses (Fig. 1A, lane 1). This tumor containedthree detectable newly acquired EcoRI viral fragments of 20,

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ti

ONCOGENESIS AND INSERTIONAL MUTAGENESIS BY MuLV 5735

19, and 14 kbp (Fig. 1A, lane 1) and six HindIII fragments of9, 8, 5, and 4 kbp (data not shown). The 13- to 16-kbp EcoRIand 5- to 9-kbp HindIII size-selected fragments from tumor1213-2 DNA were ligated to EcoRI-digested Charon 4A andHindIII-digested Charon 21 A lambda arms, respectively,and cloned. Recombinant phages were identified with the32P-labeled Mo-MuLV U3 LTR probe.

Several clones from this EcoRI library contained a 14-kbpEcoRI viral fragment, and one of them (X Mo-2A) waschosen for further analysis. Four of the six virus-specificHindIII fragments ((X Mo-2B-1, X Mo-2B-2, A Mo-2B-3, andX Mo-2B-4) were successfully cloned (data not shown); thefifth one was contained within the EcoRI fragment of clone AMo-2A. To ascertain their origin, the inserts from all of theseclones were comigrated with EcoRI- or HindIII-digestedtumor 1213-2A DNA and hybridized with a Mo-MuLV U3LTR-specific probe (data not shown). These cloned provi-ruses were subcloned into plasmid vectors and analyzedwith restriction endonucleases and the Southern blot tech-nique, using the Mo-MuLV U3 LTR probe (data not shown).The 14-kbp EcoRI fragment from clone X Mo-2A was foundto contain a solo LTR (Fig. 1B).

Cellular fragments flanking each provirus (or solo LTR)were subcloned, and those free of repetitive sequences wereused as probes to screen other tumor DNAs to search forcommon proviral integration sites. Using one of the probes(fragment 5) derived from clone Mo-2A (Fig. 1B), we foundrearrangement in the DNA of another T-cell leukemia (seebelow). This region, which represents a common proviralintegration site, was designated Mis-2 (Mo-MuLV integra-tion site).

Restriction endonuclease map of the Mis-2 region. A cruderestriction map of the Mis-2 region was obtained by digestingnormal mouse cellular DNA with various restriction endo-nucleases, alone or in combination, and hybridizing themwith Mis-2 probes 5 and 2.6 (data not shown). This map wasconfirmed by cloning a homologous fragment from an EcoRInormal rat library constructed in Charon 4A. The predicted13.5-kbp EcoRI fragment from the normal Mis-2 region wasalso cloned. A summary of these data is presented in Fig.1B.The solo LTR present in clone pMo-2A was oriented by

restriction endonuclease analysis using the Mo-MuLV U3LTR-specific MS2 probe and a cellular probe derived fromthis clone (fragment 2.6). Double digestion of pMo-2A withEcoRI-XbaI generated two fragments of 11 and 3.3 kbp, andonly the 11-kbp fragment hybridized with the MS2 probe,flanking the U3 sequences at the left of the XbaI site.Similarly, digestion of the same DNA with EcoRI-SacIgenerated two fragments containing LTR sequences, a 3.8-kbp SacI fragment and a 3.2-kbp EcoRI-SacI fragment. Onlythe 3.8-kbp Sacl fragment hybridized with the MS2 probe,confirming that the U3 region of the LTR was at the left ofthe SacI site. In addition, the 3.2-kbp EcoRI-SacI fragmentalso hybridized with the cellular Mis-2 probe 2.6, as ex-pected. This analysis showed that the LTR was orientedfrom left to right in the Mis-2 region (Fig. 1B).

Detection of provirus integrated within the Mis-2 region ofMuLV-induced T-cell leukemias. The frequency at whichMis-2 was occupied by a provirus in other Mo-MuLV-induced thymomas was determined by screening 40 addi-tional KInI-digested tumor DNAs by the Southern tech-nique, using the 32P-labeled Mis-2 fragment 4 probe. KpnIwas chosen because it allowed the screening of a relativelylong region (-20 kbp) and because it cleaved within theMo-MuLV LTR. With the original tumor 1213-2A (Fig. 1A,

A 1 29

H HPROBE. FR5 U3

5 6 7 8

KbpKb*;G - G

logoN u

6.4

toa-

RV RV R R/H R/RV RIXFR5 U3 FR5

BR213-19 RV p RV R

PROVIRUS X*KX B H BB KX

4PROBES 5

R Rd-_ 9 --

6.4 RV--6.5 - - H

FIG. 2. Localization and orientation of the Moloney provirusinserted within the Mis-2 region in rat thymoma 1213-19. (A) DNA(20 ,ug) from tumor 1213-19 was digested with restriction endonu-cleases, electrophoresed on 1% agarose gels, transferred on nitro-cellulose membranes by the Southern technique, and hybridizedwith 32P-labeled Mis-2 probe fragment 5 (FR5) (lanes 1, 3, and 5 to8). The filters shown in lanes 1 and 3 were washed and rehybridizedwith the U3 LTR probe (U3) (lanes 2 and 4, respectively). Digestionwas with HindIlI (H) (lanes 1 and 2), EcoRV (RV) (lanes 3 and 4),EcoRI (R) (lane 5), EcoRI-HindIII (lane 6), EcoRI-EcoRV (lane 7),and EcoRI-XbaI (lane 8). 32P-labeled HindIII-digested A DNA wasused as markers. Asterisks mark germ line fragments (G); arrowsshow the fragments hybridizing with both probes. (B) Correspond-ing partial restriction endonuclease map of the novel Mis-2 DNAfragments in tumor 1213-19. Line, Mis-2 cellular sequences; openbox, LTR; open bar, viral sequences; closed bars, unique-copyprobes. Restriction endonucleases: B, BamHI; H, HindIII; K,KpnI; P, PstI; R, EcoRI; RV, EcoRV; X, XbaI. Lengths areindicated in kilobase pairs.

lane 1), one other tumor (1213-19) (3%) showed a noveltumor-specific fragment in addition to the germ line fragmentof the normal allele (Fig. 2A).To determine whether this rearrangement reflected the

integration of a provirus and if so to map it more precisely,we performed a restriction analysis using Mis-2 (fragment 5and fragment 2.6) and U3 LTR probes. The novel EcoRVand HindIII fragments in tumor 1213-19 were found tocomigrate with a provirus-containing fragment of the sametumor DNA (Fig. 2A, lanes 1 to 4), indicating that theserearranged fragments contained a provirus.

Digestion of thymoma 1213-19 DNA with EcoRI generateda novel 22-kbp rearranged fragment (Fig. 2A, lane 5), com-patible with the insertion of an 8.8-kbp nondeleted provirusin this normal fragment (14 kbp). Digestion of the same DNAwith EcoRI-HindIII, EcoRI-EcoRV, and EcoRV-XbaI gen-erated novel rearranged fragments of, respectively, 9.8, 6.5,and 6.4 kbp in length (Fig. 2A, lanes 6, 7, and 8, respective-ly), allowing us to map the precise integration site at 6 kbpdownstream of the left EcoRI fragment and to identify itstranscription orientation.

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Kbp

A B C1 2 3 1 2 3 4 5 6 7 8 M

460360-'4290 -210'110 -

E1 2 3 4 5 6

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7 8 9 Kbp- 460- 360- 290- 210

242-41'k* 0 C. f

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w

110 - *,,ot

- 48.5 -

- 33.4 -

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GCHROMOSOME 10

-100 -50 -20 0 50 100 150 200 250 Kbp

180 - 40

- 160

M Sf1

B S

)Ism Sm

Myb

MlSm

Sm N Sf

Mis-2

S M

E Sm

FIG. 3. Long-range mapping of the Mis-2 region. Normal mouse DNA, prepared and digested with various restriction endonucleases as

described in Materials and Methods, was separated by pulsed-field gel electrophoresis, blotted onto nylon membranes, and hybridized a

32P-labeled myb-specific probe (A, C, and E) or Mis-2-specific probe 5 (B, D, and F). (A and B) Digestion with NruI (lanes 1), SfiI (lanes 2),and NotI-SfiI (lanes 3). (C and D) Digestion with SacII (lanes 4), MluI-SacII (lanes 5), BssHII (lanes 6), BssHII-SacII (lanes 7), andBssHII-MluI (lanes 8). Lane M, bacteriophage multimers as size markers. (E and F) Digestion with SfiI (lanes 1), MluI (lanes 2), NotI-SfiI(lanes 3), NotI-MluI (lanes 4), NotI-SmaI (lanes 5), MluI-SfiI (lanes 6), Sfil-SmaI (lanes 7), MluI-SacII (lanes 8), and MluI-SmaI (lanes 9).(G) Long-range restriction map of the mouse myb/Mis-2 region. Positions of myb and the Mis-2 region are shown. The length of the Mis-2locus remains unknown and may be shorter or longer than the hatched box shown. Restriction endonucleases: B, BssHII; M, MluI; N, NotI;S, SacII; Sf, Sfil; Sm, SmaI. Lengths are indicated in kilobase pairs.

Surprisingly, the two proviruses detected within the Mis-2region are inserted in the opposite transcriptional orienta-tion.Although the frequency of integration within this region is

low, these results indicated that Mis-2 is a common proviralintegration site. Considering the low probability of twoindependent proviral integration events occurring at randomin the same 20-kbp genomic fragment, this event remainshighly significant (22).Mis-2 maps to mouse chromosome 10. To map the Mis-2

region to a specific mouse chromosome, a series of Chinese

hamster-mouse somatic cell hybrids, each containing a dif-ferent subset of murine chromosomes (12, 14), was firstscreened. DNAs from these cell hybrids were cleaved withEcoRI and analyzed by the Southern technique with theMis-2-specific probe 5. The murine fragment (14 kbp) couldbe distinguished from the homologous hamster fragment (3.3kbp) by its length (data not shown). A total of 26 Chinesehamster-mouse hybrid cell DNAs were analyzed and scoredas negative or positive for the presence of the mouse-specificfragment. This analysis revealed that Mis-2 maps to mousechromosome 10 (data not shown). The myb and gamma

D4 5 6 7 8

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4.

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interferon genes and the Ahi-1 common provirus integrationsite have been mapped to chromosome 10. We screened theA Mo-2A cloned DNA with these probes and found no

homology (data not shown).To position Mis-2 on the mouse linkage map, the progeny

of a multilocus cross were typed for restriction enzyme

variants of Mis-2 by Southern blotting. Digestion withBamHI produced fragments of 12.8 kbp in parental NFS/Nand C58/J mouse DNAs and 6.6 kbp in M. musculus mus-

culus. Inheritance of the 12.8-kbp fragment was comparedwith inheritance of other chromosome 10 markers typed inthis cross. No recombinants between Mis-2 and myb in 88mice were observed, which suggests that at the 95% confi-dence interval, Mis-2 and myb lie within 3.4 centimorgans(8).

Physical mapping of the Mis-2 region downstream of myb.Since the genetic studies located the Mis-2 locus close tomyb, it was of interest to determine the proximity of theseregions more precisely by using pulsed-field gel electro-phoresis. This analysis was performed with myb cDNA andrat Mis-2 (fragment 5) probes. The rat Mis-2 probe ishomologous to mouse Mis-2 DNA. Previous results haverevealed the presence of SacII, BssHII, and SmaI sites atthe 5' end of the myb gene and of SmaI sites within the mybgene. We first confirmed that these sites were cleaved incellular DNA (data not shown).

Digestion of normal mouse cellular DNA with severalrestriction endonucleases generated distinct fragments afterhybridization with Mis-2 probe 5: NruI (380 kbp) (Fig. 3B,lane 1), SfiI (220 kbp) (Fig. 3B, lane 2), SacII (230 kbp) (Fig.3D, lane 4), BssHII (230 kbp) (Fig. 3D, lane 6), BssHII-SacII(230 kbp) (Fig. 3D, lane 7), and BssHII-SfiI (160 kbp) (datanot shown). The same fragments were detected with thec-myb probe (Fig. 3A, lanes 1 and 2; Fig. 3C, lanes 4, 6, and7). Moreover, the c-myb and Mis-2 probes detected commonpartial SfiI restriction fragments of 240, 320, and 380 kbp(Fig. 3E and F, lanes 1). These results indicated that the myband Mis-2 regions are located within a 160-kbp DNA frag-ment.

Digestion of DNA with MluI and hybridization with thec-myb probe revealed a completely digested 240-kbp frag-ment and a partially digested 360-kbp fragment (Fig. 3E, lane2). The same blot hybridized with the Mis-2 probe detectedthe 360-kbp partially digested fragment and an additional120-kbp fragment (Fig. 3F, lane 2), suggesting that the myband the Mis-2 regions are separated by a single MluI site andthat the 360-kbp partially digested MluI fragment representsthe combined 240-kbp myb and 120-kbp Mis-2 fragments.A SfiI-MluI double digestion revealed a 40-kbp fragment

hybridizing with Mis-2 fragment 5 probe (Fig. 3F, lane 6) anda 180-kbp fragment hybridizing with the myb probe (Fig. 3E,lane 6), indicating that one MluI restriction site separatesboth regions. DNA digested with NotI generated very longfragments (>1,500 kbp) hybridizing with both the myb andMis-2 (fragment 5) probes (data not shown). However, theSfiI-NotI double digestion revealed a 35-kbp fragment hy-bridizing with the Mis-2 probe (Fig. 3F, lane 3) and a 190-kbpfragment hybridizing with the myb probe (Fig. 3E, lane 3),thus placing a NotI site within the SfiI 220-kbp fragmentclose to the MluI site. This was confirmed by the MluI-NotIdouble digestion, which showed that the 120-kbp MluI Mis-2fragment was not recut by NotI (Fig. 3F, lane 4).The myb gene could be mapped on a shorter fragment, as

120-kbp MluI-SacII and MluI-BssHII bands could be de-tected with the c-myb probe (lanes 8 in Fig. 3E and C,respectively).

The 14- and 60-kbp SmaI myb fragments (Fig. 3E, lane 9)and the 120-kbp SmaI Mis-2 fragment (Fig. 3F, lane 9) werenot altered by further digestion with MluI. The fact that a 5'myb probe and a 3' myb probe could detect the same 240-kbpMluI fragment (data not shown) confirmed the absence of anMluI site between the 14- and 60-kbp SmaI fragmentsrepresenting the 5' and 3' ends of the myb gene. Moreover,cleavage of the 220-kbp Sfil fragment by MluI resulted in thegeneration of a 40-kbp Mis-2 fragment (Fig. 3F, lane 6) andof a 180-kbp myb fragment (Fig. 3E, lane 6). Since Mis-2 andmyb share a common Sfi1 fragment and since an MluI sitelies between the Mis-2 region and the myb gene, the tworegions can thus be localized to both sides of an MluI site.Moreover, since the 240-kbp c-myb MluI fragment is re-duced to 120 kbp with BssHII (Fig. 3C, lane 8) and SacII(Fig. 3C, lane 5) and since the BssHII and SacII sites areknown from sequencing data to be located 5' upstream of thefirst exon of myb and were shown to cleave at this site incellular DNA (data not shown), the Mis-2 region must belocated at about 160 kbp downstream of the first myb exon.Data from additional double digestions of DNA by NotI-SmaI (Fig. 3E and F, lanes 5) and by other enzymes (Table1) further confirmed these results. A restriction map sum-marizing these data is presented in Fig. 3G.

DISCUSSION

One of the important mechanisms by which Mo-MuLVinduces T-cell leukemia appears to be through the integra-tion of its provirus within or around genes involved ingrowth control (15, 22, 34). Several genes and severalregions of the genome have been identified as being thetargets of provirus integration. In MuLV-induced tumors,the proto-oncogene myc appears to be one of the mostfrequent genes to be activated by such a provirus integrationevent (2, 29). Using this provirus insertion mutagenesisapproach, we have now identified a novel common provirusintegration site, Mis-2, in Mo-MuLV-induced rat thymoma.The frequency of provirus occupancy of this region was

TABLE 1. Summary of restriction fragments homologousto c-myb and Mis-2 probes

Length of fragment (kbp)Enzyme(s)

Mis-2 probe myb probe

ApaI > 1,500 > 1,500BssHII 230 230MluI 120, 360Pa 240, 360PNotI > 1,500 > 1,500NruI 380 380SacII 230 230SalI 220 220SfiI 220, 240P, 320P, 380P 220, 240P, 320P, 380PSmaI 120 14MluI-BssHII 120, 230P 120, 230PMluI-NotI 120 240MluI-SacII 130 120MluI-SfiI 40 180MluI-SmaI 120 14NotI-SfiI 35 190NotI-SacII 120 120NotI-SmaI 120 14SacII-SmaI 120 14BssHII-SacII 230 230BssHII-SfiI 160 160

a p indicates a partially digested fragment.

VOL. 67, 1993


found to be low (-3%). A low frequency of provirusoccupancy has also been observed for many other loci ofgreat biological significance (15, 22, 34). However, theidentification of the novel common integration site Mis-2remains highly significant, since the probability of two suchintegration events occurring independently in two differenttumors in a short 5-kbp fragment is very low, around 10-10for random integration (22). This site is unlikely to corre-

spond to the preferred integration targets reported by Shih etal. (31), since these targets were found to occur at preciselythe same base and at a lower frequency (once per 4,000integrations) than the one found for Mis-2. This observationsuggests that integration of the viral genome within the Mis-2region somehow confers a growth advantage to the cells inwhich it occurs. Presumably, these inserted proviruses areactivating a gene, which may behave as an oncogene, or

inactivating a tumor suppressor gene.Interestingly, Mis-2 was found to map on mouse chromo-

some 10, close to myb. Our long-range mapping of thisregion has allowed us to locate Mis-2 at about 160 kbpdownstream of myb. The myb gene has previously beenfound to be activated by provirus insertion in plasmocytoidlymphosarcomas or in acute myeloid leukemia, but in thesetumors, the proviruses were all integrated at the 5' end of thegene, very often truncating the N-terminal coding region ofthe gene (16, 20, 30, 37). In humans, several chromosomalabnormalities have been reported in the 6q region, aroundthe myb locus, but long-range mapping of the myb gene intumors harboring these abnormalities has shown that norearrangements or deletions occurred within 1 Mbp of myb(21). Therefore, the gene involved in these human translo-cations is very likely distinct from Mis-2.

Since long-distance activation of myc by provirus integra-tion in Mlvi-1 and Mlvi-4 loci has previously been reported(17), we considered the possibility that provirus integrationwithin Mis-2 could influence myb transcription. We testedthis hypothesis and found no significant alteration of the mybRNA levels in tumors harboring a Mis-2 rearrangementcompared with those with no Mis-2 rearrangement (data notshown). Interestingly, the Mis-2 region also appears distinctfrom another gene, Ahi-1, initially identified as a commonprovirus integration site in Abelson MuLV-induced pre-B-cell lymphoma (24). We have recently mapped the Ahi-1gene within the same myb/Mis-2 region, at about 35 kbpdownstream of myb (unpublished data). Therefore, it ap-pears that the Mis-2 region may be involved in oncogenesisindependently of the myb and Ahi-1 genes.

It will be interesting to identify the putative Mis-2 genewhich may be affected by provirus integration. The presenceof rare restriction sites associated with a CpG island aroundthe Mis-2 region may suggest the existence of a transcrip-tional unit in this region.

ACKNOWLEDGMENTS

This work was supported by grants to P.J. from the MedicalResearch Council of Canada and the National Cancer Institute ofCanada. X.J. was the recipient of a studentship from McGillUniversity and from Hydro-Quebec.We are grateful to T. J. Gonda and L. Wolff for providing the myb

probes. We thank Benoit Laganiere for excellent technical assis-tance and Marie Bernier for typing the manuscript.

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VOL. 67, 1993

long-range mapping of mis-2, a common provirus integration site

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