Proc. Nati. Acad. Sci. USA Vol. 77, No. 6, pp. 3307-3311, June 1980 Biochemistry Structure of Moloney murine leukemia viral DNA: Nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences (strong-stop DNA/recombinant DNA/DNA sequence determination/inverted repeat/transposons) CHARLES VAN BEVEREN*, JANICE G. GODDARD*, ANTON BERNSt, AND INDER M. VERMA* *Tumor Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138; and tLaboratory of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands Communicated by E. Peter Geiduschek, March 31, 1980

ABSTRACT Some unintegrated and all integrated forms tRNAPrO viral DNA contain terminal of murine leukemia long repeats 5' gag pol end @ ib3 (LT s). The entire nucleotide sequence of the LTR and adjacent L-,, i Genomic terminal cellular sequences at the 5' end of a cloned integrated proviral ViralRredundancy DNA obtained from BALB/Mo mouse has been determined. Viral RNA (45-66cbp) It was compared to the nucleotide sequence of the LTR at the Reverse transcription 3' end. The results indicate: (i) a direct 517-nucleotide repeat at yT the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end ent 3' 5 {Cellular DNA of the primer tRNA (strong-stop DNA); (ifi) an li-nucleotide ,-Strong-stop DNA , -Strong-stop DNA inverted repeat is present at the ends of the 5'-LTR and a total (145 bp) of 17 out of21 nucleotides at the termini are inverted repeats; 5'-LTR (145 bp) 3'-LTR-LTR (iv) sequences CAATAAAAG (at positions -24 to -31) and Integrated viral DNA CAATAAAC (at positions +46 to +53) resembling the hypo- thetical DNA-dependent RNA polymerase II promoter site can FIG. 1. Diagram of the viral DNA containing two long terminal be identified in the 5'-LTR; (v) the sequence GAAA appears to repeats (LTRs). Empty box, strong-stop DNA (145 bp); hatched box, be repeated on both sides of the junction of viral and cellular sequences at the 3' end ofthe genome (372 nucleotides); 0, terminal sequences; and (vi) in analogy with the bacterial transposons, redundancy in the genomic RNA (45-66 bp); and A, cellular DNA. the presence of an inverted reat seuence at the termini of 5'-LTR suggests that M-MLV also has the integration properties (13, 14,4i) Thus, if the genomic RNA is about 8.2 kb long, some of a transposon. of the unintegrated and all of the integrated viral DNAs have additional nucleotides at 3' and 5' ends, which appear to be Infection by retroviruses requires the conversion of viral direct repeats (10, 11, 15-18, #) genomic RNA into DNA by reverse transcriptase (RNA-de- In this communication, we report the nucleotide sequence pendent DNA polymerase) (1, 2). Viral DNA synthesis initiates of the entire long terminal repeat (LTR) located at the 5' end at the 3'-OH end of the tRNA primer located near the 5' end of the cloned integrated proviral DNA obtained from BALB/ of the genomic RNA (3). In the case of Moloney murine leu- Mo mouse (19). Furthermore, the nucleotide sequence of the kemia virus (M-MLV), 19 nucleotides at the 3' end of the viral-cellular junction at the 5' end has also been determined. primer tRNAPrO are hydrogen bonded to the genomic RNA (4). In ref. 20, the nucleotide sequence of the LTR at the 3' end is The 3'-A-OH of the primer tRNAPrO is not hydrogen bonded reported. The results indicate: (i) The LTR is 517 nucleotides and is located 146 nucleotides from the 5'-CAP nucleotide of long; over 99% of the sequences at the 5'-LTR are identical to the genomic RNA. It forms a phosphodiester bond with the first the sequence in the 3'-LTR. (fi) Of the 517 nucleotides present deoxynucleotide triphosphate, and the synthesis of the com- at the 5'-LTR, 145 nucleotides are present in the strong-stop plementary DNA proceeds until it reaches the 5' nucleotide of DNA. (iii) Eleven nucleotides at each terminus of the 5'-LTR the genomic RNA. The 145-nucleotide-long cDNA (strong-stop are inverted repeats. (iv) The sequence CAATAAAAG, which DNA), covalently linked to the tRNAPrO, dissociates and hy- resembles the hypothetical promoter site, is present between bridizes to terminally redundant sequences at the 3' end of the -24 and -31 nucleotides from the 5'-CAP nucleotide. (v) The genomic RNA (5). The synthesis of DNA then proceeds from sequence CAATAAAC is present at positions +46 to +53 from the 3' end of the RNA toward its 5' end. A 600 base pair (bp)- the 5'-CAP nucleotide. (vi) The 18-nucleotide complement to long DNA fragment of opposite polarity (+) can be observed the 3' end of the tRNAPrO is present. after the synthesis of 0.5-1.0 kilobase (kb) of the cDNA (-) transcript (6-9). The double-stranded DNA synthesized either MATERIALS AND METHODS in vitro or in vivo has two types of genome-length molecules: Synthesis of Strong-Stop DNA. The synthesis of strong-stop (i) those that contain the 5'-end genomic sequences repeated DNA was carried out as described (21, 22). at their 3' end (i.e., 5'-3'5') and (ii) those that, in addition to Molecular Cloning of DNA Fragments. The isolation of the having 5' genomic RNA sequences repeated at their 3' end, also 24-kb EcoRI fragment containing the endogenous Moloney contain 3' genomic RNA sequences repeated at their 5' end genome from BALB/Mo mouse liver DNA has been described (3'5'-3'5') (Fig. 1) (10-12). Analysis of the integrated murine (19). The 24-kb EcoRI fragment was cleaved with HindIII, proviral DNA shows structures containing 3'5'-3'5' sequences which cuts once in the M-MLV genome (23). A 9-kb HindIII

The publication costs of this article were defrayed in part by page Abbreviations: LTR, long terminal repeat; M-MLV, Moloney murine charge payment. This article must therefore be hereby marked "ad- leukemia virus; bp, base pair(s); kb, kilobase pair(s). vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t A. Berns, M.-H. T. Lai, R. A. Bosselman, M. A. McKennett, M. Van this fact. der Putten, and I. M. Verma, unpublished. 3307 Downloaded by guest on October 1, 2021 3308 Biochemistry: Van Beveren et al. Proc. Natl. Acad. Sci. USA 77 (1980) fragment, containing 5.3 kb of proviral DNA, corresponding shows that bands 1, 2 + 3, and 4, upon treatment with alkali, to the 5' portion of the genome with the LTR, and 3.7 kb of migrate faster on the gel. The size difference between the un- adjacent cellular sequences was cloned in the HindIII site of treated and alkali-hydrolyzed bands is about 70-80 nucleotides, phage X21a as described.t The 9-kb fragment was then sub- which is the approximate size of the primer tRNAPro (4). In cloned in the HindIII site of plasmid pBR322 (24). In addition, order to establish that the DNAs in the bands (Fig. 2A) are co- the 2.35-kb Kpn I subfragment, containing 0.4 kb of the 5'-LTR valently linked to the primer tRNA, ai-32P atom transfer ex- with 1.95 kb of cellular sequences, was subcloned in the Pst I periments were performed (27). DNA synthesis was carried out site of pBR322 by the dG-dC-tailing method (25). The National under conditions in which the limiting dNTP was [a-32P]dATP. Institutes of Health guidelines for recombinant DNA research If the first dNTP incorporated is dA, then, upon hydrolysis with were followed during the entire cloning procedure. alkali, the ribonucleotide participating in the formation of the Sequence Determination Procedures. Restriction endo- phosphodiester band will have an a-32P atom transferred to the nuclease-cleaved DNA was labeled at the 5' termini with ribose moiety. Because the 3'-OH of tRNAPrO is A-OH, the polynucleotide kinase, or at the 3' termini with terminal transfer should occur from [a-32P]dATP to rA. Fig. 2C shows deoxynucleotidyltransferase, and sequence analysis was per- that in bands 1, 2 + 3, and 4 only 32P-labeled AMP can be de- formed as described (26). Sequence ladders were displayed on tected. Thus, the synthesis of bands 1, 2 + 3, and 4 is initiated 8% polyacrylamide/8 M urea 20 X 40 X 0.04 cm or 30 X 40 X at the 3'-A-OH of the primer tRNA. Fig. 2D shows the nucle- 0.04 cm gels, or 6% polyacrylamide/8 M urea 18 X 78 X 0.04 otide sequence of band 1. It contains 145 nucleotides and cm gels. reaches to the 5'-CAP oligonucleotide GCG reported earlier (28). Bands 2 + 3 and 4 are premature termination products and RESULTS represent a subset of strong-stop DNA (unpublished data). Structure of the Strong-Stop DNA. Strong-stop DNA rep- Characterization of Cloned DNA Fragments. A 9.0-kb resents sequences between the 3'-OH nucleotide of the primer HindIl fragment obtained from BALB/Mo mouse cellular tRNA and the 5'-CAP nucleotide of the genomic RNA. The DNA was inserted into the unique HindIII site of the X Charon primer tRNA is covalently linked to the strong-stop DNA. If 21A molecularly cloned.t It was subsequently inserted into the the viral DNA synthesis is carried out under conditions of HindIII site of the plasmid pBR322 and subcloned. The re- limiting substrate concentration, a large portion of the DNA combinant plasmid pMLV1nt-1 contained a 5.3-kb viral se- synthesized appears to be strong-stop DNA (21). Fig. 2A shows quence and a 3.7-kb adjacent cellular sequence (Fig. 3). In the synthesis of strong-stop DNA, using purified M-MLV vir- addition, it contained the entire LTR. The pMLVint-1 was ions. In addition to the major band (band 1), several other bands cleaved with restriction endonuclease Kpn I and the two migrating slower and faster than it can also be seen. Fig. 2B fragments of sizes corresponding to 2.9 kb and 2.35 kb were separated on an agarose gel. The 2.35-kb fragment containing the viral and cellular sequences was subcloned in pBR322 by A B C using the dC-dG-tailing method (25). Restriction endonuclease Origin Band 1 2 + 3 4 Band' 1 2+ 3 4 Kpn I cleaves at the site GGTAdC, leaving a 3' extended end tI -_ _7 .bpbp_ (29). Thus, addition of dC residues on the Kpn I-cleaved DNA (,. fragment and hybridizing to Pst I-cut dG-tailed pBR322 allows the regeneration of not only the Pst I sites (25) but also the Kpn 250-_W I sites. 150- _

Bond - 1 80- BALB/Mo 24.0-kb EcoRI fragment A. EcoRI HindIII HindIII EcoRI I ------I 4 60- 5 5,AA&%A4VVVAAAAA----AAAAAA13 Cell A M-MLV - Bond i 2 +3

Bond' - & | .- -ORIGIN n ICloned in X Charon 21A 50 5 AATGAAAGAC CCCCGCTGAC GGGTAGTCAA TCACTCAGAG GAGACOCCTOC i00 CAAGGAACAG CGAGACCACA AGTCGGATGC AACTGCAAGA GGGTTTTATTG 145 HindIII 5'-LTR Hindull GATACACGGG TACCCGGGCG ACTCAGTCAA TCGGAGGACT GGCGC 3 I

KpntI KpnI KpnIt FIG. 2. Characterization of the M-MLV strong-stop DNA. (A) Kpn I Kpn I Kpn I Analysis onr a 10% acrylamide gel of cDNA synthesized at limiting [a-32PJdATP substrate concentration. (B) Bands 1, 2 + 3, and 4 were excised from the gel and eluted as described (22). A portion of each Subcloned in pBR322 fraction was hydrolyzed with alkali (+) and analyzed on a 10% acrylamide gel along with an unhydrolyzed sample (-). The standard HindIII-HindIII fragment Kpn I-Kpn I fragment molecular weight markers were denatured prior to electrophoresis. HindIII site (C) A portion of each band after hydrolysis with alkali was analyzed dC-dG-tailing by high-voltage paper electrophoresis at pH 3.5 in a buffer containing acetic acid and pyridine (27). The standards C, A, G, and U were pMLVint~- pMLVint-I 1 visualized under a UV lamp. On the left, the arrow points to the FIG. 3. Molecular cloning of pMLVit-1 and pMLVint-11. A 32P-labeled AMP residue and the charge polarity (+, -) is indicated. schematic drawing of the source of cellular DNA and molecular (D) Nucleotide sequence of strong-stop DNA. The first nucleotide, cloning procedures used is shown. The details of cloning of these dA, was determined by the a-32P atom transfer experiment shown fragments are described eleswhere.t -, viral sequences; -A, cell in C. sequences. Downloaded by guest on October 1, 2021 Biochemistry: Van Beveren et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3309 5 -CAP A B -400: -300 -200 -100 -1 +1 +100 +200 RNAIgenom 3' G A>C T+C C 51 G A>C T4-C C Cell Virus ~~~~~~~~~i RNA genome ._._o_ [Sau3A]K -189- _ [BstNIJK 9 IPvu 111K rAva IIK .[Xba I"K .m [SaL 3A]K [KpnIIT -157- --

_ofr"'_..wl ~ _ of I[BstNl'K .,.Oqg.t O-Olk....,..W-do", IPVu MIT . [Ava IIK [Kpn UIT

[PVUII K [Xba IJK [Sau3A]K [Sst 'JK

FIG. 4. Sequenced regions ofthe integrated M-MLV 5'-LTR. The 4020~~ heavy lines show the portions of the upper (U) and lower (L) strands whose sequences were determined. The arrows indicate the extent and direction of the sequence obtained by cleaving the DNA with the -189- indicated restriction endonuclease and labeling either the 5' terminus -157- _0 with polynucleotide kinase (K) or the 3' terminus with terminal transferase (T). 5' _ 3

FIG. 6. Autoradiographs of DNA sequencing gels of a portion of DNA Sequence Analysis. Fig. 4 shows the overall strategy 5'-LTR. (A) pMLVit-11 DNA was labeled at the 5' termini of the used to determine the nucleotide sequence of the various re- Xba I site. After Pst I cleavage, the chemical degradation products (26) were on a 12% M urea striction fragments. In general, the 5'- 3' sequences were de- separated polyacrylamide/8 gel. (B) The purified pMLVit -11 insert DNA was labeled at the 3' termini of the termined by labeling with polynucleotide kinase and [fy- Pvu II site with cordycepin [a-32P]triphosphate (New England Nu- 32P]ATP. The 3' 5' sequences were determined by labeling clear). After Sst I cleavage and partial degradation (26), the products the 3' end with terminal transferase, either with a-32P-labeled were separated on an 8% polyacrylamide/8 M urea gel. The sequence cordycepin triphosphate or [a-32P]rNTP. Over 80% of the se- of the complementary strand to the sequence in Fig. 6 can be quence shown here was determined on both strands. A repre- read from positions -157 to -189 in the 5' - 3' direction (bottom sentative gel showing the sequence of a DNA fragment whose to top in A, top to bottom in B) as GCTGAGGGCTGGA- CCGCATCTGGGGACCATCTG. sequence was determined on one strand in both directions is shown in Fig. 5. The entire sequence of the strong-stop DNA, base differences between the 5'- and 3'-LTRs may be due to LTR, and 113 nucleotides of adjacent cellular DNA sequences errors in the sequence determination procedure or errors in- is shown in Fig. 6. Table 1 shows the location of various re- troduced during reverse transcription (30). We conclude that striction endonuclease sites in this DNA. The 5'-LTR contains the point of sequence divergence (-372) is the virus-cell 145 nucleotides corresponding to the strong-stop DNA followed junction sequence. It is possible that divergent sequences are by 372 nucleotides until the beginning of the cellular se- due to a deletion in the 3'-LTR sequence. However, the cloned quences. DNA used to determine the sequence of 3'-LTR appeared to DISCUSSION be a faithful transcript of the genomic RNA as judged by its restriction endonuclease mapping. The viral sequence 3'-

Structure of the 5'-LTR. Although the genome of M-MLV proximal to the cell junction is 5'-TGAAAG .. .-3' and the is 8.2 in or vivo kb, DNA synthesized vitro in is about 8.8 kb. cellular sequence at the junction is 5'-. . . GAGAAAATTAC All integrated forms of viral DNA so contain identified far di- . . .-3'. GAAA appears to be the only sequence repeated on ei- rect repeats at their termini as determined by restriction en- ther side of the junction. donucleases or by electron microscopy (13, 14, 16, 18, :). As Transcription of Proviral DNA. The viral DNA has been already mentioned, the 3' and 5' halves of the M-MLV proviral shown to be transcribed by cellular DNA-dependent RNA DNA contain sequences from the 5' and from the 3' ends of the polymerase II by virtue of its sensitivity to the drug a-amanitin genomic RNA, and thus can be described in the 5'--3' direction (31). No large precursor of M-MLV genomic RNA has been as 3'5'-3'5' (Fig. 1). We have determined the nucleotide se- detected in the infected cells (32, 33). It thus appears that in- quence of the entire long terminal repeat at the 5' end of mo- tegrated proviral DNA either contains its own promoter or is lecularly cloned portions of integrated BALB/Mo viral DNA. integrated near a cellular promoter. If the 5'-LTR contains the The sequence of the long terminal repeat of a cloned DNA viral promoter for transcription, genome-length DNA tran- fragment containing sequences from the 3'-end is presented scripts can be obtained. A hypothetical promoter site (Hog- in ref. 20. ness-Goldberg box) containing a sequence of NTATAAAN, The extent of terminal repeat was determined by comparing located 25-30 nucleotides from the 5'-CAP nucleotide, has been the sequence of the 5'-LTR to 3'-LTR. Over 99% of the se- identified in several eukaryotic systems (34). Analysis of the quences in the 3'-LTR are homologous to 5'-LTR. They both nucleotide sequence of 5'-LTR shows a sequence of CAA- contain the entire strong-stop DNA (except the 3'-LTR did not TAAAA located at positions -24 to -31 from the 5'-CAP nu- contain the first two A residues) and then a stretch of 372 cleotide. This could be the presumptive promoter site for the identical nucleotides (with single base changes at three positions: initiation of synthesis of the viral genomic RNA. However, a -28, A instead of T; -133, G instead of T; and -287, A instead similar sequence CAATAAA is also located between positions of G) before showing sequence diversity. It should be noted that +46 and +52. In the 3'-LTR, this latter sequence is also present the 3'-LTR sequence is derived from a cDNA clone. The three at the same position (20). However, the sequence CAATAAAA Downloaded by guest on October 1, 2021 3310 Biochemistry: Van Beveren et al. Proc. Natl. Acad. Sci. USA 77 (1980)

5'1 -480 -470 -460 AACCT ATGATCTCCT TTTCCTTAAT CTTGCTGTTC CELL I VIRUS -450 -440 -430 -420 -410 -400 -390 -380 1 -370 CCCTCCCCCA TATGTTTACC TACTGAACAT CACTTGGGGT TGTAGAAACT ATTGGGAACT TGTCCTGGAG AAAATTAICTG AAAGACCCCA I_- -360 -350 -340 -330 -320 -310 -300 -290 -280 CCTGTAGGTT TGGCAAGCTA GCTTAAGTAA CGCCATTTTG CAAGGCATGG AAAAATACAT AACTGAGAAT AGAGAAGTTC AGATCAAGGT A -270 -260 -250 -240 -230 -220 -210 -200 -190 CAGGAACAGA TGGAACAGCT GAATATGGGC CAAACAGGAT ATCTGTGGTA AGCAGTTCCT GCCCCCGCTC AGGGCCAAGA ACAGATGGTC

-180 -170 -160 -150 -140 -130 -120 -110 -100 CCCAGATGCG GTCCAGCCCT CAGCAGTTTC TAGAGAACCA TCAGATGTTT CCAGGGTGCC CCAAGGACCT GAAATGACCC TGTGCCTTAT AL G

-90 -80 -70 -60 -50 -40 -30 -20 -10 TTGAACTAAC CAATCAGTTC GCTTCTCGCT TCTGTTCGCG CGCTTCTGCT CC.CCGAGCT AATAAAA G CCCACAACCC CTCACTCGGG 5'-CAP i 10 20 30 40 50 60 70 80 90 GCGCCAGTCC TCCGATTGAC TGAGTCGCCC GGGTACCCGT GTATCAT~A~ACCCTCTTC, CAGTTGCATC CGACTTGTGG TCTCGCTGTT

100 110 120 130 140 150 160 170 3 ' CCTTGGGAGG GTCTCCTCTG AGTGATTGAC TACCCGTCAG CGCGGGTCTT TCATTTGGGG GCTCGTCCGG GATCCGGAGA CCCCT

FIG. 6. Nucleotide sequences of the integrated M-MLV 5'-LTR and adjacent cellular sequences. The sequence of the strand having the same polarity as the M-MLV genomic RNA is given, with nucleotide 1 being that which corresponds to the 5'-CAP RNA nucleotide. The sequence from nucleotide -372 through +143 corresponds to that in the lower strand in ref. 20 from position 515 through 1. Interesting features include: (i) the sequence (nucleotides 145-1, underlined) corresponding to the RNA template for strong-stop cDNA (Fig. 2D); (ii) the junction between viral and cellular DNA at position -372; (iii) the 11-base inverted repeat (heavily underlined) at the termini ofthe LTR, with the longer, incomplete inverted repeat shown with broken lines; (iv) two sequences (in boxes) that could function as either promoters or poly(A) addition signals; (v) the 18-nucleotide complement (bracket) to the 3' end of the tRNAPro; and (vi) the three nucleotide substitutions (As) by which the sequence of the unintegrated 3'-LTR (20) differs from the sequence presented here. The two open arrows show the positions of the two A residues missing in the 3'-LTR (20). observed between positions -24 and -31, upstream from the Implication for Integration. Retroviruses integrate at 5'-CAP nucleotide, has undergone one base change multiple sites in the host DNA. Although the unintegrated forms (CAAAAAAA instead of CATAAAA) (20). of viral DNA contain either one or two copies of LTR, the in- In several eukaryotic systems, a sequence AAATAAA or tegrated proviral DNA contains two copies of LTR at its ter- AATAAA (35) has been shown to be a signal for addition of mini. It is not known if the circular or the linear form of viral poly(A) sequences. Presumably, the poly(A) addition could be DNA is integrated. The presence of inverted repeats at the signaled by one or both of the CAATAAA sequences in the termini of either 5'-LTR or 3'-LTR suggests an analogy with LTR. the bacterial transposons (36). A circular form of the uninte- Presence of Inverted Repeats. The 3' and 5' termini of the grated viral DNA containing one copy of LTR can integrate 5'-LTR contain an li-nucleotide-long inverted repeat (marked by staggered cuts in the inverted repeats and generate two by solid lines in Fig. 6). A total of 17 out of the 21 nucleotides copies of LTR according to the model described by Shapiro at the termini are inverted repeats (marked by broken lines). (37). A similar model for integration of M-MLV has been pro- Similar inverted repeats can also be seen in the 3'-LTR (20). It posed by Shoemaker et al. (38). However, in the studies per- is interesting to note that the 3'-LTR does not contain the first formed with molecularly cloned Moloney murine sarcoma virus two nucleotides (AA) of the strong-stop DNA at its 5' end (20). DNA (24) and in vitro reconstructed cloned M-MLV DNA,t However, the 3' terminus of 3'-LTR does contain the two A we have observed that only those molecules that contain two residues (positions 514 and 515) (20). The loss of two A residues copies of LTR are able to transform or infect cells. could be an artifact of the cloning procedure (generated during The "transposon model of M-MLV integration" predicts: (i) SI nuclease digestion) or natural sites for integration because Multiple sites of integration; M-MLV has been shown to have the virus-cell junction at the 5' end also shows the loss of AA as many as 5-10 sites of integration (39, 40). (ii) Duplication of residues. cellular sequences at both termini of integrated proviral DNA.

Table 1. Restriction endonuclease cleavage sites within the 5'-LTR Acy I Alu I Asu I Ava I EcoRII Hae III Hha I HinfI Hpa II Kpn I Pvu II Sau3A Sma I Sst I Xba I +1 -344 -244 -39 -130 -242 +3 +22 -207 +36 -253 -280 +30 -32 -152 -340 -199 -6 -197 +29 -253 -184 +28 -34 -171 -116 The nucleotide positions indicated for each restriction endonuclease are those immediately 5' to point of cleavage in the genome sense strand, shown in Fig. 6. Downloaded by guest on October 1, 2021 Biochemistry: Van Beveren et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3311 In order to test this prediction the nucleotide sequences of the 16. Sabran, J. L., Hsu, T. W., Yeater, C., Kaji, A., Mason, W. S. & 5' and 3' viral-cell junctions should be determined. The nu- Taylor, J. M. (1979) J. Virol. 29, 170-178. cleotide sequence at the 5' junction is reported here. (iii) 17. Shank, P. R., Hughes, S. M., Kung, H.-J., Majors, J. E., Quintrell, DNA of the N., Guntaka, R. V., Bishop, J. M. & Varmus, H. E. (1978) Cell 15, Identification in uninfected cell unique cellular 1383-1395. sequence that underwent duplication after insertion of M-MLV 18. Hughes, S. H., Shank, P. R., Spector, D. H., Kung, H.-J., Bishop, DNA. The cellular DNA sequences adjacent to 5' viral-cell J. M., Varmus, H. E., Vogt, P. K. & Breitman, M. L. (1978) Cell junctions can be used as probes to isolate from uninfected cells 15, 1397-1410. a DNA fragment in which the viral DNA integrates upon in- 19. Van der Putten, H., Terwindt, E., Berns, A. & Jaenisch, R. (1979) fection. Cell 18, 109-116. 20. Sutcliffe, J. G., Shinnick, T. M., Verma, I. M. & Lerner, R. A. We are very grateful to A. Ohtsuka, P. Deininger, and T. Friedmann (1980) Proc. Natl. Acad. Sci. USA 77,3302-3306. for help in determining the sequence of the strong-stop DNA. We 21. Haseltine, W. A. & Baltimore, D. (1976) in Animal Virology, thank M. A. McKennett for excellent technical help and B. Sefton for ICN-UCLA Symposia on Molecular and Cellular Biology, eds. critical review of the manuscript. We thank J. Simon for illustrations Baltimore, D., Huang, A. S. & Fox, C. F. (Academic, New York), and Maureen Brennan for preparing the manuscript. We are partic- Vol. L, pp. 175-213. ularly indebted toJ. Rose for constant help and encouragement during 22. Fan, H. & Verma, I. M. (1978) J. Virol. 26,468-478. this work, and to S. Brennan for a gift of polynucleotide kinase. This 23. Verma, I. M. & McKennett, M. (1978) J. Virol. 26,630-645. work was supported by Grant CA 16561 and CA 21408 from the Na- 24. Verma, I. M., Lai, M.-H. T., Bosselman, R. A., McKennett, M. tional Cancer Institute and VC297 from the American Cancer So- A., Fan, H. & Berns, A. (1980) Proc. Natl. Acad. Sct. USA 77, ciety. 1773-1777. 1. Bishop, J. M. (1978) Annu. Rev. Blochem. 47,37-88. 25. Chang, A. C. Y., Nunberg, J. H., Kaufman, R. J., Erlich, H. A., 2. Verma, I. M. (1977) Biochim. Biophys. Acta 473,1-38. 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Monroy, G., Jacquet, M., Groner, Y. & Hurwitz, J. (1979) Cold 9. Dina, D. & Benz, E. W., Jr. (1980) J. Virol. 33, 377-389. Spring Harbor Symp. Quant. Biol. 39,1033-1041. 10. Gilboa, E., Goff, S., Shields, A., Yoshimura, F., Mitra, S. & Bal- 32. Haseltine, W. A. & Baltimore, D. (1976) J. Virol. 19,331-37. timore, D. (1979) Cell 16,863-874. 33. Fan, H. (1977) Cell 11, 297-305. 11. Bosselman, R. A. & Verma, 1. M. (1980) J. Virol. 33, 487- 34. Ziff, E. B. & Evans, R. M. (1978) Cell 15, 1463-1475. 493. 35. Proudfoot, N. J. & Brownlee, G. G. (1976) Nature (London) 263, 12. Yoshimura, F. & Weinberg, R. A. (1979) Cell 16,323-332. 211-214. 13. Vande Woude, G. F., Oskarsson, M., Enquist, L. W., Nomura, 36. Kleckner, N. (1977) Cell 11, 11-23. S., Sullivan, M. & Fischinger, P. J. (1979) Proc. Natl. Acad. Sci. 37. Shapiro, J. A. (1979) Proc. Natl. Acad. Sci. USA 76, 1933- USA 76, 4464-4468. 1937. 14. Lowy, D. R., Rands, E., Chattopadhyay, S. K., Garon, C. F. & 38. Shoemaker, C., Goff, S., Gilboa, E., Paskind, M., Mitra, S. H. & Hager, G. L. (1980) Proc. Natl. Acad. Sci. USA 77,614-618. Baltimore, D. (1980) Proc. Natl. Acad. Sci. USA 77, in press. 15. Hsu, T. W., Sabran, J. L., Mark, G. E., Guntaka, R. V. & Taylor, 39. Steffen, D. & Weinberg, R. A. (1978) Cell 15,1003-1010. J. M. (1978) J. Virol. 28, 810-818. 40. Bacheler, L. T. & Fan, H. (1979) J. Virol. 30,657-667. Downloaded by guest on October 1, 2021