Vol. 11, No. 3MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1500-15070270-7306/91/031500-08$02.00/0

A Novel c-fgr Exon Utilized in Epstein-Barr Virus-InfectedB Lymphocytes but Not in Normal Monocytes

J. SILVIO GUTKIND,1 DANIEL C. LINK,2 SHIGERU KATAMINE,1 PEDRO LACAL,1 TORU MIKI,3TIMOTHY J. LEY,2 AND KEITH C. ROBBINS'*

Laboratory of Cellular Development and Oncology, National Institute of Dental Research,1 and Laboratory ofCellular and Molecular Biology, National Cancer Institute,3 Bethesda, Maryland 20892, and Division of

Hematology-Oncology, Departments of Medicine and Genetics, Jewish Hospital atWashington University Medical Center, St. Louis, Missouri 631102

Received 16 August 1990/Accepted 19 December 1990

Thefgr proto-oncogene encodes a nonreceptor protein-tyrosine kinase, designated p55c-fg.r In this study, wehave isolated human fgr cDNA molecules from normal monocyte mRNA templates. Nucleotide sequence

analysis of the longestfgr cDNA revealed a 5' untranslated region of 927 bp which included two Alu-like repeatsas well as three translation stop codons immediately upstream of the initiator for p55c-fgr synthesis. Withingenomic DNA, these sequences were distributed over 13 kbp as three distinct 5' untranslated exons. Previousstudies have shown that Epstein-Barr virus (EBV) increases c-fgr mRNA levels in B lymphocytes. Bycomparing the nucleotide sequence reported for transcripts isolated from EBV-infected B lymphocytes withthose of our monocyte cDNA as well as genomic DNA, we identified a novel untranslated exon utilized only inEBV-infected cells. The transcriptional initiation sites of fgr mRNA expressed in EBV-converted cells were

mapped and shown to reside within a region identified as an intron for fgr mRNA that is expressed in normalmyelomonocytic cells. Furthermore, the region of thefgr locus upstream of the novel exon displayed propertiesof a transcriptional promoter when transfected into heterologous cells. We conclude from all of these findingsthat activation of the fgr gene by EBV is achieved by mechanisms distinct from those normally regulating itsprogrammed expression in myelomonocytic cells.

Thefgr gene belongs to the family of proto-oncogenes thatencode cytoplasmic protein-tyrosine kinases (9). Initially,fgr was described within the context of an oncogenic retro-virus, Gardner-Rasheed feline sarcoma virus (GR-FeSV)(18, 21). The nucleotide sequence of GR-FeSV revealed theexpected virus components as well as portions of twocell-derived genes, ,B-actin and the v-fgr oncogene (18).Deletion mutagenesis experiments have shown that the,B-actin moiety does not contribute to the oncogenicity ofGR-FeSV but instead inhibits its biologic potency (24). Thev-fgr oncogene is truncated with respect to its normalcellular counterpart, c-fgr (13), in that the v-fgr translationalproduct lacks 127 and 12 amino acid residues found at theamino and carboxyl termini, respectively, of the c-fgr geneproduct p55c-fgr (13). A unique amino-terminal domain ofapproximately 70 amino acid residues represents the majorstructural feature distinguishing p55c-fgr from proteins en-coded by other members of the src family.

Expression surveys have revealed the presence of c-fgrmRNA in Epstein-Barr virus (EBV)-infected B lymphocytesbut not in a number of lines derived from sarcomas orcarcinomas (2). Whereas normal B lymphocytes do notcontain detectable c-fgr transcripts, abundant mRNA isfound in circulating monocytes, granulocytes, tissue macro-phages, and natural killer cells (8, 11, 16). Furthermore,differentiation of myelomonocytic HL60 or U937 cells isaccompanied by a dramatic increase in c-fgr mRNA andprotein levels (16, 19). Thus, c-fgr expression is lineagespecific and developmentally regulated.Although cDNA clones containing the p55cfgr_coding re-

gion have been described, such clones lack all but a few

* Corresponding author.

nucleotides of 5' untranslated sequence (13). In this study,we have isolated normal fgr cDNAs containing sequencesupstream of the codon for initiation of p55c-fgr synthesis. Thenucleotide sequence of the longest fgr cDNA was deter-mined, as was the sequence of corresponding genomic DNA.Comparison of the genomic sequence with that of cDNAsisolated from monocytes or EBV-infected B lymphocytesrevealed a number of 5' untranslatedfgr exons, one of whichwas utilized exclusively in EBV-infected B lymphocytes.

MATERIALS AND METHODS

Cells. Continuous NIH 3T3 (12) and IM-9 (6) cell lineshave been described. Burkitt's lymphoma cell lines BJABand RAMOS as well as their EBV-infected counterpartsBJAB-GC and RAMOS-AW (4, 14) were also used. Mono-nuclear cells were prepared from normal human peripheralblood by Ficoll-Hypaque centrifugation. Human monocyteswere purified from mononuclear cell preparations by elutri-ation.DNA libraries. EMBL3 (Clontech, San Francisco, Calif.)

and K-Fix (Stratagene, La Jolla, Calif.) human genomic DNAlibraries were screened with fgr probes as described else-where (13). Complementary DNA was prepared from puri-fied human monocyte poly(A)+ mRNA templates, and thelibrary was constructed in XpCEV9 as recently described(17).

Nucleotide sequence determination. Restriction enzymefragments derived from cDNA or genomic DNA clones weretransferred into the polylinker regions of M13mpl8 and -19.Sequencing reaction products were analyzed in 6% poly-acrylamide gels. In each case, the nucleotide sequence forboth strands was determined. Sequences were determinedwith the aid of the Intelligenetics GEL program.

1500

DISTINCT c-fgr mRNA INDUCED BY EBV 1501

mRNA preparation and detection. RNA was isolated bylysing cells in a guanidinium thiocyanate-sarcosyl solution,followed by phenol extraction and ethanol precipitation aspreviously described (3). For Northern (RNA) blotting, aDNA probe representing intron 2 of c-fgr was derived fromplasmid pc4101 (13) and contained nucleotides 557 to 921.The c-fgr exon 2 probe contained nucleotides 121 to 382 ofpc41/22 (13), and the exon A probe contained nucleotides1998 to 2244 of c-fgr genomic DNA (see Fig. 4). RNAs wereanalyzed as described previously (13).

Si protection analysis. To develop a probe for c-fgrmRNAs containing exon A, complementary DNA was syn-thesized from total cellular RNA templates isolated fromIM-9 cells by using a c-fgr exon 3 primer (nucleotides 421 to402; 5'-CATAGTCATACAGGGCAATG-3') (10) and avianmyeloblastosis virus reverse transcriptase. RNA was dis-solved in 10 ,ul of distilled H20, heated to 65°C for 5 min, andadded to a reaction mixture containing 30 ,M primer, 1.25mM each deoxynucleoside triphosphate, 1 mM dithiothrei-tol, 10 ,ug of bovine serum albumin per ml, 10 U of RNasin(Promega), and 10 U of reverse transcriptase (BethesdaResearch Laboratories, Gaithersburg, Md.). DNA tran-scripts were used as templates in a polymerase chain reac-tion which contained 25 pmol of the reverse c-fgr primer and25 pmol of a forward exon A c-fgr primer (nucleotides 1 to20); 5'-TGGAGACCAAAGCACTGATG-3) (10). After 35cycles, amplified products were blunted with Klenow DNApolymerase and separated by electrophoresis on a 5% poly-acrylamide gel. The expected band of 421 bp was eluted andsubcloned into pUC9. The identity of the cDNA clone wasconfirmed by restriction enzyme digestion. An end-labeledprobe was prepared by performing restriction endonucleasecleavage at a BstE2 site in exon 3, followed by treatmentwith calf intestinal phosphatase (Pharmacia Fine Chemicals)and labeling with [_y-32P]ATP (>5,000 Ci/mmol; ICN Phar-maceuticals) and polynucleotide kinase (U.S. Biochemical).A genomic c-fgr fragment (SmaI-BamHI), extending frompositions -110 to +118 with respect to the 5'-most c-fgrmRNA containing exon A, was subcloned into pUC9 justupstream of a 2.34-kb HindIII cassette containing the neo-mycin phosphotransferase gene. The resulting DNA clonewas designated fgr-neo. The 1-actin probe was generatedfrom a human ,-actin genomic clone containing portions ofintron 2 and exon 3; the probe was end labeled at an AvaIsite in exon 3. Hybridization conditions and Si nucleaseprotection analyses have been described elsewhere (1, 15).All hybridizations were performed in probe excess.

Immunoblotting. Cells were disrupted in a buffer contain-ing 10 mM sodium phosphate (pH 7.4), 100 mM NaCl, 1%Triton X-100, 0.5% sodium deoxycholate, 0.1% sodiumdodecyl sulfate, 1% Trasylol, 1 mM phenylmethylsulfonylfluoride, and 1 mM sodium orthovanadate. Lysates repre-senting 40 ,ug of protein were fractionated by sodium dode-cyl sulfate-polyacrylamide gel electrophoresis and trans-ferred to nitrocellulose filters as described previously (8).Immunodetection using antipeptide antibodies has been de-tailed previously (8). Briefly, filters were incubated for 2 h atroom temperature with antibody, washed, and treated with1251I-labeled protein A (Amersham Corp., Arlington Heights,Ill.). Protein size was estimated by comparison with '4C-labeled protein molecular mass standards (Bethesda Re-search Laboratories). Bands were visualized by autoradiog-raphy at -80°C.

RESULTS

Isolation of normal human c-fgr cDNA clones. Previousstudies have shown that expression of the human fgr proto-oncogene is limited to mature monocytes, granulocytes,tissue macrophages, and natural killer cells (8, 11, 16). Thus,we chose to prepare mRNA templates from monocytes, themost readily available of these normal sources. Complemen-tary DNAs were cloned into XpCEV9, a vector developedfor high-efficiency, directional cDNA cloning (17). The li-brary consisted of 2 x 106 clones which were first screenedwith an exon 2 probe; 210 clones scored as positive. Since30% of c-fgr mRNAs contain introns (13), 20 clones werefurther tested for hybridization with a probe that representedc-fgr intron 2 (13). Six clones scored as positive with theintron 2 probe and were not analyzed further. The remaining14 clones were propagated in bacteria and subjected torestriction enzyme mapping. The clones were overlappingand ranged in size from 2.1 to 3.1 kbp. A cluster ofrestriction enzyme sites previously known to reside withinhuman fgr cDNA was present in each of the 14 cDNAsexamined (data not shown). Thus, each of these clonesrepresented transcripts from the fgr proto-oncogene. Thelongest cDNA clone, pcFGR 15, was chosen for furtheranalysis.

Nucleotide sequence analysis of pcFGR 15. We determinedthe nucleotide sequence of pcFGR 15 by using the dideoxy-chain termination method. The clone was 3,137 bp in length,and its sequence was identical to that of our previouslydescribed cDNA clones (13) from positions 852 to 3137. Thesequence from 1 to 852 was unique among known nucleotidesequences. The most notable feature of this sequence wasthe presence of two stretches, approximately 300 bp inlength, that were 63 and 69% related to members of the Alufamily of repeats (5) (Fig. 1). This suggestion of repeatedsequences was verified by Southern blotting experiments inwhich DNA probes from various regions of this sequencedetected a large number of bands in genomic DNA isolatedfrom human but not mouse cells (data not shown). The exon2 sequence began at position 915, and the codon previouslydescribed as the initiator of p55c-fgr synthesis was located atpositions 928 to 930. Translational stop codons in all threereading frames at positions 890 to 892, 901 to 903, and 909 to911 provide further evidence that protein synthesis begins aspreviously described within exon 2 (13).

Isolation of genomic sequences upstream of c-fgr exon 2. Todetermine the organization of thefgr locus upstream of exon2, genomic DNA libraries were screened by using an exon 2probe. An EMBL3 clone, designated X-fgrl, was identifiedby virtue of its hybridization to exon 2 as well as to cDNAsequences upstream of exon 2. However, X-fgrl did nothybridize with a probe, designated p55UT, which containedthe first 336 bp of the pcFGR 15 insert. Thus, an additionalgenomic DNA clone, designated X-fgr2, was isolated from aX-Fix genomic DNA library, using the 5'-most 2.3-kbpSalI-EcoRI fragment from X-fgrl as a probe. The pSSUTprobe detected A-fgr2 by hydridization. Our genomic clonesoverlapped and contained 16 kbp of DNA lying upstream offgr exon 2 (Fig. 2).To localize genomic DNA fragments containing previ-

ously unidentified fgr exons, we synthesized a 20-baseoligonucleotide representing the sequence of pcFGR 15immediately upstream of exon 2. This probe hybridized witha 2.9-kbp EcoRI-PstI fragment from X-fgrl (Fig. 2). Thus,the 2.9-kbp fragment was isolated and its nucleotide se-quence was determined (Fig. 1; positions 901 to 3801). By

VOL . 1 l, 1991

1502 GUTKIND ET AL.

GCCACCACGC CCGGCTAATT TTTGATTTTT AGTAGAGATG GGCTTTCACT GTGTTAGCCAGGACGGTCTT GATCTCCTAA CATCGTGATC CA CL¢ £::::GCC.CT.C. ::::A0T. TC.

rz~~~~~~~~C C. .GOC

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ACTGCGCTTT........... ......................TTTGGA CCGCGG GA TCAG TGAGT

MOL. CELL. BIOL.

EXON Ml(1-728)

--------------------------9 kbp----------------------------------

GGCCAACATA GTGAAAACCCGGGTGCCTGT AATCCCAGCTGGCGGAGGTT GCAGTGAGCTAAACTCCCTC TCAAAATAAAGTGGATAAAA TGAGATAATAGCGCTATAAA TTTTGCTATTGTTCATGTGC ATGCACATCTGTGTGTGTAC ACTAGAAGATGCATGAGGCA GCTGAGGTAATGGGTATCAG CATGGTGCGCv^vvorrsoroo<rovvvg

ATCGCTACTA AAAATACAGA AATTAGCCTGACTCAGGAGG CTGAGGCAAG AGAATGGCTTGAGATCATGC CATTGCACTC CAGCCTGGGTTAAATTAATA ATAATAATAC TACTTAAATCTAGGTAAAGT GCTTAATGCA GTGGCCAGCGCTTTTATAGA GGATGTGTGT GTGTGTGTGTATGTGCAGAC GGATGAGGGC TGGAGGTGTGGTTTGTGCTA GGAATCTGTG CAGAGGAGCCTGTGCCAACT GAGAAAGGGG TCTGAAGGGCCATTTGAGGT GTGTATGTGT GTCTTCCTGTrvqvrmrqmro mqmrhahhman m4--14EM29P imUAUAAUU(;i7l 1^AL7V17UAkAv_ Ui7XUU vss7 sss ssT^wYM":aCeeA¢g:gTT& -AS Mtet £ckEQt att.XB.g$TGGGTG

CCATCAGGAC TGGCTAATTA AAAAAAGATT TTTTTTTATA GAGACGGGGT GTCCCTATGTTGCTCAGGCT GGTCTCAAGC TCTGGGCTGA AGCAATCATC TGCTCGGCTA CAAAGTCTGGATAGACGTGT GAACCACTGT GCCGCCTCAC CCTTGTTTTT GTATCAGCCC CATCTCTCTTTTCACCAGTT CCTGAAATCC CTCCGCTGGG CCCTGGATGG CTTCCAGTCC TCCACCTCTATTTTCTGCCC TGCTCTAACT AGCCCTGTAG CATCCTGGGG CGTTTTAGAC ACAGTGGTTTCATCCCCAGG GAGGGGGTCC CGGGGCAAAG GTCTCAGCAG GGCCCAGTGA ACAGGGGCTATTTTAGGGCA GGCTTCTCAC CACAGCCCGC CCCACAGTTC ACCACATGGG TGTGATGCCCCCACCCCCAC CCAATACACA CATGAGAGAT CACTTAGAGC AAAGGGTGAG AGGGGCAGGTGGGGCTAGGG 41.a.Xa.te.X.eeKB...TG-.$ .C-£.^M:':A-MmZ:^CGA||atGG=CD:.GAAG¢££.g¢GGASAGGTGGTGTGCTAGGTAGAA AGGATAGGAC

CCAGAGAGAA GAGGAAGAGA ATATCTGTAA GGATGACTGG ACTGGGGATC GAGAGAGAGAAGCTGGGGGC CCTTTCTTCT AGGACCTTGG GGCCCTCTGG AATCAGGGTT CACAAGGTTGGCCCCACCCT AAACTCTCCA TTCTCACATC TTAGGAAACC AAGCCCTCTC ACCAGTCGGTTCCTCTCTGA GTGTTGCAAT GTTTCTGGCA GGGTGTGGGG GACCTTGCTC AATGACCTCCTGCCCTGTTG CTCAGAGGAT ACCGCTGCCC AGAAAAGGGT TGGCTCATTG TGGGGCTTCCCAAGGTATCT CTGGTAGCCC CCAGCTTCTG ACCTGGTCCT TTCTCTGGTA TGGGGATAGGAGGAGAGCTC CGGAGTAGGT ATCCACTCTC ACTCAGCCAC CACATGGAAC CCTAGGGTGGCTGGGAGCAC AGCAGGGTTC AGAGGAAGGA CTGTTTTTTG TTTGTTTGTT TGTTTGTTTTTGAGATGGAG TCTTGCTCTG TCACCCGGGC TGGAGTGCAG TGGTGCGATC TCGGCTCACTGCAAGCTCCA CCTCCCAGGT TCAAGTGATT CTGCTGCCTC GGCCTCCCAA GTAGCTGGGACTACAGGCGC CCACCTCCAC GTCTGGCTAA TTTTTGTATT TTTAGTAGAG ACGGGGTTTCACCATATTGG CCAGGCTGGT CTCGAACTCC TGACCTTGTG ATCCACTCAC CCTCGGTCTTCCCAAAGTGC TGGGATTATA GGCGTGAGCC ACTGCGCCTG GCCGGAAGAA CTGGTTTTTAGGAGATGGTG ACTGGGGACT GTGAGGGAGC TGAGCATGGC TTGATAGAAA TCCTGTTAGAGAGATGATTA TAATGTTCAA AATCATGTGT GTCTGAGTGT GTTCGTCTGT TAACCTGGCAGGCACCCCAT GTATATGTGC ATGTGTATGT GTGTGTGCTA TTGTGAGCTT GGGCTTGTTAGAGCCTGTAT TGGCGTGTGA TGGGGTTGGC ACGCACACTC ATGCAAATAT ATGCTGTGAGTGTTATTGTG TGACTGTGCT GGTGGGTCAG GTGAGTATGG AGTGTGAAAG AGAGCTGGGTGTGGGTGGTT TGCCCTATGT GACGGGGGTT GTGTAAGTGT GCCAGGGGTG ATAGGAAGGAAAAGTGAAGG CAGAAGTCAT GCTGGGCAGA GCCCAGGCCT TCTGGCTTCC TGAAGAGGGCAGGAGCTGGG CAGGGGGCTG CTGACAGAAA CATTGGCAGA GACTTCATCT TCCTTGTCCTTCTGTCTCAC CCTCATTtt:e....2t.e.^SAU.... A.¢B*¢ek£$:t:T

GGTGGGGGTG GCCTTCCTGA ACCCCACAAC TCCTCACAGC CTCCTCCTCC TACAAGGACCCTGTTGCTAG GTAACGGATG GGGGAGCCAG AATGAGGCAG CTTGAGAGGC TGAAGGCTGG

EXON M2(729-851)

EXON A

EXON 1(852-914)

FIG. 1. Nucleotide sequences of pcFGR 15 cDNA and corresponding genomic DNA. DNA fragments derived from X-fgrl (2.9-kbpEcoRI-PstI) or X-fgr2 (1.2-kbp BamHI-BamHI and 1.4-kbp BamHI-BamHI) were subcloned into M13 vectors and sequenced. Alu-like repeatsare bracketed. The 9-kbp stretch between exons M1 and M2 was not sequenced. Exons are shown as shaded boxes.

comparing genomic with cDNA sequences, two additionalexons, designated 1 and M2, were identified within thegenomic DNA fragment. Exons 1 and M2 were 63 and 123 bpin length, respectively, and their sequences completelymatched analogous stretches within pcFGR 15 cDNA. Con-sensus splicing signals bordered these exons. We haveconcluded previously that the sequence of pc4l cDNAimmediately upstream of exon 2 represented fgr intron 1(13). Identification of these sequences in genomic DNA (datanot shown) verifies our earlier interpretation. The arrange-ment of these exons within the fgr locus is shown in Fig. 2.The region of A-fgr2 containing the most upstream stretch

of pcFGR 15 cDNA was also subjected to nucleotide se-

quence analysis. Its sequence revealed a fgr exon compris-ing the 5' end of pcFGR 15 cDNA and stretching 728 bpdownstream. This exon, designated M1, accounted for the

remaining sequences present in pcFGR 15 cDNA. We con-

clude that the fgr locus contains three 5' untranslated exons

which account for 914 bases of thefgr transcript. We suspectthat the transcriptional initiation site for this fgr mRNAspecies lies very close to the 5' end of pcFGR 15 cDNA.

c-fgr mRNA molecules containing exons Ml and M2 are

rarely detected in myelomonocytic cells. To determine the

relative abundance offgr mRNA molecules containing exons

161

121181241301361421481541601661721781841

GAATTCGCCTGCATGGTGTTGAACCCAGGAGACAAGAGTGATAGGGTTGTCATATAGTAAGTGTGTGTGTTGTATATGGGTGGAGCCTGTTGTGTGGATCrwrAsAflflfrrwr

901961102110811141120112611321138114411501156116211681174118011861192119812041212121812241230123612421248125412601266127212781284 12.90129613021308131413201326133213381344135013561362136813 74 1

DISTINCT c-fgr mRNA INDUCED BY EBV 1503

2 kbp-I

1 728

Ml

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l-fgr 1,

729 851 852 914 915 928

M2 A 1 2

B B E

B B EJ,J, 4

B E E B4 44 4

B E E B444 4

FIG. 2. Organization of c-fgr upstream exons. Sites of restriction enzyme cleavage within A phage clones are shown. Nucleotide positionscorresponding to those of pcFGR 15 cDNA are shown for each exon. The 3' end of the 2.9-kbp EcoRI-PstI fragment sequenced in this studyis shown on the X-fgrl map terminating with the only PstI site shown. Abbreviations: B, BamHI; E, EcoRl; P, PstI.

M1 and M2, we prepared a cDNA probe end labeled at theBstE2 site within exon 3 and extending to exon M1 (Fig. 3C).RNA derived from normal human mononuclear cells (15%monocytes, 15% neutrophils, and 70% lymphocytes) or

normal human neutrophils (>95% pure) protected a predom-inant probe fragment of 315 nucleotides (nt) from Si diges-tion, indicating that exons M1 and M2 are rarely utilized inthese cells. A second band at 321 nt suggested the presenceof another unique 5' untranslated exon, lying upstream ofexon 1 and sharing a region of identity at its 3' end with exonM2. Neutrophil RNA also protected a trace amount of a437-nt probe fragment, indicating that rare fgr mRNA mol-ecules contain exon M2 but not M1. RNA derived from IM-9cells protected a probe fragment of 315 nt from Si digestion,indicating that exons M1 and M2 are also rarely a part of c-fgrmRNA molecules in these cells. As expected, RNA fromK562 cells failed to protect the cDNA probe from nucleasedigestion. Since mostfgr mRNA molecules from neutrophilsor monocytes contain neither exon M1 nor M2, and sinceexon 1 is bordered by a splice acceptor sequence, additionaluncharacterized 5' untranslated exons must be predomi-nantly utilized in myelomonocytic cells and in EBV-infectedB lymphocytes. The rarity of mRNAs containing exons M1and M2 precluded our ability to map the 5' ends of this c-fgrmRNA species.

Identification of an additional fgr exon utilized in EBV-infected B cells. Human fgr cDNA molecules have also beenisolated from mRNA of two EBV-infected Burkitt's lym-phoma cell lines, IM-9 and BL2-B95/1 (10, 20). The IM-9cDNA contained exon 1 identified in this study as well asnovel sequences upstream. This latter sequence, designatedexon A, was also identified within our genomic DNA clones,lying between monocyte exon M2 and exon 1 (Fig. 1). Adonor splicing signal followed the exon A sequence, but noconsensus acceptor splice signal was detected upstream(Fig. 1). These findings suggested that fgr exon utilizationmay be affected by EBV transactivation. To determinewhether exon A was utilized when fgr expression was

normally programmed, we prepared a probe capable ofdetecting mRNAs containing correctly spliced exons A, 1,and 2 (Fig. 3D). A predominant probe fragment, 396 nt inlength, was protected from nuclease digestion by RNA fromIM-9 cells (Fig. 3B). In contrast, neutrophil or mononuclearcell RNAs protected a probe fragment of 315 nt. A faint bandof 396 nt was detected when the probe was annealed withRNA from mononuclear cells, perhaps reflecting natural

EBV infection of a small number of B lymphocytes presentin the mononuclear cell sample or a small number of naturalkiller cells. In any case, we conclude that fgr mRNAexpressed in IM-9 cells contains exon A sequences immedi-ately upstream of fgr exon 1, whereas neutrophils andmononuclear cells express a fgr mRNA species having a

different structure.To determine whether exon A was present in fgr tran-

scripts isolated from other sources, RNA was prepared frommonocytes, other B-lymphocyte cell lines, some of whichwere infected with EBV, and HL60 cells induced to expressc-fgr mRNA by treatment with retinoic acid (19). Whenexamined by Northern blotting, the exon A probe detectedtranscripts of 2.8 kb in EBV-converted human B cells (Fig.4) but not in RNA samples prepared from monocytes or

induced HL60 cells. An unidentified band of around 4 kb wasdetected in control murine fibroblasts. The 1-actin probereadily detected a 1.8-kb mRNA in human cells but onlyweakly hybridized with murine actin mRNA. Our exon 2probe detected transcripts of 2.8 kb in monocytes andmononuclear cells as well as EBV-infected B cell lines, butno such transcripts were detectable in RNA from controlfibroblasts or EBV-negative B cells (Fig. 4). These findingsdemonstrate that exon A is utilized in EBV-converted Bcells but not in myelomonocytic cells. Furthermore, the sizeof the predominant fgr mRNA species detected (2.8 kb) was

consistent with our findings that fgr mRNA molecules of 3.1kb are rare.

Expression of p55C-fgr in EBV-converted B cells. Our findingthat normal c-fgr transcripts differed in the 5' untranslatedregions from those induced by EBV raised the possibilitythat exon A sequences directly or indirectly affect expres-sion of p55c-fgr in B lymphocytes. To address this question,cell lysates were examined by immunoblotting using p55c-fgrantibodies. p55c-fgr was readily detected in control mononu-clear cells, induced HL60 cells, and each of the EBV-converted B-cell lines examined (Fig. 5). As expected, no

p55c-fgr was detected in RAMOS or BJAB cell lines (Fig. 5).From these findings, we conclude that c-fgr transcriptscontaining exon A direct the synthesis of p55c-fgr.The genomic region just upstream fromfgr exon A possesses

properties of a transcriptional promoter. The 5' ends of twodistinct cDNA clones isolated from IM-9 cells differ by onlya few nucleotides in length (10, 20). Genomic sequences justupstream from exon A reveal no consensus splice acceptorsites. Using S1 protection assays, Patel et al. (20) have

-< A-fgr 2

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VOL. 11, 1991

1504 GUTKIND ET AL.

A

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FIG. 4. Detection of exon A sequences in EBV-converted Blymphocytes. Total cellular RNA was extracted from NIH 3T3fibroblasts (lane a), purified human monocytes (lane b), humanmononuclear cells (lane c), B-lymphocyte cell lines IM-9 (lane d),BJAB (lane e), BJAB-GC (lane f), RAMOS (lane g), andRAMOS-AW (lane h), retinoic acid-treated HL60 cells (lane i), andpurified human granulocytes (lane j). Aliquots (10 ,ug) of each RNAwere fractionated by electrophoresis, blotted onto nitrocellulose,and hybridized with labeled DNA fragments representing c-fgr exon2, exon A, or f-actin. Bands were visualized by autoradiography.Molecular size standards are shown on the right.

aw***S --3- ,X

540

FIG. 3. Abundance of various c-fgr mRNA species in hemato-poietic cells. (A) Total RNA (20 p.g) from each indicated source washybridized with approximately 100,000 cpm (-5 ng) of a c-fgr cDNAprobe end labeled at a unique BstE2 site in exon 3 as shown (C).Exon 2 is 246 bp in length, and the BstE2 site is 6 bp downstreamfrom the exon 2-exon 3 border. Samples were treated with S1nuclease and electrophoresed as previously described (1). The sizes(in nucleotides) of protected probe fragments corresponding to c-fgrmRNAs containing exons 1 and 2 or containing additional exons areshown. All RNA samples were cohybridized with an end-labeled13-actin probe. Correctly spliced P-actin mRNA protects a fragmentof 177 nt. The sizes (in nucleotides) of HaeIII-digested 4OX174 DNAfragments are shown on the left and right. (B) Total RNA (20 ,ug)from each indicated source was hybridized with a c-fgr cDNA probecontaining exon A linked with exon 1 and exon 2 (D). Probefragmefits protected from S1 nuclease digestion by c-fgr mRNAwere visualized by autoradiography after electrophoresis on an 8%sequencing gel.

shown that c-fgr mRNA from IM-9 cells is initiated justupstream from exon A. To confirm these results, we endlabeled a genomic c-fgr probe at the BamHI site in exon Aand hybridized this probe with RNAs derived from IM-9cells or 12-O-tetradecanoylphorbol-13-acetate (TPA)-in-duced U937 myelomonocytic cells (16). c-fgr mRNA fromIM-9 cells protected probe fragments ranging in size from 72to 118 nt (Fig. 6), indicating that fgr mRNA transcriptionalinitiation sites are distributed over a stretch of around 50 nt.

The pattern of c-fgr bands is identical to that described byPatel et al. (20). In contrast, c-fgr mRNA from induced U937cells did not protect the same probe fragments, even thoughthe total abundance of c-fgr mRNA is approximately thesame in IM-9 and TPA-induced U937 cells. Although weattempted to confirm the transcriptional initiation sites offgrmRNA expressed in IM-9 cells by primer extension, wewere unable to obtain sufficient signal to interpret the

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.

68

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FIG. 5. Expression of p55c-fgr in EBV-converted B lymphocytes.Protein extracts were prepared from mononuclear (lane a), IM-9(lane b), BJAB (lane c), BJAB-GC (lane d), RAMOS (lane e),RAMOS-AW (lane f), or retinoic acid-induced HL60 (lane g) cells.Aliquots (50 jig) of each were fractionated by electrophoresis andimmunoblotted with anti-fgr C serum in the presence (B) or absence(A) of homologous peptide. Electrophoretic mobilities of molecularweight standards are shown.

MOL. CELL. BIOL.

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DISTINCT c-fgr mRNA INDUCED BY EBV 1505

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FIG. 6. S1 mapping of the 5' ends of c-fgr mRNA speciescontaining exon A. Total cellular RNAs (20 ,ug) isolated from IM-9cells and TPA-treated U937 cells (U937+), as well as a samplecontaining no RNA, were hybridized with approximately 100,000cpm of a genomic c-fgr probe end labeled at a unique BamHI site inexon A (B). Probe fragments protected from S1 nuclease digestionby c-fgr mRNA were visualized by autoradiography after electro-phoresis on an 8% sequencing gel. The sizes (in nucleotides) ofHaeIII-digested 4X174 DNA fragments are shown at the left.

results. We attribute this fact to the large number of startsites diluting the primer extension signal. Thus, to date notranscriptional initiation sites for fgr mRNAs containingexon A have been described by primer extension.To determine whether the region upstream from exon A

possessed properties of a transcriptional promoter, welinked a genomic c-fgr fragment extending from -110 to theBamHI site in exon A (Fig. 7B) to a neomycin phosphotrans-ferase reporter gene. The construct was transfected intoU937, K562, or BJAB cells, which were then selected for theability to grow in medium containing G418. Total cellularRNA derived from pools of G418-resistant cells was ana-lyzed with an end-labeled fgr-neo probe. In U937 cells, theregion upstream of exon A directed the synthesis offgr-neotranscripts initiating exactly at the same sites detected forfgrmRNA from IM-9 cells (Fig. 7A). Similar results wereobtained when K562 or BJAB cells were used as recipientsfor the fgr-neo plasmid. Thus, it would appear that negative

194-9

174- **

MMM 2 3 4

B-iO+Io 8 BglII

* fgr-neo probe-412-458 fqr-neo

325 RSV-neo

FIG. 7. Promoter function of the c-fgr genomic sequence imme-diately upstream of exon A. (A) U937 cells were transfected withplasmid fgr-neo or RSV-neo and subjected to selection with G418.Total cellular RNA (20 ,ug) was extracted from each indicatedsource and hybridized with an end-labeled fgr-neo probe. Hybridmolecules were examined by S1 nuclease protection. Bands repre-senting correctly initiated fgr-neo transcripts (probe fragments of412 to 458 nt in length) as well as RSV-neo transcripts (a probefragment of 325 nt) are shown (B).

regulatory sequences affected by EBV transactivation do notlie within the stretch of the c-fgr locus extending from -110to the BamHI site in exon A. Collectively, these resultsdemonstrate that the region upstream from exon A is indeedcapable of behaving as a promoter and support the idea thatthis promoter is elevated to functional status in EBV-infected B lymphocytes.

B5'/t" Exon A

118 721 1 *

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1506 GUTKIND ET AL.

DISCUSSION

In this study, we have isolated a 3,137-bp human fgrcDNA molecule by using normal monocyte mRNA as a

template. Nucleotide sequence analysis of this fgr cDNArevealed a 5' untranslated region of 927 bp which includedtwo Alu-like repeats as well as three translation stop codonsimmediately upstream of the initiator for p55c-fgr synthesis.Sequences from positions 852 to 3137 were identical to thoseof previously described cDNAs (10, 13, 20). The first 914 ntwere arranged in human genomic DNA as three 5' untrans-lated exons, distributed over 13 kbp of genomic DNA.Shorter c-fgr cDNAs (16a) also isolated from our monocytelibrary have revealed a number of additional 5' untranslatedfgr exons. The locations and arrangement of these exons

suggest that both alternative splicing and differential pro-

moter utilization account for the diversity observed in the 5'untranslated region of fgr mRNA. Moreover, the mostabundantfgr mRNA found in monocytes is 2.8 kb in length.This finding is consistent with our observation that the3.1-kb species described in this report represents a rare fgrmRNA.

Previous studies have shown that EBV transactivatesexpression of c-fgr mRNA in B lymphocytes (2, 15). Bycomparing transcripts isolated from EBV-infected B cellswith our monocyte cDNA, we identified a novel untranslatedexon utilized only in EBV-infected cells. Furthermore, fgrtranscripts presumably arising from EBV transactivation inIM-9 cells were initiated just upstream from exon A. Similarresults have been obtained by others (20). Thus, it appears

that a cryptic promoter located immediately upstream ofexon A is elevated to functional status in EBV-infected cells.Experiments showing that the region immediately upstreamof exon A can function as a promoter in heterologous cellslend further support to this idea. Thus, this study demon-strates that activation of the fgr gene by EBV in B lympho-cytes is achieved by mechanisms distinct from those thatnormally regulate its programmed expression in myelomono-cytic cells.As demonstrated in this study, exon A-containing tran-

scripts are effectively translated in EBV-converted B lym-phocytes. However, the biologic effects of p55c-fgr expres-sion in B cells remains an open question. Previous studieshave shown that both immortalizing and nonimmortalizingstrains of EBV induce expression of c-fgr mRNA (2, 15).EBV is thought to play a role in the etiology of Burkitt'slymphomas even though this neoplasm also arises in theabsence of EBV, especially in the Americas. Thus, there isno direct evidence that c-fgr expression is involved in B-cellimmortalization or in the etiology of Burkitt's lymphomas.However, c-fgr is normally expressed in myelomonocyticcells but not B lymphocytes whether resting or activated (8,16, 19; unpublished observations). Thus, p55c-fgr is not anormal constituent of B lymphocytes and may serve toelevate basal protein phosphorylation on tyrosine in EBV-infected cells, thereby contributing to the process of malig-nant transformation.The Ick gene represents the only member of the human src

gene family with a well-characterized 5' end. Ick is expressedas two distinct mRNA species which differ only in their 5'untranslated regions (27). Type I Ick transcripts are foundpredominantly in immature thymocytes (22) and nonlym-phoid neoplasms (23), but type II Ick transcripts are foundpredominantly in normal human peripheral T lymphocytesand T-cell lymphomas (23, 25). DNA flanking the 5' end ofthe Ick gene contains two promoter regions that are greater

than 9 kbp apart. Each transcript arises from a distinctpromoter. The nucleotide sequences of these promotersreveals the absence of known eukaryotic cis-acting elementssuch as TATA, CCAAT, or GC boxes. The Ick transcriptsinitiate over a span of 10 to 20 nt, typical of TATA-lesspromoters (25). In the c-fgr locus, the sequence immediatelyupstream of exon A does not contain consensus TATA orCCAAT boxes, but several AP-2 sites and a single SP-1consensus binding site were observed (20). Sequence com-parison of this region with the Ick promoters revealed nosignificant homology. Work is in progress to define theregulatory elements of the c-fgr exon A promoter and toidentify the promoters utilized for the normal expression offgr proto-oncogene mRNA.

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20. Patel, M., S. J. Leevers, and P. M. Brickell. 1990. Structure ofthe complete human c-fgr proto-oncogene and identification ofmultiple transcriptional start sites. Oncogene 5:201-206.

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25. Takadera, T., S. Leung, A. Gernone, Y. Koga, Y. Takihara,N. G. Miyamoto, and T. W. Mak. 1989. Structure of the twopromoters of the human Ick gene: differential accumulation oftwo classes of Ick transcripts in T cells. Mol. Cell. Biol.9:2173-2180.

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