Patterns of Proviral Insertion and Deletion in Avian Leukosis Virus- Induced Lymphomas

Transcription

1 JOURNAL OF VIROLOGY, Jn. 1986, p X/86/ $02.00/0 Copyright C 1986, Americn Society for Microbiology Vol. 57, No. 1 Ptterns of Provirl Insertion nd Deletion in Avin Leukosis Virus- Induced Lymphoms H. L. ROBINSON* AND G. C. GAGNON Worcester Foundtion for Experimentl Biology, Shrewsbury, Msschusetts Received 12 July 1985/Accepted 26 September 1985 Sixty-eight lymphoms induced by eight different vin leukosis viruses hve been nlyzed on Southern blots for virus-induced muttions in the chicken c-myc gene. Sixty-six of the lymphoms exhibited provirl insertion in c-myc, wheres one exhibited new trnsduction of c-myc. Sixty-four of the provirl insertions were in the sme trnscriptionl orienttion s c-myc. Two were in the opposite trnscriptionl orienttion. All of the insertions were upstrem of the protein-coding sequences of c-myc, with most residing in the first exon or the first intron of c-myc. All of the lymphom-inducing proviruses hd deletions tht included either sequences ner the 5' long terminl repet (LTR) or n LTR. The most frequent lymphom-inducing provirus ppered to hve retined both of its LTRs, but hd lost sequences ner its 5' LTR. The second nd third most frequent lymphom-inducing proviruses consisted of solo LTRs or of proviruses tht hd lost the 5' LTR s well s some internl sequences. Twenty-four insertions were mpped in c-myc. Ech of these mpped to within 150 bse pirs of one of the five DNse I-hypersensitive sites tht occur in 3-kilobse region immeditely 5' to the protein-coding sequences of c-myc. One lymphom contined new c-myc trnsducing virus. This virus, MYC-3475, cused rpid-onset myelocytomtosis. The induction of B-cell lymphom by vin leukosis viruses (ALVs) is ssocited with provirl insertions in the chicken c-myc gene (5, 9, 21). Virtully ll of these insertions increse the expression of c-myc either by the initition of trnscripts in provirl long terminl repet (LTR) or by the enhncement of the expression of norml trnscripts (5, 9, 14, 21). In no cse hs lymphom-inducing insertion truncted the c-myc protein product. Consequently, insertions re thought to cuse lymphom by up-regulting the trnscription of c-myc. In this mnuscript we report dt obtined from the nlysis of 68 ALV-induced lymphoms for provirl insertions in c-myc. In greement with the work of others, we find tht the chicken c-myc is frequent trget for lymphominducing insertions nd tht lymphom-inducing proviruses hve sustined deletions (4, 5, 9, 17, 21, 22, 29). However, in contrst to the implied results of others, we find tht the deletions in lymphom-inducing proviruses do not necessrily include ll of one of the provirl LTRs. Rther, the most frequent form of lymphom-inducing provirus hs retined sequences in both of its LTRs but sustined n internl deletion. These internl deletions pper to include t lest 500 bse pirs (bp) ner the 5' LTR. MATERIALS AND METHODS Viruses. Ech of the eight ALVs used to induce lymphoms contined the gg (core proteins), pol (RNA-directed DNA polymerse), nd env (envelope glycoproteins) genes nd LTR sequences tht re chrcteristic of repliction competent ALVs. None contined trnsduced host sequences. Rous ssocited virus type (RAV-1) (28) ws obtined from L. B. Crittenden of the Regionl Poultry Reserch Lbortory, Est Lnsing, Mich. The RAV-60s were obtined from H. nd T. Hnfus, Rockefeller University, New York, N.Y. The RAV-60s represent four * Corresponding uthor. different recombinnts between RAV-1 or RAV-2 nd endogenous ALVs (26). Fujinmi-ssocited virus ws isolted from stock of Fujinmi srcom virus provided by H. Hnfus. This isolte of Fujinmi-ssocited virus hs n EcoRI site in its LTR. Nontrnsforming subgroup E virus strin 2 is recombinnt of the Prgue strin of Rous srcom virus type B nd RAV-0 (35). It ws obtined from J. Coffin, Tufts University School of Medicine, Boston, Mss. A moleculrly cloned isolte of RAV-1 (prav-1) ws obtined from J. M. Bishop nd H. E. Vrmus. Virus recovered from this clone is considered to be different from tht obtined from L. B. Crittenden, since RAVs mintined in different lbortories frequently hve different sequences in their enhncer regions (2, 10, 11, 40). Virus stocks were obtined by hrvesting the culture medium of infected chicken or turkey cells. Lymphoms. Lymphoms were hrvested from K28 or (K28 x 151) x K28 chickens tht hd been intrvenously inoculted t 1 dy of ge with -106 IU of n ALV (24-26). Tumor nd control tissues were hrvested from moribund or recently decesed chickens. A portion of these tissues ws fixed in Formlin for subsequent histologicl nlyses. The bulk of the tissue ws quick-frozen nd stored t -80 C to preserve the tumor DNA, RNA, nd proteins for biochemicl nlyses. Anlysis of tumor DNA. DNA ws hrvested from tumor tissues, digested with restriction endonucleses, nd nlyzed on Southern blots s described by Miles nd Robinson (15). DNA blots hybridized with the MYC, SMA, nd IN2 probes were wshed t higher stringencies thn blots hybridized with other probes. These high-stringency wshes (0.3 x SSC [lx SSC is 0.15 M NCl plus M sodium citrte], 0.1% sodium pyrophosphte, 0.1% sodium dodecyl sulfte; 65 C) served to remove much of the nonspecific hybridiztion observed for probes with the G+C-rich regions of c-myc. Mny of the Southern blots underwent sequentil hybridiztions with severl probes. Probes were melted from blots by wshing the blot for 15 min t 45 C in 0.3 x SSC plus 28

2 VOL. 57, 1986 c-myc RAV-I R H S SmSmnSmS PHC R HS 11 J IJ.. II. 11 RSBB R B B R R provirus I I L I I PVA PVB I m U - SMA IN2EX3 MYC kilobses FIG. 1. Restriction endonuclese sites nd probes for c-myc nd RAV-1 sequences. Symbols: 0, c-myc exons; _, probes; O, provirl LTR (the entire LTR ws used s probe for LTR sequences, the filled re 3' to the EcoRI site represents sequences used for the 3' LTR probe). Restriction endonucleses: B, BmHI; C, ClI; H, HindIll; P, PstI; R, EcoRI; S, ScI; Sm, SmI. Sites for ClI nd PstI hve not been mpped throughout the c-myc sequence. Positions of exons nd introns in c-myc re from Wtson et l. (39), Shih et l. (32), nd Linil nd Groudine (14). 50% formmide followed by wshing the blot for 5 min in 0.3 x SSC-0.1% sodium dodecyl sulfte. Blots were then ir dried nd verified to be free of 32P-lbeled DNA by utordiogrphy. DNAs used for hybridiztion probes. The DNAs used for hybridiztion with c-myc were obtined from the recombinnt plsmid pcmcbam (38). Subclones of pcmcbam were used to detect sequences contining the first exon, the second intron, nd the third exon of c-myc (Fig. 1). The subclone of sequences in the third exon ws kind gift of M. Linil. On some blots, SlI frgment of cloned v-myc sequences (37) ws used s probe for third-exon sequences. This probe hybridizes with sequences in the 16- kilobse (kb) EcoRI frgment of c-myc tht contins the first-, second-, nd third-exon sequences s well s -20-kb frgment of c-myc tht contins only third-exon sequences. The SMA nd IN2 DNAs were prepred by isolting pproprite restriction endonuclese frgments of pcmcbam from low-melting-point grose gels nd cloning them into puc plsmids. The IN2 DNA ws isolted from its cloning vector before nick trnsltion. The PVA probe for provirl gg sequences is the frgment bounded by BmHI sites t bp -550 nd of provirl DNA. A pbr322 clone contining PVA sequences in the BmHI site of pbr322 ws kindly provided by workers in the lbortories of J. M. Bishop nd H. E. Vrmus. The PVB probe is the 3.8-kb EcoRI frgment of prav-1 DNA. This frgment is flnked by the EcoRI sites t bp nd in provirl DNA. PVB DNA s well s sequences representing one complete LTR (internl EcoRI frgment from the tndem LTRs in prav-1) were isolted from EcoRI-digested prav-1 DNA. The 3' LTR probe comprises sequences 3' to the EcoRI site in the LTR. The isoltion of this probe from p53, cdna clone of PrRSV-C, is described by Miles nd Robinson (15). Sizing of DNA frgments. Frgments detected on Southern blots were sized by compring the mobility of the frgment with tht offrgments of HindIII-digested lmbd DNA. The positions of norml c-myc nd provirl frgments (30, 39) were lso used in the construction of the sizing curve. LYMPHOMA-INDUCING PROVIRAL INSERTIONS 29 DNse I-treted DNA. DNA isolted from DNse I-treted chromtin from the burs of 2-week-old chickens ws kind gift of Mrk Groudine nd co-workers (29). RESULTS Screening of ALV-induced lymphoms for provirl insertions in c-myc. Lymphoms were screened for provirl insertions in c-myc by sequentilly hybridizing Southern blots of EcoRI-digested DNAs with probes for the third exon of c-myc (EX3 probe), for sequences 3' to the EcoRI site in the LTR (3' LTR probe), nd for sequences within the most 5' EcoRI frgment of the provirus (PVA probe) (Fig. 1). Hybridiztion of lymphom-specific frgment with the EX3 probe provided evidence for the involvement of c-myc in the genertion of the lymphom. Hybridiztion of the sme frgment with the 3' LTR probe suggested tht the tumor ws induced by n insertion in the sme trnscriptionl orienttion s c-myc. Filure of the tumor-specific bnd to hybridize with the PVA probe suggested tht the 3' LTR of provirus ws t the junction of provirl nd c-myc sequences. The initil screen detected novel EcoRI frgments of c-myc in 67 of the 68 lymphoms (Tble 1); 80% of these were 2.9- to 4.0-kb EcoRI frgments tht hybridized with the EX3 nd 3' LTR probes, but not with the PVA probe. Herefter, such frgments re termed typicl frgments, wheres frgments of other sizes or ptterns of hybridiztion re termed typicl. Detiled mpping of selected lymphom-inducing insertions. All of the RAV-1-induced lymphoms s well s ll of the lymphoms with typicl novel frgments were tested for 5' s well s 3' junctions of provirl nd c-myc sequences, for deletions in provirl sequences, nd for deletions or rerrngements in flnking host sequences. The gols of these studies were to define the ptterns of lymphominducing insertions ssocited with virus tht induces high incidence of lymphom nd to obtin number of exmples of less frequent ptterns of insertions. 5' nd 3' junctions of provirl nd c-myc sequences. Junction frgments 'of provirl nd c-myc sequences were identified in EcoRI-digested DNA s well s in HindIII-EcoRIdigested DNAs (see Fig. 1 for reltive positions of EcoRI nd Hindlll sites in c-myc). Southern blots of these digests were sequentilly hybridized with the 3' LTR, the LTR, the EX3 (EcoRI-digested DNAs), the IN2 (EcoRI-HindIIIdigested DNAs), the SMA, nd the MYC probes (Fig. 1). This series of hybridiztions served to define both the 5' nd TABLE 1. Lymphoms tested for novel c-myc frgments No. with novel c-myc frgments Inducing virus Typicl Atypicl None RAV NY201RAV NY202RAV NY203RAV NY204RAV Fujinmi-ssocited virus Nontrnsforming subgroup E virus strin 2 Recovered from prav A typicl frgment is 2.8- to 4.0-kb EcoRI frgment tht hybridized with the EX3 nd 3' LTR but not the PVA probes. An typicl frgment exhibited size or hybridiztion pttern tht ws not frequently observed. None indictes tht the lymphom did not exhibit virl integrtion between the EcoRI site 5' of c-myc nd BgII site -4 kb 3' of c-myc (Fig. 1).

3 30 ROBINSON AND GAGNON J. VIROL. TABLE 2. Orienttions nd ptterns of deletion of lymphom-inducing proviruses in series of 32 RAV-1-induced lymphoms Orienttion frgmentb Provirl deletion Host deletion, No. Tble" Sme Sequences ner 5' LTR Unlikely 14 3 Sme All except one LTR Unlikely 6 4 Sme Including 5' LTR Likely 5 5 Sme Including 3' LTR Likely 2 6 Sme 3.2 sequences ner 5' LTR, Likely 2 7 LTR (?) Sme 10.5, 12.5? Likely 2 8 Opposite 3.5, 7.5 Including 5' LTR Likely 1 9 Trnscriptionl orienttion of the provirus with respect to the trnscriptionl orienttion of c-myc. b Size in kilobses of novel frgment tht hybridized with EX3 probe. c Tble in which ech clss is presented in detil. 3' junctions of provirl nd c-myc sequences, to mp first- lymphom DNAs with SmI, n enzyme tht genertes n exon sequences in these junction frgments, nd to identify -1-kb frgment contining first-exon sequences (Fig. 1). The LTR sequences tht might be present in junction frgments SmI-digested DNAs were hybridized on Southern blots (Fig. 2). with the SMA probe. SmI-digested DNAs displyed fint Deletions or rerrngements in provirl or c-myc sequences. s well s intense novel frgments. The fint bnds could be Deletions or rerrngements were identified by hybridizing due to incomplete digests (SmI is methyl-sensitive en- Southern blots of ScI-digested DNAs with the MYC probe zyme) (Lym3095 nd 1789; see Tble 3) or limited sequence (Fig. 1). Scl ws chosen for these digests since 7-kb Scl homology of novel frgment with the SMA probe frgment of c-myc encompssed the pprent cceptor sites (Lym3029; see Tble 5). Fint bnds were not observed for for virtully ll of the lymphom-inducing insertions nd other digests. since there is only one Scl clevge site in provirl DNA. If Seven different clsses of lymphom-inducing proviruses. this site were present in provirus, the insertion should The bove nlyses reveled seven different clsses of result in two tumor-specific Scl frgments. If this site is not lymphom-inducing proviruses (Tble 2). Six of the clsses present, the insertion should result in only one tumor- contin proviruses in the sme trnscriptionl orienttion s specific Scl frgment. In either instnce, the size of the c-myc, wheres the seventh contins proviruses in the novel ScI frgment or frgments provides n estimte of the opposite trnscriptionl orienttion to c-myc. All of the totl mount of provirl (7.8-kb) nd c-myc (7.0-kb) se- clsses contin proviruses inserted 5' to the first coding exon quences retined in the locus which hs sustined n inser- (second exon) of c-myc. No proviruses were observed tion. downstrem of c-myc. Test for perturbtions in first-exon sequences. Since most The next seven sections of the Results describe the of the lymphom-inducing insertions ppered to reside in different clsses of lymphom-inducing insertions. Tbles 3 the first intron or exon of c-myc, the integrity of the first through 9 summrize the DNA frgments used to clssify the exon ( non-protein-coding exon) ws exmined by digesting insertions, wheres Fig. 2 through 4 present restriction DNA TABLE 3. Lymphom-inducing proviruses: sme trnscriptionl orienttion, internl deletions ner 5' LTR HindIII-EcoRI ScIS E1 5' (LTR, MYC) 3' (LTR, 3' LTR, 5' (LTR, MYC) 3'(LTR, 3' LTR) (MYC) Smi (EXI) ~~MYC) ~~ MYC, IN2) MYC, EX3) Lyml NT 2.9NT 7.5NT NT Lym NT 314NT14NT NT Lym None Lym None Lym None Lym None Lym None Lym None Lym None Lyml None Lyml None Lym NT NT 9 NT NT Lym , (2.4), 6.2 Lym ? 1.6, (2.7), 6.5 Lyml486b ? 3.9k , (3.0) All proviruses re RAV-1 except where otherwise indicted. + nd - superscripts indicte hybridiztion nd no hybridiztion, respectively, with the SMA probe for the EcoRI digest nd with the PVA probe for the Scl digest. NT indictes not tested. Prentheses indicte fint bnds. b NY201RAV-60 provirus. EcoRI

4 VOL. 57, 1986 A. Lym 1801 B. Lym 1961 C. Lym 3020 D. Lym 5535 E. Lym 1524 R H S SmSm S H R NS LpII Wt R R delefion SocI site mincudoo R R HS 8 SmSm S H RHS I 11 1I?cel lulr R H S B Sm Sm S H R HS I I I I dd I R R R? virl 7eel lulr R HMS B SmSm S H R HS R?virol R? cellulr R H S B SmS H R HS I FIG. 2. Schemtics of lymphom-inducing proviruses. Ech schemtic represents likely restriction mp. A, Lyml801, two-ltr provirus in the sme trnscriptionl orienttion s c-myc tht hs sustined n internl deletion (Tble 3). B, Lyml961, solo LTR in the sme trnscriptionl orienttion s c-myc (Tble 4). C, Lym3O2O, 5' LTR-deleted provirus in the sme trnscriptionl orienttion s c-myc (Tble 5). D, Lym5535; 5' LTR-deleted provirus in the opposite trn5criptionl orienttion to c-myc (Tble 9), E, Lyml524, 3' LTR-deleted provirus in the sme trnscriptionl orienttion s c-myc (Tble 6). Prentheses indicte deletion tht includes the Scl site. Other symbols nd designtions re s in Fig. 1. endonuclese mps nd frgments of representtive insertions. The results section ends with the mpping of 24 of the insertions with respect to DNse-I hypersensitive sites in c-myc (see Fig. 5). Lymphom-inducing proviruses with internl deletions. The most frequently observed lymphom-inducing provirus retined sequences in both of its LTRs but hd deleted sequences ner its 5' LTR. This clss of proviruses represented 44% of the RAV-1 insertions (Tble 2). Southern blots of ech of the 15 proviruses in this clss exhibited two novel EcoRI-HindIII frgments (Tble 3, Fig. 2A, Fig. 3). One of these, the 3' junction of provirl nd c-myc sequences, hybridized with the IN2 nd 3' LTR probes (Fig. 3, lnes 1 nd 2). The other, the 5' junction frgment, hybridized with the MYC nd the LTR probes (Fig. 3, lnes 3 nd 4). In individul lymphoms, the sum of the sizes of the 5' nd 3'junction frgments rnged from 9.0 to 9.3 kb (Tble 3). This sum is close to wht would be expected for frgments with one provirl LTR (-0.3 kb) plus the sequences present in the 8.8-kb HindIII frgment of c-myc (Fig. 1). I' LYMPHOMA-INDUCING PROVIRAL INSERTIONS 31 Similr nlyses of EcoRI digests of ech of the internlly deleted insertions confirmed the presence of LTR sequences t the 5' nd 3' junctions of provirl nd c-myc sequences (Tble 3; Fig. 3, lnes 5 through 9). Agin, the sum of these junction frgments pproximted the size of one LTR plus the sequences present in the -16-kb EcoRI frgment of c-myc (Tble 3). Hybridiztion of blots of EcoRI-digested DNA with the SMA probe indicted tht nine of the internlly deleted proviruses resided 3' to the first exon of c-myc (the SMA probe hybridized with the 5' junction frgment), wheres two resided within the SmI frgment tht contins first-exon sequences (the SMA probe hybridized with both the 5' nd 3' junction frgments). Despite the presence of both LTRs nd pprently unperturbed cellulr sequences in ech of these lymphoms, Scl digests reveled only one tumor-specific frgment (Tble 3, Fig. 2A, Fig. 3). In ech cse the tumor-specific Scl frgment ws lrger thn the 7.0-kb Scl frgment of c-myc nd smller thn the -15-kb frgment expected for complete provirus with point muttion in its Scl site. Twelve of the novel Scl frgments were tested for hybridiztion with the PVA probe. Ten exhibited no discernible hybridiztion. Thus, most of the deletions ppered to include sequences from 155 bp (the ScI site) to -1,750 bp (the 3' boundry of the PVA probe) immeditely 3' to the 5' LTR. The lrgest deletions resulted in proviruses tht ppered to retin little but two LTRs (lymphoms 1814 nd 1486), wheres the smllest deletions ppered to encompss only 500 to 1,000 bp of provirl sequences (lymphoms 4446, 3211, 1801, 3095, nd 1789). These striking results suggest tht two LTR proviruses induce lymphoms when nd only when they hve deleted sequences ner their 5' LTR. Lymphom-inducing proviruses with solo LTRs. Nineteen percent of lymphom-inducing RAV-1 proviruses (Tble 2) ppered to hve lost ll of their sequences except for one LTR (Tble 4, Fig. 2B). This clss, referred to s the solo LTR clss, ws presumbly generted by homologous recombintion between the LTRs of n intct provirus (3, 36). HindIII-EcoRI s well s EcoRI digests of DNAs contining solo LTRs reveled LTR nd pprently unperturbed host sequences t both the 5' nd 3' junctions of provirl nd c-myc sequences. The Scl digests of these DNAs did not revel tumor-specific frgment. Presumbly this ws due to the similrity in size of the Scl frgment of c-myc (7.0 kb) nd the predicted ScI frgment of c-myc with solo LTR (7.3 kb). Lymphom-inducing proviruses with 5' LTR deletions. Sixteen percent of lymphom-inducing RAV-1 proviruses (Tble 2) hd sustined deletions of their 5' LTR s well s some of their internl sequences (Tble 5, Fig. 2C). HindIII- EcoRI s well s EcoRI frgments of these proviruses contined LTR sequences in their 3' junction frgments but not their 5' junction frgments. Since the 5' junction frgments of these proviruses contined undefined mounts of virl sequences, the sizes of these frgments cn not be used to estimte their content of c-myc sequences. However, four of the five 5' LTR deleted proviruses hd 5' junction frgments tht were either not detected or shorter thn would be predicted from the restriction mp of c-myc. In ech of these cses, it is pprent tht host sequences 5' to the provirus hve undergone mjor deletions or rerrngements. In two of the lymphoms (3018 nd 4566) c-myc sequences 3' s well s 5' to the inducing provirus pper to hve undergone deletions or rerrngements. Thus deletions tht included 5' LTR s well s some internl sequences

5 32 ROBINSON AND GAGNON J. VIROL. Hindin: + EcoRI 3' Jnct 5'Jnct 'Jnct Eco RI 5'Jnct 8 9 ScI io * ~*13- Wm_ 13* * _w ~~~~~~85 i _~~~~~~ _ * * S * 2.3 ti.e * EX3, 3'LTR, PVA SMA,LTR MYC,PVA I N2,3'LTR.MYC,LT R FIG. 3. Restriction endonuclese frgments used in the mpping of lymphom-inducing provirus with n internl deletion ner its 5' LTR. Dt re for the lymphom-inducing provirus in Lyml801 (Tble 3). For schemtic digrm of this provirus, see Fig. 2A. Ech lne presents dt from utordiogrphs of Southern blots. Lnes obtined from sequentil hybridiztions of the sme Southern blot re grouped. The restriction endonucleses used to digest Lyml801 DNA re indicted bove the lnes, nd DNA frgments used s hybridiztion probes re indicted below the lnes. Sizes of frgments re given in kilobses. Asterisks indicte tumor-ssocited frgments. Other frgments represent unperturbed c-myc sequences, the endogenous virus tht resides t ev-1, or proviruses tht were not integrted in c-myc. Hybridiztion of the 3' junction frgments with the MYC probe gve bnds tht were discernible in the originl utordiogrphs, but not in the reproductions. Cler evidence for these frgments is seen in the hybridiztion ptterns of the sme blot (HindIII-EcoRI digest) or of different blots (EcoRI digest) with the EX3 probe. tended to be ccompnied by deletions or rerrngements in host sequences (Tble 5) (36). Lymphom-inducing proviruses with 3' LTR deletions; new trnsduction of c-myc. Five of the proviruses in the sme trnscriptionl orienttion s c-myc hd recombined c-myc sequences with sequetces internl to the provirus. This ws evidenced by the 3' junction frgments hybridizing with the PVA or PVB probes (Tble 6, Fig. 2E, Fig. 4). Three proviruses ppered to hve undergone recombintion of provirl nd c-myc sequences 5' to the EcoRI site t bp 2400 of provirl DNA (proviruses in lymphoms 3055, 1524, nd 3475; Fig. 2E nd 4A), nd two to hve undergone recombintion of provirl nd c-myc sequences 3' to this EcoRI site (proviruses in lymphoms 1463 nd 1926; Fig. 4B). To determine whether ny of these 3' LTR-deleted provi- TABLE 4. Lymphom-inducing proviruses": sme trnscriptionl orienttion, solo LTRs HindIII-EcoRI EcoRI DNA 5, 3' (LTR, 5' 3' (LTR, Sc SmI (LTR, 3'LTR,YC 3MLCR, (LTR, MYC, 3' MYC, LTR, MYC) (SMA) A IN2) SMA) EX3) Lyml Lyml Lyml Lym Lyml Lym All of the proviruses re RAV-1 proviruses. ruses represented new trnsduction of c-myc, tumorspecific EcoRI frgments were nlyzed for sequences in the second intron of c-myc. The IN2 probe hybridized with ll of the 3' junction frgments except for tht in lymphom 3475 (Fig. 4C). Thus the novel provirus in lymphom 3475 ws cndidte for new trnsduction of c-myc. Proof tht this provirus represented trnsducing virus ws obtined by intrvenous inocultion of filtered homogente of lymphom 3475 into 1-week-old chickens. Three of the four inoculted chickens succumbed to myelocytomtosis between 12 nd 14 weeks fter inocultion. DNAs prepred from these tumors exhibited the sme novel 2.9-kb EcoRI frgment s lymphom 3475 (dt not shown). Thus the TABLE 5. Lymphom-inducing proviruses: sme trnscriptionl orienttion, deletion includes the 5' LTR HindIII-EcoRI EcoRI DNA 3' (LTR, 3' (LTR, Scl SmI 5' 3' LTR, 5' 3' LTR, (MYC) (SMA) (MYC) MYC, (MYC) MYC' IN2) EX3) LymR None None Lym3O2O None Lym3018 None 2.55 None Lym3029 None (1.4) Lym1549b NT None Lym4566 NT NT None All of the proviruses re RAV-1, except where otherwise indicted. Superscripts, NT, nd prentheses re explined in footnote of Tble 3. b NY204RAV-60 provirus.

6 VOL. 57, 1986 LYMPHOMA-INDUCING PROVIRAL INSERTIONS 33 TABLE 6. DNA Lymphom-inducing proviruses: sme trnscriptionl orienttion, deletion includes the 3' LTR 5' EcoRI junction 3' EcoRI junction frgment frgment MYC LTR SMA EX3 IN2 3' LTR PVA PVB SMA Lyml463b Lym Lym3055 NTC NT + Lym1524d NT - Lym3475e NT NT NT Lym1926 nd Lym3055 contin RAV-1 proviruses. b NY202RAV-60 provirus. c NT, Not tested. d NY203RAV-60 provirus. e Nontrnsforming subgroup E virus strin provirus. A ' Jnct %V _ Af X3,VLTt,PVA B 'Jnct 3' Jnc' s~ - *6.5-t t 6.5* 13- I. EX3.3LTR,PVB -3.8 * -2.4 SMA novel c-myc frgment in lymphom 3475 represents new c-myc trnsducing virus. This virus hs been nmed MYC Lymphom-inducing proviruses with internl deletions nd deletions or rerrngements of flnking host sequences. Two of the lymphom-inducing proviruses ppered to hve ipteml deletions s well s deletions or rerrngements in flnking host sequences (Tble 7). One of these (in lymphom 4591) hd novel ScI frgment tht ws smller thn the cceptor ScI frgment of c-myc. The other (in lymphom 2963) hd EcoRI-HindIII frgments tht were lrger thn would hve been expected for one LTR plus the cceptor c-myc frgment. Lymphom-inducing proviruses with unusully lrge 3' junction frgments. Two lymphom-inducing proviruses hd unusully lrge 3' junction frgments (Tble 2). These nd 10.5-kb EcoRI frgments hybridized with the EX3 nd 3' LTR probes, but did not hybridize with the PVA probe (Tble 8). Since the novel frgments did not hybridize with the PVA probe, they ppered to represent 3' LTR sequences linked to c-myc sequences. If this configurtion is correct, then one would expect no perturbtion in the size of the 7.0-kb ScI frgment of c-myc (Fig. 1). However, ScI digests of these tumors displyed novel frgments tht were 4 to 6 kb lrger thn the norml ScI frgment of c-myc. The position nd sequence content of these insertions re uncler. Lymphom-inducing proviruses with opposite trnscriptionl orienttion. The lest frequent clss of lymphominducing proviruses (Tble 2) contined insertions in the opposite trnscriptionl orienttion to c-myc (Tble 9, Fig. 2D). Interestingly, the two proviruses in this clss hd undergone deletions which included their 5' LTRs. One of these deletions (in lymphom 4586) ppers to hve resulted in the recombintion of sequences 5' to the EcoRI site t bp in provirl DNA with c-myc, wheres the other C )- " 40, AN _ 5.4- %* 4.5- &1- EX3 _# t IN2 FIG. 4. Restriction endonuclese frgments used in the mpping of lymphom-inducing proviruses with 3' LTR deletions. A, Dt used in the mpping of the lymphom-inducing provirus in Lym3475 (Tble 6). B, Dt used in the mpping of the lymphom-inducing provirus in Lym1463 (Tble 6). C, Dt for the mpping of intron 2 sequences in lymphom-inducing proviruses with 3' LTR deletions (Tble 6). All of the dt re for EcoRI-digested DNAs. The DNA being nlyzed is indicted bove the lnes. Other designtions re s in Fig. 2. The dt in C come from three sequentil hybridiztions of Southern blot without removl of the probe between hybridiztions. The first hybridiztion ws with PstI-HindIII frgment of MYC tht is 3' to the coding sequences for c-myc. This probe hybridized with the -20-kb frgment. The second hybridiztion ws with the IN2 probe. The third hybridiztion ws with the EX3 probe. The IN2 nd EX3 probes hybridize with the 16-kb c-myc frgment s well s with tumor-ssocited frgments. ppers to hve resulted in the recombintion of sequences 3' to this site with c-myc. Mpping of provirl insertions with respect to DNse I- hypersensitive sites. The 24 lymphom-inducing insertions with no discernible deletions or rerrngements in their 3' junction frgments (lymphoms in Tbles 3 nd 4; lymphoms R326, 3020, 3029 nd 1549 in Tble 5) could be TABLE 7. Lymphom-inducing proviruses: sme trnscriptionl orienttion, poorly defined deletions of virl nd host sequences DNA HindIII-EcoRI EcoRI Scl SmI 5' (LTR, MYC) 3' (LTR, 3' LTR, 5' (LTR, MYC) 3' (LTR, 3' LTR, MYC, 1N2) MYC, (MYC) EX3) (SMA) Lym NT NT 4.8 NT Lym None (2.6), (2.1) Both of the proviruses re RAV-1 proviruses. Superscripts, NT, nd prentheses re explined in footnote of Tble 3.

7 34 ROBINSON AND GAGNON TABLE 8. Lymphom-inducing proviruses : pprent integrtion site -6 kb 5' of c-myc DNA EcoRI EX3 SMA 3' LTR PVA Scl MYC PVA Lym Lym ? Both of the proviruses re RAV-1. mpped in c-myc (Fig. 5). In HindIII-EcoRI digests of these insertions, the 3' junction frgment is bounded by the HindlIl site in the second intron of c-myc nd the EcoRI site in the provirl LTR (Fig. 2A through C). Therefore the distnce of the insertion from the HindIII site is the size of the 3' junction frgment minus the -150 bp of virl sequences tht lie 3' to the EcoRI site in the LTR. The positions of open chromtin structures in c-myc (29) were mpped reltive to the sites of lymphom-inducing insertions by using HindIII-EcoRI junction frgments s size mrkers for HindIll frgments of DNse I-treted DNAs. The positions of the DNse I-hypersensitive sites re indicted by sterisks in Fig. 5. Interestingly, ech of the 24 mpped insertions ws within 150 bp of one of the five DNAse I-hypersensitive sites tht lie immeditely 5' to the coding sequences for c-myc. Since only 1,350 of the bses in this 2,750-bse region re within 150 bp of n open chromtin structure, the probbility tht integrtions occurred next to hypersensitive sites by chnce is (1,350/2,750)24 or <106. DISCUSSION Sixty-eight ALV-induced lymphoms hve been nlyzed for provirl insertions into or new trnsductions of c-myc (Tble 1). The most frequent form of lymphom-inducing provirus hd retined sequences in both of its LTRs, but lost sequences ner its 5' LTR (Tble 2). Thus, contrry to ccepted belief, ALV proviruses do not hve to lose n LTR to ctivte the trnscription of downstrem host sequences. Ptterns of deletion in lymphom-inducing proviruses. More thn 75 lymphom-inducing proviruses tht re upstrem of nd in the sme trnscriptionl orienttion, 7 tht re upstrem of but in the opposite trnscriptionl orienttion, nd 1 tht is downstrem of c-myc hve been nlyzed for deletions (Tbles 3 through 9) (22, 27, 29). Ech of these hs lost sequences tht include n LTR or sequences ner the 5' LTR. Among insertions in the sme trnscriptionl orienttion, the most frequent deletion is the deletion of sequences ner the 5' LTR (Tble 2). Among insertions in the opposite trnscriptionl orienttion, four hve lost sequences tht include the 5' LTR (Tble 9) (22), two hve lost sequences ner the 5' LTR (21, 22, 40), nd one hs lost the 3' LTR (29). The one downstrem provirus hs lost sequences tht include the 5' LTR (21). Before our study, ll upstrem proviruses in the sme trnscriptionl orienttion s c-myc hd been ssumed to hve lost one LTR. This erroneous ssumption ws bsed on the bsence of the Scl site ner the 5' LTR (27). The loss of sequences ner the 5' LTR in ll two LTR lymphom-inducing proviruses suggests tht this deletion is required for two LTR proviruses to up-regulte the trnscription of djcent host sequences. In norml two-ltr proviruses, the 5' nd 3' LTRs hve distinct ctivities, with the 5' LTR inititing nd the 3' LTR providing the polydenyltion signl for trnscripts (J. Coffin nd S. Hermn, personl communiction). This speciliztion of the LTRs fvors trnscription of virl rther thn flnking host sequences (8). Known functions of the selectively deleted sequence include splice donor site (30), signls for the pckging of RNA into virions (31), nd the 5' sequences of ll of the virl proteins (30). The selectively deleted sequence lso ppers to be region with high secondry structure s evidenced by the presence of two hypersensitive sites in nuclese Si-treted chromtin (7). Which, if ny, of these fetures my be relevnt to the selection for 5' deletions in two LTR lymphom-inducing proviruses is not known. All of the mpping of lymphom-inducing insertions is bsed on Southern blot nlyses which do not rigorously define frgment sizes or sequence contents. Proviruses tht re clssified s solo LTRs my hve some internl sequences, nd proviruses tht re clssified s two LTR proviruses with internl deletions my hve deletions tht extend into the 5' LTR. However, we feel tht most of the clssifictions will be correct, since lymphom-inducing ALVs tht hve been sequenced include true solo LTR nd true two LTR provirus with deletion ner its 5' LTR (40). Deletions nd rerrngements in flnking host sequences. The vst mjority of solo nd two LTR proviruses were not ssocited with deletions or rerrngements in flnking host sequences (Tbles 3 nd 4). J. VIROL. Interestingly, these proviruses were inserted downstrem of the mjor hypersensitive site tht lies immeditely 5' to the first exon of c-myc (hypersensitive site II) (Fig. 5) (29). In contrst, the six insertions upstrem of hypersensitive site II were ssocited with esily detected deletions or rerrngements in host sequences. These six insertions included three 5' LTR-deleted proviruses (lymphoms 1549 nd 4566, Tble 5; lymphom 4586, Tble 9), one 3' LTR-deleted provirus (lymphom 1463, Tble 6), nd both of the pprently fr upstrem proviruses (Tble 8). Hence, deletions in host sequences my be necessry for insertions tht re upstrem of hypersensitive site II to ctivte c-myc. Sites of integrtion of lymphom-inducing proviruses. The DNA TABLE 9. Lymphom-inducing proviruses: opposite trnscriptionl orienttion, deletion includes the 5' LTR Hindlll-EcoRI 5' (3' LTR, 3' (MYC, 5' (3' 3 MYC) IN2) LTR, MYC) MYC, EX3 LTR PVA PVB SMA LymS535b Lym None NT + Lym4586 contins n RAV-1 provirus. Symbols re s in Tble 3. b Provirus encoded by the virus recovered from prav-1. EcoRI

8 VOL. 57, 1986 LYMPHOMA-INDUCING PROVIRAL INSERTIONS 35 m I I I z It tll I I It I Hind= * * * Kiloboses i.3 0 FIG. 5. Position of DNse I-hypersensitive sites nd lymphom-inducing RAV-1 proviruses in c-myc. Symbols: 4, provirl insertion; *, DNAse I-hypersensitive site. The mpping of the DNse I-hypersensitive sites ginst our sized tumor frgments resulted in positions of hypersensitive sites tht were within 100 bp of those reported by Schubch nd Groudine (29). sites of 24 of the lymphom-inducing insertions could be mpped in c-myc (Fig. 5). The mpping of these insertions ws in good greement with prior studies (32), with most of the insertions mpping in the 3' end of the first intron. Since it hd been proposed tht retrovirl DNAs integrte ner regions of open chromtin structure (1, 29), the sites of insertions were lso mpped with respect to hypersensitive sites in DNse I-treted bursl chromtin. Remrkbly, the sites of ech of the 24 insertions mpped within 150 bp of one of the five DNse I-hypersensitive sites tht lie in 2,600-bp region immeditely 5' to the first coding exon of c-myc (29). None of the integrtions occurred in the 1,400 bp in this region tht lie more thn 150 bp from DNse I- hypersensitive site. Frequency of genertion of new c-myc-trnsducing viruses. One lymphom ws found to hve new c-myc-trnsducing virus, wheres four were found to hve proviruses with 3' LTR deletions tht were upstrem of nd in the sme trnscriptionl orienttion s c-myc (Tble 6). Since new trnsductions cn be initited by 3' LTR-deleted proviruses (6), we nticipted tht severl of these proviruses might hve generted c-myc-trnsducing viruses. To our surprise, most such proviruses were not ssocited with new trnsductions. Thus, the low incidence of new myctrnsducing viruses my reflect the genertion of trnsducing viruses by only minority of 3' LTR-deleted proviruses (Tble 6) s well s the infrequent occurrence of such proviruses in lymphoms (Tble 2). Frequency of induction of lymphom by proviruses in the opposite trnscriptionl orienttion to c-myc. Only one of the 32 RAV-1-induced lymphoms contined provirus in the opposite trnscriptionl orienttion to c-myc (Tble 2). This is in contrst to the occurrence of four proviruses in the opposite trnscriptionl orienttion to c-myc in series of eight RAV-2-incuded lymphoms (22). The probbility tht this difference in frequency ws due to chnce is <0.01. Since lymphom induction in such cses is due to the enhncement of c-myc trnscription (21), it seems possible tht the enhncer sequences in virus determine the frequency with which stock cuses lymphom by insertions in the opposite trnscriptionl orienttion to c-myc. Other lymphom-inducing proviruses. Reticuloendotheliosis viruses, murine leukemi viruses, nd feline leukemi viruses lso induce lymphoms by insertions in c-myc (13, 18, 19, 33). Lymphom-inducing reticuloendotheliosis virus insertions pper to be very similr to lymphominducing ALV insertions. The vst mjority of these re deleted proviruses in the sme trnscriptionl orienttion s c-myc. The most frequent pttern of provirl deletion results in two LTR proviruses tht hve lost sequences ner the 5' LTR, the second most frequent pttern is proviruses tht hve deleted sequences including the 5' LTR, nd the third most frequent pttern is solo LTRs (23, 34; H.-J. Kung, personl communiction). The similrities in position, orienttion, nd ptterns of deletion of lymphom-inducing insertions in these two fmilies of viruses suggest tht ALV nd reticuloendotheliosis virus ctivte the expression of the chicken c-myc gene by similr mechnisms. In contrst, lymphom-inducing murine leukemi virus proviruses tend to be integrted in the opposite trnscriptionl orienttion to c-myc, reside lmost exclusively upstrem of the first exon of c-myc, nd hve typiclly not sustined deletions (13, 33). At present little is known bout lymphom-inducing insertions of feline leukemi viruses. However, in contrst to ALV, reticuloendotheliosis virus, nd murine leukemi virus insertions, feline leukemi virus insertions my trnsduce c-myc t high frequency s evidenced by the isoltion of myc-contining viruses from number of T-cell lymphoms in pet cts (12, 16, 18). ACKNOWLEDGMENTS We thnk M. Groudine for DNA isolted from DNse I-treted bursl chromtin, H.-J. Kung nd J. Coffin for the permission to cite unpublished results, nd D. Steffen, S. Wdsworth, S. Vijy, D. Gmett, M. Groudine, nd H.-J. Kung for comments on the mnuscript. We re indebted to B. P. Blis for expert nd invluble ssistnce with the cre nd identifiction of disesed chickens nd to M. Linil, G. Pyne, D. Sheiness, J. M. Bishop, nd H. E. Vrmus for DNAs used either s probes or for the isoltion of probes. This work ws supported by Public Helth Service reserch grnts CA nd CA from the Ntionl Institutes of Helth, by Cncer Center core grnt P30 CA 12708, nd by the W. J. Tnnenberg Fund. LITERATURE CITED 1. Breindl, M., K. Hrbers, nd R. Jenisch Retrovirusinduced lethl muttion in collgen I gene of mice is ssocited with n ltered chromtin structure. Cell 38: Coffin, J. M Genome structure, p In R. Weiss, N. Teich, H. Vrmus, nd J. Coffin (ed.), Moleculr biology of RNA tumor viruses II (supplement). Cold Spring Hrbor Lbortory, Cold Spring Hrbor, N.Y. 3. Copelnd, N. G., K. W. Hutchison, nd N. A. Jenkins Excision of the DBA ecotropic provirus in dilute cot-color revertnts of mice occurs by homologous recombintion involving the virl LTRs. Cell 33: Fung, Y.-K. T., L. B. Crittenden, nd H.-J. 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Csey Isoltion of feline leukemi provirus contining the oncogene myc from feline lymphosrcom. Nture (London) 308: Li, Y., C. A. Hollnd, J. W. Hrtley, nd N. Hopkins Virl integrtions ner c-myc in 10-20% of MCF247 induced AKR lymphoms. Proc. Ntl. Acd. Sci. USA 81: Linil, M., nd M. Groudine Trnscription of three c-myc clones is enhnced in chicken bursl lymphom cell lines. Proc. Ntl. Acd. Sci. USA 82: Miles, B. D., nd H. L. Robinson High-frequency trnsduction of c-erbb in vin leukosis virus-induced erythroblstosis. J. Virol. 54: Mullins, J. I., D. S. Brody, R. C. Binri, Jr., nd S. M. Cotter Virl trnsduction of c-myc gene in nturlly occurring feline leukemis. Nture (London) 308: Neel, B. G., W. S. Hywrd, H. L. Robinson, J. Fng, nd S. Astrin Avin leukosis virus-induced tumors hve common provirl integrtion sites nd synthesize discrete new RNAs: oncogenesis by promoter insertion. Cell 23: Neil, J. C., D. Hughes, R. McFrlne, N. M. Wilkie, D. E. Onions, G. Lees, nd 0. 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Fujit In vitro trnscription nlysis of the virl promoter involved in c-myc ctivtion in chicken B lymphoms: detection nd mpping of two RNA initition sites within the reticuloendotheliosis virus long terminl repet. J. Virol. 54: Robinson, H. L., B. P. Blis, P. N. Tsichlis, nd J. M. Coffin At lest two regions of the virl genome determine the J. VIROL. oncogenic potentil of vin leukosis viruses. Proc. Ntl. Acd. Sci. USA 79: Robinson, H. L., L. Jensen, nd J. M. Coffin Sequences outside of the long terminl repet determine the lymphomogenic potentil of Rous-ssocited virus type 1. J. Virol. 55: Robinson, H. L., M. N. Person, D. W. DeSimone, P. N. Tsichlis, nd J. M. Coffin Subgroup E vin leukosis virus ssocited disese in chickens. Cold Spring Hrbor Symp. Qunt. Biol. 44: Rovigtti, V., C. Rogler, B. Neel, W. Hywrd, nd S. Astrin Expression of endogenous oncogenes in tumor cells. Results, p In A. H. Owens, D. S. Cffey, nd S. B. Bylin (ed.), Tumor cell heterogeneity. 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