Proc. Natl. Acad. Sci. USAVol. 84, pp. 2824-2828, May 1987Genetics

MYC oncogene involved in a t(8;22) chromosome translocation is notaltered in its putative regulatory regions

(Burkitt lymphoma/oncogene deregulation/immunoglobulin gene rearrangement/somatic cell hybrids)

LOUISE C. SHOWE*, ROBERT C. A. MOORE, JAN ERIKSON, AND CARLO M. CROCEThe Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104

Communicated by Peter C. Nowell, December 18, 1986

ABSTRACT We have cloned the translocation-associatedMYC gene from the Burkitt lymphoma cell line (BL2) with at(8;22) chromosomal translocation and have determined thenucleotide sequence of the first exon and of the 3' and 5'flanking regions, where sequences with putative regulatoryfunctions have been identified. The nucleotide sequence of the5' flanking region, which contains regions of DNase hypersen-sitivity and binding sites for putative regulatory proteins, is thesame as that of the normal MYC. Accordingly, mutations inthese regulatory regions are not required for the transcrip-tional deregulation ofMYC in the BL2 cell line. The nucleotidesequence of the first exon is similar to that of the normal MYC[Gazin, C., Dupont de Direchin, S., Hampe, A., Masson,J. M., Martin, P., Stehelin, D. & Galibert, F. (1984)EMBO J.3, 383-387] and has the coding capacity for a 188-residuepolypeptide. However, six nucleotide changes that occur in themiddle of this reading frame could result in amino acidsubstitutions. We also have cloned and sequenced the t(8;22)chromosomal breakpoint that is located 10 kilobases 3' of theMYC exon 3 and near the CA3 gene on chromosome 22.Sequences with homology to immunoglobulin joining signalsoccur close to the breakpoint both on chromosome 8 and 22,providing further evidence that the immunoglobulin joiningenzymes may be involved in the recombinations associated witha variety of chromosomal translocations in B and T cells.

In most Burkitt lymphomas, the MYC gene on the normalchromosome 8 is transcriptionally silent, while the translo-cation-associated gene is transcriptionally active (1, 2). It hasbeen suggested that this phenomenon of allelic exclusionresults from the disruption or mutation of negative regulatoryelements within the first exon and/or the 5' flanking region ofthe translocated gene (3). Some of us have suggested (1, 2)that the deregulation of the MYC gene in Burkitt lymphomaresults from the juxtaposition of the MYC locus and anactivated immunoglobulin locus.BL2 is a Burkitt lymphoma cell line that carries a t(8;22)

chromosomal translocation that involves the MYC gene onchromosome 8 and the immunoglobulin A light chain locus onchromosome 22. No gross alterations in the gene are detectedby Southern analysis with probes for introns 1, 2, or 3 (4). Thetranslocation occurs 10 kilobases (kb) 3' of MYC exon 3 (5)and outside of any region implicated in transcriptional regu-lation. The normal MYC allele is transcriptionally silent (1).Transcription from the translocation-associated allele resultsin steady-state levels ofMYC transcripts that are 3-4 timeshigher than levels in lymphoblastoid controls (6). NucleaseS1 cleavage-protection studies of the MYC transcripts in BL2cells with probes for exon 1 yield protected fragments of theexpected size (2), suggesting that the first exon of BL2 is nothighly mutated. Because of the apparent normal structure of

this gene, we were interested in determining whether minorchanges might have occurred that could be involved in thistranscriptional deregulation.

MATERIALS AND METHODSNucleotide Sequences. Nucleotide sequences were deter-

mined by the dideoxy chain-termination method of Sanger etal. (7) using phage M13 and the Clontech M13 sequencingprimer or synthetic primers derived from the nucleotidesequence.Genomic DNA Libraries. The genomic DNA library used to

isolate the 3' breakpoint region was prepared from a completeEcoRI digest of high molecular weight BL2 DNA, andappropriate fractions from sucrose gradients were cloned intothe bacteriophage A vector EMBL 4 (8). The genomic libraryfor the isolation of the translocation-associated MYC genewas prepared from a somatic cell hybrid between BL2 and themurine fusion partner NP3, which contained the derivativechromosome 8 but not the normal human allele (4). Thislibrary was constructed in the bacteriophage A vector EMBL3a.Genomic DNA libraries were screened as described (8)

with the exception that screenings with 32P-end-labeled withsynthetic oligonucleotides had final washes with 0.9 MNaCl/0.09 M sodium citrate, pH 7, at 37°C for 1-2 hr.

Probes. The 3' humanMYC probes CA 1.7S and pPA1.3SBare derived from regions 2 kb and 12 kb 3' of MYC,respectively (6). The CA probe is an 8-kb genomic probe thatincludes the immunoglobulin A light chain constant (C) regiongenes CA and CA3 (9). Ryc7.4 is a 1.1-kb cDNA clone thatcodes for the human MYC exon 3 and part of exon 2 (10).

RESULTSIsolation of the Translocation-Associated MYC Gene. While

no rearrangements could be detected within the MYC genewith the Ryc7.4 probe on Southern blots, a rearrangementwas detected with the 3' pCA1.7S probe (6) and was found tooccur - 10 kb 3' ofMYC exon 3 and 19 kb 3' of the P1 and P2promoter regions (5). A rearrangement was also detectedwith the pPA1.3SB probe derived from a region 3' of thebreakpoint. This rearranged fragment segregated with the22q- chromosome and indicated that no gross deletionoccurred as a result of the translocation (data not shown).Since the distance between the 3' MYC rearrangement andputative regulatory sequences of interest 5' of the gene madeit unlikely that both the identifying breakpoint junction andthe 5' region of interest would be found in a single bacteri-ophage A genomic clone, it was necessary to ensure that theMYC gene being analyzed was the translocation-associatedgene and not the normal gene. Accordingly, a genomic DNAlibrary was constructed from a somatic cell hybrid (17-6)

Abbreviations: C, constant; J, joining; V, variable.*To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 84 (1987) 2825

derived from the fusion of the BL2 cell line and the murineplasmacytoma cell line NP3 (4). The 17-6 hybrid has beencharacterized cytogenetically and by Southern blotting andcontains the derivative but not the normal human chromo-some 8 (4).High molecular weight DNA was first digested to comple-

tion with the restriction enzyme BamHI. This digests themurine c-myc gene to a size that is too small (5.6 kb) to beefficiently cloned into the EMBL 3a phage vector, whereasthe BamHI restriction fragment derived from human MYCgene is too large (28 kb) to be cloned in this vector. This largeBamHI restriction fragment was subsequently subjected to alimited digestion with the restriction enzyme Sau3A to trimthe fragment to a clonable size. Six MYC-positive cloneswere isolated from 100,000 recombinants, and one of these,BL2R, which extended sufficiently far 5' to encompassputative regulatory regions (3, 11, 12), was subcloned fornucleotide sequencing (Fig. 1). The nucleotide sequence (Fig.2) was determined for the entire first exon and part of intron1 as well as 1.65 kb 5' of exon 1.

Nucleotide Sequence of the 5' Flanking Region. The nucle-otide sequence for the 1.65 kb of DNA 5' of exon 1 iscompared in Fig. 2 to that of a normal MYC gene (13, 14).Three nucleotide differences occur between nucleotides870-874, and additional changes occur at 1965 and 2134.However, these changes are in agreement with the nucleotidesequence for 870-874 as determined by Battey et al. (15) andfor 1965 and 2134 as determined by Bentley and Groudine(14). Therefore, these differences most likely representsequencing differences or polymorphic changes rather thanmutations. This 1.65-kb region ofDNA contains three of thefive major DNase 1-hypersensitive sites that have beenidentified in the MYC locus and two sequences that arehomologous to nuclear factor 1 protein binding sites,-all ofwhich have been suggested to regulate MYC gene expression(3). The sequence through the MYC major promoters P1 andP2 (14) as well as the recently described minor promoterregion P0 (14) is also normal. Thus, the exclusive expressionof the translocation-associated MYC gene in BL2 cells is notdue to the alteration (as a result of somatic mutation) oftranscriptional regulatory sequences that are located withinthis region and that might regulate transcription from eitherP0, P1, or P2.

Nucleotide Sequence of MYC Exon 1. The nucleotide se-quence of the first exon (Fig. 2) extends from nucleotides2325 to 2880 as numbered by Gazin et al. (13). The regionwithin exon 1 to which DNase hypersensitive site II12 hasbeen mapped is identical to the reference sequence. The extranucleotidfes in the first exon sequence described by Watt etal. (10, 16) that introduce stop codons in all three readingframes are not present in the BL2 MYC sequence, and thecoding capacity for a 188-amino acid peptide, which has beensuggested by Gazin et al. (13), is intact in this MYC gene. Sixnucleotide changes, which cluster in a 166-base-pair (bp)region within the middle of exon 1, would each result in anamino acid substitution. A 32-kDa polypeptide has now beenidentified in cell extracts by using antisera prepared againstseveral synthetic peptides deduced by the translation of this

5-

A

B

0

H P

1000 2000cwvvu Xh Pv u'K P PV pi I p2 S

648

*4 * 4 * 4

500 bp

open reading frame (17), although it is not yet known whetherthe 188-amino acid polypeptide has any biological function.The phenomenon of MYC truncation as a result of trans-

location and the observed accumulation of somatic mutationsthat frequently surround the first exon of translocated geneshas focused on possible implications of these alterations fortranscriptional regulation. The 3' end of exon 1 also has beenimplicated in the regulation of MYC gene expression; inparticular, the region around the Pvu II site (Fig. 2) seems tobe altered in many samples derived from the endemic AfricanBurkitt lymphomas (18, 19). There are no alterations in the200 nucleotides to either side of this Pvu II site in the BL2MYC gene. The region 200 bp from the Pvu II site into the 5'end of intron 1 (in particular the thymidine runs T7, T4, T7)has been suggested to regulate MYC RNA at the level oftranscription (11, 18). There are five single nucleotidechanges within the subsequent 370 bp of intron 1, some ofwhich may represent polymorphic changes or sequencingdifferences. This is also the region where the breakpoints forseveral sporadic Burkitt lymphomas occur. No regulatoryfunction has been demonstrated for this region specifically,although there are three putative binding sites (CCCGCC) forthe Spl transcription factor that are unaffected by the fivenucleotide changes. Thus, mutation of the Pvu II region,which has been implicated by others in the deregulation ofMYC in the endemic Burkitt lymphomas, does not appear tobe required for the deregulation seen in BL2.The BL2 Breakpoint. The 18-kb normal and the 12-kb

rearranged EcoRI restriction fragments from the 3' flankingregion of MYC were isolated from the BL2 genomic libraryin EMBL 4. The restriction maps are shown in Fig. 3 A andC. The maps diverge in the region of a Kpn I site located 3 kbfrom the 3' end of the rearranged EcoRI fragment. Asexpected, this region cross-hybridized with the 8.0-kb C,genomic probe (Fig. 3D). The restriction map for the 3' endof the 8.0-kb CA EcoRI fragment (Fig. 3D) is identical to thatfor the 3' end of the BL2 breakpoint clone (Fig. 3C). The twomaps diverge within the region of a previously mapped X lightchain joining (J) region gene JA (6, 9).

Recognition Signals Associated with the Breakpoint. Thefrequent association of MYC translocations with regionsinvolved in immunoglobulin gene rearrangements [J or vari-able (V) regions] or immunoglobulin class-switching [heavychain switch regions] suggests that mechanisms involved inimmunoglobulin gene rearrangements may also take part inthe recombinatorial events associated with chromosomaltranslocations in B- and T-cell neoplasias. Evidence tosupport this hypothesis has come from the identification ofnucleotide sequences with homology to the heptamer-nonamer signal sequences, which are utilized in immuno-globulin gene rearrangements, close to chromosomal break-points within the nonimmunoglobulin loci involved in chro-mosomal translocations in B- and T-cell neoplasias (20-22).The nucleotide sequence through the breakpoint regions on

chromosomes 8 and 22 and the sequence through the sameregion on the normal chromosome 8 are shown in Fig. 4. Thebreakpoint sequence diverges from the normal chromosome

FIG. 1. Sequencing strategy forMYC exon3510 31l and flanking regions. (A) The HindIII-Xba IXb restriction fragment (H-Xb) from the translo-

I cation-associated gene was cloned into M133507 mpl8 and -19. The black bar denotes exon 1.

H,HindIII;P,PstI;Pv,PvuII; K, KpnI; S,Sau3A; Xb, Xba I; pl and p2, promoters. (B)The nucleotide sequence was determined ei-ther by primer extension from synthetic oli-gonucleotides on the cloned H-Xb fragmentor from subcloned restriction fragments ofH-Xb.

I

Genetics: Showe et al.

_ 3000

2826 Genetics: Showe et al. Proc. Natl. Acad. Sci. USA 84 (1987)

myc 648 CCCCTTTCCCCCGAATTGTTTTCTCTTTTGGAGGTGGTGGAGGGAGAGMMAGTTTACTTMMTGCCTTTGGGTGAGGGACCAAGGATGAGMGMTGTTTTTTGTTTTb12 1

myc 758 TCATGCCGTGGMTAACACMAATAMMATCCCGAGGGAATATACATTATATATTMMTATAGATCATTTCAGGGAGCMACAAATCATGTGTGGGGCTGGGCAACTAGb12 111

myc 868 CTGATG-CGAAGCGTMATAMATGTGAATACACGTTTGCGGGTTACATACAGTGCACTTTCACTAGTATTCAGA TTGTGAGTCAGTGMCTAGGMMTTMTGCCb12 221 X A T

myc 978 TGGAAGGCAGCCAAATTTTAATTAGCTCMGACTCCCCCCCCCCCCCAMAAAAGGCACGGMGTMTACTCCTCTCCTCTTCTTTGATCAGMTCGATGCATTTTTTGTb12 331 X

myc 1088 GCATGACCGCATTTCCAATMTAAAAGGGGAMGAGGACCTGGMAGGMTTMACGTCCGGTTTGTCCGGGGAGGAMGAGTTAACGGTTTTTTTCACMGGGTCTCTGbl2 440

myc 1198 CTGACTCCCCCGGCTCGGTCCACAAGCTCTCCACTTGCCCCTTTTAGGMGTCCGGTCCCGCGGTTCGGGTACCCCCTGCCCCTCCCATATTCTCCCGTCTAGCACCTTTb12 550

myc 1308 GATTTCTCCCAMCCCGGCAGCCCGAGACTGTTGCMMCCGGCGCCACAGGGCGCMMGGGGATTTGTCTCTTCTGMACCTGGCTGAGAMTTGGGMCTCCGTGTGGGb12 660

myc 1418 AGGCGTGGGGGTGGGACGGTGGGGTACAGACTGGCAGAGAGCAGGCMCCTCCCTCTCGCCCTAGCCCAGCTCTGGMCAGGCAGACACATCTCAGGGCTMMCAGACGCb12 770

myc 1528 CTCCCGCACGGGGCCCCACGGMGCCTGAGCAGGCGGGGCAGGAGGGGCGGTATCTGCTGCTTTGGCAGCMATTGGGGGACTCAGTCTGGGTGGMGGTATCCMTCCAb12 880

myc 1638 GATAGCTGTGCATACATAATGCATAATACATGACTCCCCCCAACMMTGCMTGGGAGTTTATTCATAACGCGCTCTCCAAGTATACGTGGCMTGCGTTGCTGGGTTATb12 990

myc 1748 TTTMTCATTCTAGGCATCGTTTTCCTCCTTATGCCTCTATCATTCCTCCCTATCTACACTMCATCCCACGCTCTGMCGCGCGCCCATTAATACCCTTCTTTCCTCCAb12 1100

myc 1858 CTCTCCCTGGGACTCTTGATCMAGCGCGGCCCTTTCCCCAGCCTTAGCGAGGCGCCCTGCAGCCTGGTACGCGCGTGGCGTGGCGGTGGGCGCGCAGTGCGTTCTCTGTb12 1210 G

myc 1968 GTGGAGGGCAGCTGTTCCGCCTGCGATGATTTATACTCACAGGACAAGGATGCGGTTTGTCAAACAGTACTGCTACGGAGGAGCAGCAGAGAAAGGGAGAGGGTTTGAGAbi2 1320

* ***** . 1111Imyc 2078 GGGAGCAAMGMMATGGTAGGCGCGCGTAGTTAATTCATGCGGCTCTCTTACTCTCTTTACATCCTAGAGCTAGAGTGCTCGGCTGCCCGGCTGAGTCTCCTCCCCACCb12 1430 G

myc 2188 TTCCCCACCCTCCCCACCCTCCCCATAAGCGCCCCTCCCGGGTTCCCAAAGCAGAGGGCGTGGGGGAMAGAAAAAAGATCCTCTCTCGCTMTCTCCGCCCACCGGCCCb12 1540

P1*-- >.

m 2298 TTTATA ATG CGA GGG TCT GGA CGG CTG AGG ACC CCC GAG CTG TGC TGC TCG CGG CCG CCA CCG CCG GGC CCC GGC CGT CCC TGG CTC CCCMet Arg Gly Ser Gly Arg Leu Arg Thr Pro Glu Leu Cys Cys Ser Arg Pro Pro Pro Pro Gly Pro Gly Arg Pro Trp Leu Pro

b 1650

m 2388 TCC TGC CTC GAG AAG GGC AGG GCT TCT CAG AGG C T GGC GGG AAA MG MC GGA GGG AGG GAT CGC GCT GAG TAT AM AGC CGG TTT TCGSer Cys Leu Glu Lys Gly Arg Ala Ser Gln Arg Leu Gly Gly Lys Lys Asn Gly Gly Arg Asp Arg Ala Glu Tyr Lys Ser Arg Phe Ser

b 1740P2-.-

m 2478 GGG CTT TAT CTA ACT CGC TGT AGT AAT TCC AGC GAG AGG CAG AGG GAG CGA GCG GGC GGC CGG CTA GGG TGG AAG AGC CGG GCG AGC AGAGly Leu Tyr Leu Thr Arg Cys Ser Asn Ser Ser Glu Arg Gln Arg Glu Arg Ala Gly Gly Arg Leu Gly Trp Lys Ser Arg Ala Ser Arg

b 1830

m 2568 GCT GCG CTG CGG GCG TCC TGG GAA GGG AGA TCC GGA GCG MT AGG GGG CTT CGC CTC TGG CCC AGC CCT CCC GCT GAT CCC CCA GCC AGCAla Ala Leu Arg Ala Ser Trp Glu Gly Arg Ser Gly Ala Asn Arg Gly Leu Arg Leu Trp Pro Ser Pro Pro Ala Asp Pro Pro Ala Ser

b 1920 T ATrp Thr

m 2658 GGT CCG CM CCC TTG CCG CAT CCA CGA AAC TTT GCC CAT AGC AGC GGG CGG GCA CTT TGC ACT GGA ACT TAC MC ACC CGA GCA AGG ACGGly Pro Gln Pro Leu Pro His Pro Arg Asn Phe Ala His Ser Ser Gly Arg Ala Leu Cys Thr Gly Thr Tyr Asn Thr Arg Ala Arg Thrb 2010 T T A A

Ser Val Thr Lys

m 2748 CGA CTC TCC CGA CGC GGG GAG GCT ATT CTG CCC ATT TGG GGA CAC TTC CCC GCC GCT GCC AGG ACC CGC TTC TCT GM AGG CTC TCC TTGArg Leu Ser Arg Arg Gly Glu Ala Ile Leu Pro Ile Trp Gly His Phe Pro Ala Ala Ala Arg Thr Arg Phe Ser Glu Arg Leu Ser Leub 2100PvuII

m 2838 CAG CTG CTT AGA CGC TGG ATT TTT TTC GGG TAG TGGAMACCAGGTAAGCACCGAAGTCCACTTGCCTTTTMTTTATTTTTTTATCACTTTAATGCTGAGln Leu Leu Arg Arg Trp Ile Phe Phe Gly ---

b 2190

myc 2938 GATGAGTCGMTGCCTAMTAGGGTGTCTTTTCTCCCATTCCTGCGCTATTGACACTTTTCTCAGAGTAGTTATGGTMCTGGGGCTGGGGTGGGGGGTMTCCAGMCTb12 2290 T

myc 3048 GGATCGGGGTMMGTGACTTGTCMGATGGGAGAGGAGMGGCAGAGGGMMCGGGMTGGTTTTTMGACTACCCTTTCGAGATTTCTGCCTTATGMTATATTCACGb12 2400 C

myc 3158 CTGACTCCCGGCCGGTCGGACATTCCTGCTTTATTGTGTTAATTGCTCTCTGGGTTTTGGGGGGCTGGGGGTTGCTTTGCGGTGGGCAGAMGCCCCTTGCATCCTGAGCbl2 2510 C

myc 3268 TCCTTGGAGTAGGGACCGCATATCGCCTGTGTGAGCCAGATCGCTCCGCAGCCGCTGACTTGTCCCCGTCTCCGGGAGGGCATTTMMTTTCGGCTCACCGCATTTCTGAb12 2620

myc 3378 CAGCCGGAGACGGACACTGCGGCGCGTCCCGCCCGCCTGTCCCCGCGGCGATTCCMCCCGCCCTGATCCTTTTMGMGTTGGCATTTGGCTTTTTMMMGCMTAATb12 2730 G A

myc 3488 ACAATTTAAAACCTGGGTCTCTAGAGGTGTTAGGACGTGGTGTTGGGTAGGCGb12 2840

FIG. 2. Nucleotide sequence of MYC exon 1 and flanking regions. The top line is the sequence of Gazin et al. (14) and begins 648 bp fromthe 5' end of that sequence. The lower line is the BL2 sequence and only regions of sequence differences are indicated. DNase I-hypersensitivesites are indicated by Roman numerals, and putative nuclear factor I binding sites are boxed. The exon 1 reading frame is that proposed by Gazinet al. (14), and changes in the proposed BL2 sequence are indicated. The Pvu II site is indicated at the end of exon 1.

8 sequence around position 448. The boxed sequence 30 bp of seven match with the consensus sequence for a heptamer3' of the breakpoint on the normal chromosome 8 has a five recombination signal. A heptamer with a six of seven match

Proc. Natl. Acad. Sci. USA 84 (1987) 2827

5' SBg BH E 3

Bg q~g iEI SI I K|Bg B lSI BCh. 8

1 2 3 probe . probe

pCA1.7S J . pPA1.3SB

BCh.8K " * - nBB Ch.~

C K H B B Bg B ECI I I Ch.22

-40.O. *-4-

Ch. 22

D ,g B BI *

B Bg B EI

3.3 kbLWI

FIG. 3. Restriction map of the BL2 breakpoint.(A) Restriction map of the normal MYC gene and 3'flanking region. The position of 3' MYC probes is

0.3 kb indicated; the breakpoint occurs within the insertshown in B. (B) Expanded restriction map of theBL2 breakpoint region on the normal chromosome8. (C) Restriction map of the BL2 breakpoint clone.(D) Restriction map of the normal chromosome 22region involved in the translocation. B, BamHI; Bg,Bgi II; E, EcoRI; K, Kpn I; H, HindIII; S, Sst I.Arrows indicate regions sequenced; asterisks indi-cate breakpoint positions; and the solid boxes areexons 1, 2, and 3.

occurs 12 bp 3' of the breakpoint within the sequence derived twice is present 100 bp from the breakpoint on chromosomefrom chromosome 22. In addition, we find the GAGG or 8. The significance of this repeat is not known.CCTC tetranucleotide occurring several times 5' of the The matched sequence 161 bp 3' of the breakpoint isbreakpoint on chromosome 8 and 3' of the breakpoint on identical to the published sequences for a JA3 gene (25, 26).chromosome 22. This tetranucleotide is frequently found in However, the sequence 5' of the J gene is not consistent withclose association with the chromosomal breakpoints that the JA 5' flanking region (data not shown) and lacks both thehave been characterized in mouse plasmacytomas (23) and heptamer and nonamer signal sequences, which should beoccurs with a significant frequency in both the MYC locus present 5' of an unrearranged JA gene, suggesting a V-Jand the murine and human immunoglobulin loci (24). A rearrangement has occurred. An open reading frame (Fig. 4)nine-nucleotide sequence, GACCTCAGG, that is repeated that extends from the breakpoint and continues in frame

A

ch .8ch .8 GGTACCTAGTCAGACCCAATTACACGGTTGAATAT TGCAGTCGGTGTGCTCAGGTGCTGAGATCCATGGCCAGAAAGAGTCTGGTGATAGATTTATGCCCGGGTAGT

jct. GGTACCTAGTCAGACCC CTTCACGTCGTTTTGAGCGTGTGCTCAGGTGCTGAGATCCATGGCCAGAAAGAGTCTGGTGATAGATTTATGCCCC GGGTAGT

cht.8 l TTGTACCTGCCTGGTTGCTTGAGGGTTAAAATGGGTTAGMTATAGGATGATGACTCAGAGCTGGAGACGCCTTAGCATTCGCTTTCCAGATG TGGCCCTGGAGMGA

ct . 5l5l TTGTACCTGCCTGGTTGCTTGACMGTAGTGGGTGTAAAATATAGGATGATGACTCAGAGCTGGAAGACCTATAGCATTCGCATTTCCAGATGT GCCCCTGGAGGA

....

ch.8 221 CGCATCTTCCTGAGATCCCTCTACTGGCTTTTGGCTGAATTAGCTTATGACCTCAGGGCCCTGGTTGCATCCTACTGATTATC CGCAAGTAAGACCTCAGGTCTTCT

jct. 221 CGCAATCTTCCTGAGATCCCTCTACTGGCTTTTGGCTGMTTAGCTTATGACCTCAGGGCCCTGGTTGCATCCTMCTGATTATCMGCMGTMGACCTCAGGTCTTCT

ch .8 331 GAGATGCAGGATGCTTACTTGCCTTACTACTMAGATGMAGCTTTCAMACTCACCCCGTTTCCMAGTCCATGCCAGGTCTTATGCCCACACCCAGACTGGCCCAGTCMACA...22..CC T.

jct. 331 GAGATGCAGGATGCTTACTTGCCTTACTACTMAGATGMAGCTTTCMAACTCACCCCGTTTCCMTCGCATTAGCAACAATGCATMA

ch.8 441 AATATTTATCGGGCACCTCCGATGACAAAGGAGGCACCCGTGTGGMGGMCAGATTCC CAGTCTAGTAGAGAAGAGTGGCATGATACMGTATTTACATACATMTT

jct. 441 GATGATCCTGMGATGCTTAACAGTGATGGGCTTCCCAGCTTCTGGGGGAGCTGAGCGCTACCTCACCATCTCCA

ch.8 551 ACCTMMACAGATGCMMAGGCACMMAGTAGTGGGGTGAGGMAGTACAGAGTATGTGGGGGCATCTGACAGCCTAGGAGTTCCAGGMAGTTTCT

jct. 551 GCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCATTCMAGGGATGTTCGGCGGAGGGACCMAGCTGACCGTCCTAGGTGAGTCTCTTCTC

ckh.22 ........TGTTCGGCGGAGGGACCMAGCTGACCGTCCTAGGTGAGTCTCTTCTC

jct. 661 CCCTCTCCTTCCCCGCTCTCGGGACATTCTGCTGTTT

ch .22 CCCTCTCCTTCCCCGCTCTTGGGACAATTTCTGTTGT

Bjct. 448 TTG ATG MAG CTT MAC AGT GAT GGC AGC CAC AGC MAG GGG GAC

Leu Met Lys Leu Asn Ser Asp Gly Ser His Ser Lys Gly Asp

Ala Pro Leu Leu Val Ile Tyr Gly Arg Asn Asn Arg Pro Ser

V>jct. 490 GGG ATC CCT GAT CGC TTC TCA GGC TCC AGC TCT GGG GCT GAG

Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu* * * * * * * * * * * *

Gly LIle Pro Asp Arg Phe Ser Giy Ser Ser Ser Giy His Thr

jct. 532 CGC TAC CTC ACC ATC TCC AGC CTC CAG TCT GAG GAT GAG GCTArg Tyr Leu Thr Ile Ser Ser Leu Gin Ser Glu Asp Glu Ala

Ala Ser Leu Thr Ile Thr Gly Ala Gin Ala Glu Asp Glu Ala

* . ~~~J>jct. 574 GAC TAT TAC TGT CAG ACC TGG GGC ACT GGC ATT CM GGG ATG

Asp Tyr Tyr Cys Gin Thr Trp Gly Thr Gly Ile Gin Gly Met

Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Val Leu ---

jct. 616 TTC GGC GGA GGG ACC MG CTG ACC GTC CTA GGT GAG TCT CTTPhe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly* * * * * * * * * * *

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

jct. 658 CTC CCC TCT CCT TCC CCG CTC TCG GGA CAT TCT GCT GTT TT

FIG. 4. Nucleotide sequence through theBL2 breakpoint. (A) The sequence for the nor-mal MYC (lines ch. 8) is compared to that of thebreakpoint clone (lines Jct) and the normalchromosome 22 (lines ch. 22) for the 3' end ofthe sequence. (B) The deduced amino acidsequence from the region 3' of the chromosome8/chromosome 22 junction. V>, beginning ofthe sequence that is homologous to the V14 genefamily; J>, beginning of the J13 sequence. As-terisks denote homology; dots above the nucle-otide sequence indicate 10 bp; and the dashedline indicates a deletion.

Genetics: Showe et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

through the J region is highly homologous to the nucleotidesequence of the VX4 gene for 120 nucleotides 5' of J,suggesting this is actually the case. Although the readingframe continues up to the breakpoint, the deduced amino acidsequence for the 5' end of this region is not homologous tothat for any known VA gene family. The evidence that thissequence does not derive from chromosome 8 is based on thelack of hybridization of a synthetic oligonucleotide derivedfrom this region to any of our phage X clones, which extend20 kb 3' and 5' of the breakpoint on chromosome 8. We haveisolated genomic clones that contain internal restrictionfragments that hybridize with a synthetic oligonucleotidederived from the divergent sequence but not with an oligo-nucleotide derived from the sequence 20 bp further 3' that ishomologous to V4 (data not shown). Thus, the 43 bp betweenchromosome 8 and VA are not contiguous on chromosome 22.

DISCUSSIONMYC Deregulation. Since all of the regions that have been

suggested to play a role in the regulation of expression of thehuman MYC gene are normal in the BL2 gene, we cannotaccount for the exclusive expression of the translocation-associated gene as a consequence of the mutation of any ofthese regions. The only changes that do occur are clusteredin a 166-bp region within exon 1, which has not beenimplicated in transcriptional regulation. We cannot eliminatethe possibility that these changes contribute to the highsteady-state levels ofMYC transcripts by increasing messagestability. However, increased stability of MYC transcriptshas only been demonstrated for messages derived from eithertruncated genes (18) or genes in which there are extensivealterations within the first exon (27). Furthermore, whilealtered message stability could explain the high steady-statelevels of MYC RNA, it does not explain the exclusivetranscription of the translocation-associated MYC gene inmost Burkitt lymphomas. Since the nucleotide sequencethrough the putative regulatory regions within exon 1 as wellas within the 5' and 3' flanking regions are normal, this showsthat deregulation of MYC transcription does not requiresequence alterations within these regions. Although thepossibility remains that additional transcriptional regulatoryregions may exist outside of the region we sequenced, wewould suggest that the dominant effect on MYC transcriptionresults from the de novo association with an activatedimmunoglobulin locus. This would require that the influenceof the associated immunoglobulin locus be dominant over thenormal mechanisms for MYC regulation. In the case of BL2cells, the influence of the immunoglobulin locus must beexerted over a distance of =20 kb, which separates theimmunoglobulin locus and the putative 5' regulatory ele-ments.Mechanisms Involved in Translocation. Although we find

regions with homology to J signals on chromosome 8, therecombination within a V gene is not consistent with aconventional immunoglobulin gene rearrangement, whichrequires the presence of both heptamer and nonamer signalswith the appropriate 1 turn-2 turn spacing. However, singleheptamer sequences have now been shown to occur close toregions of illegitimate recombinations involving the immu-noglobulin loci in a variety of examples, including the murine(28) and human (29) K light chain-deleting elements. Recom-binations have also been demonstrated within the heavychain locus, where a V-to-V rearrangement has been pro-posed to occur via a single heptamer signal within the V genecoding sequence, resulting in a chimeric VH gene (30, 31).This suggests that a certain flexibility exists in the joiningreactions that had not been considered previously. Anexamination of published VA, nucleotide sequences revealsthe presence of multiple sequences with homologies to the

heptamer consensus sequence within the 5' ends of thesegenes (25, 26, 32). The presence of multiple heptamers withinthese sequences suggests that similar recombinations mightalso occur within VA genes. The rearrangement within the VAgene illustrated in Fig. 4 could possibly result from this typeof recombination.We thank Marina Hoffman for editing, The Wistar Institute

editorial stafffor preparing the manuscript, and Andrew Caton for hishelp in the analysis of breakpoint sequence and careful reading of themanuscript. This work was supported by National Institutes ofHealth Grant CA39860 (to C.M.C.) and American Cancer SocietyGrant CD-274 (to L.C.S.).

1. ar-Rushdi, A., Nishikura, K., Erickson, I., Watt, R., Rovera, G. &Croce, C. M. (1983) Science 222, 390-393.

2. Nishikura, K., ar-Rushdi, A., Erikson, J., Watt, R., Rovera, G. &Croce, C. M. (1983) Proc. Natl. Acad. Sci. USA 40, 4822-4826.

3. Siebenlist, U., Hennighausen, L., Battey, J. & Leder, P. (1984)Cell 37, 381-391.

4. Croce, C. M., Thierfelder, W., Erikson, J., Nishikura, K., Finan,J., Lenoir, G. M. & Nowell, P. C. (1983) Proc. Natl. Acad. Sci.USA 80, 6922-6926.

5. Erikson, J., Finger, L., Sun, L., ar-Rushdi, A., Nishikura, K.,Minowada, J., Finan, J., Emanuel, B. S., Nowell, P. C. & Croce,C. M. (1986) Science 232, 884-886.

6. Sun, L. K., Showe, L. C. & Croce, C. M. (1986) Nucleic AcidsRes. 14, 4037-4050.

7. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad.Sci. USA 74, 5463-5467.

8. Showe, L. C., Ballantine, M., Nishikura, K., Erikson, J., Kaji, H.& Croce, C. M. (1985) Mol. Cell. Biol. 5, 501-509.

9. McBride, D. W., Heiter, P. A., Hollis, G. F., Swan, D., Otey,M. C. & Leder, P. (1982) J. Exp. Med. 155, 1480-1490.

10. Watt, R., Stanton, L. W., Marcu, K. B., Gallo, R. C., Croce,C. M. & Rovera, G. (1983) Nature (London) 303, 725-728.

11. Chung, J., Sinn, E., Reed, R. & Leder, P. (1986) Proc. NatI. Acad.Sci. USA 83, 7918-7922.

12. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Fronich,S., Aaronson, S. & Leder, P. (1982) Proc. Natl. Acad. Sci. USA 79,7837-7841.

13. Gazin, C., Dupont de Direchin, S., Hampe, A., Masson, J. M.,Martin, P., Stehelin, D. & Galibert, F. (1984) EMBO J. 3, 383-387.

14. Bentley, D. L. & Groudine, M. (1986) Mol. Cell. Biol. 6,3481-3489.

15. Battey, J., Moulding, C., Taub, R., Murphy, W., Stewart, T.,Potter, H., Lenoir, G. & Leder, P. (1983) Cell 34, 779-787.

16. Watt, R., Nishikura, K., Sorrentino, J., ar-Rushdi, A., Croce,C. M. & Rovera, G. (1983) Proc. Natl. Acad. Sci. USA 80,6307-6311.

17. Gazin, C., Rigolet, M., Briand, J. P., Van Regenmortel, M. H. V.& Galibert, F. (1986) EMBO J. 5, 2241-2250.

18. Bentley, D. L. & Groudine, M. (1986) Nature (London) 321,702-706.

19. Pelicci, P. G., Knowles, D. M., II, Magrath, I. & Dalla Favera, R.(1986) Proc. Natl. Acad. Sci. USA 83, 2984-2988.

20. Tsujimoto, Y., Jaffe, E., Cossman, J., Gorham, J., Nowell, P. C. &Croce, C. M. (1985) Nature (London) 315, 340-343.

21. Haluska, F. G., Finver, S., Tsujimoto, Y. & Croce, C. M. (1986)Nature (London) 324, 158-161.

22. Finger, L. R., Harvey, R., Moore, R. C. A., Showe, L. C. &Croce, C. M. (1986) Science 234, 982-985.

23. Picolli, S. P., Carini, P. G. & Cole, M. D. (1984) Nature (L>ndon)310, 327-330.

24. Showe, L. C. & Croce, C. M. (1986) Cancer Rev. 2, 18-23.25. Denny, C. T., Hollis, G., Magrath, I. T. & Kirsch, I. R. (1985)

Mol. Cell. Biol. 5, 3199-3202.26. Sun, L. H. K., Croce, C. M. & Showe, L. C. (1985) Nucleic Acids

Res. 13, 4921-4934.27. Piechaczyk, M., Yang, J. Q., Blanchard, J. M., Jeanteur, P. &

Marcu, K. B. (1985) Cell 42, 489-597.28. Durdik, J., Moore, M. W. & Selsing, E. (1984) Nature (London)

307, 749-752.29. Siminovitch, K. A., Bakshi, A., Goldman, P. & Korsmeyer, S.

(1985) Nature (London) 316, 260-262.30. Klienfield, R., Hardy, R. R., Tarlinton, D., Dang, J., Herzenberg,

L. A. & Weigert, M. (1986) Nature (London) 322, 843-846.31. Reth, M., Gehrmann, P., Petrac, E. & Wiese, P. (1986) Nature

(London) 322, 840-842.32. Anderson, M. L. M., Szajnert, M. F., Kaplan, J. C., McColl, L. &

Young, B. D. (1984) Nucleic Acids Res. 12, 6647-6661.

2828 Genetics: Showe et al.