Proc. Nail. Acad. Sci. USAVol. 85, pp. 1492-1496, March 1988Biochemistry

Platelet-derived growth factor A chain: Gene structure,chromosomal location, and basis for alternative mRNA splicingDAVID T. BONTHRON*t, CYNTHIA C. MORTONO, STUART H. ORKIN*, AND TUCKER COLLINS*§*Division of Hematology-Oncology, Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, and*Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115

Communicated by Earl P. Benditt, November 23, 1987 (received for review July 9, 1987)

ABSTRACT Genomic clones encoding the A chain ofplatelet-derived growth factor (PDGF) have been isolated. Thegene contains seven exons spanning about 24 kilobases ofDNA. The positions of intervening sequences closely matchthose of the related B-chain (c-sis) gene on chromosome 22. Insitu hybridization was used to localize the PDGF A-chain geneto the distal portion of the short arm of chromosome 7(7p2l-p22). Within the (G+C)-rich 5' region, a single tran-scriptional start site was identified =%36 base pairs downstreamof a TATAA consensus promoter element. The three sizeclasses of A-chain mRNA probably arise by selection ofalternative poly(A) sites in exon 7, but only a single consensusAATAAA signal was identified in this region. Two functionallydifferent A-chain precursors, which differ by the presence orabsence of a basic C terminus, are generated as a result ofalternative mRNA splicing events, which include or exdudeexon 6. This and other structural features of the A-chain genesuggest that PDGF expression may be modulated at transcrip-tional and post-transcriptional levels.

Platelet-derived growth factor (PDGF) is a cationic glyco-protein of Mr 30,000 (for recent review, see ref. 1). As apotent mitogen for a variety of connective tissue cells, itinduces competence to enter S phase, which is accompaniedby transient rises in the levels of mRNA for the protoonco-genes c-fos and c-myc (2, 3). PDGF acts by way of a specificcell-surface receptor, a member of the transmembrane tyro-sine kinase family (4). As purified from platelets, PDGF is adisulfide-bonded dimer of two polypeptides, designated Aand B, which are the products of related but separate genes(5). cDNA encoding the A chain of PDGF was first isolatedfrom a malignant glioma cell line; the A-chain gene wasassigned to chromosome 7 (7pter-7q22) (5). The B-chainprecursor is encoded by the c-sis gene, located in chromo-somal region 22ql2.3-ql3.1 (6). The product of the simiansarcoma virus oncogene (v-sis) is a B-chain-related mitogenthat accounts for the viral transforming activity (7-9).

Although a variety of tumor-derived cells also secretePDGF-like mitogens, some express only A- or B-chainmRNA (5). This implies that A- and B-chain precursors canassociate to form functional homodimers. Cultured humanendothelial cells express A- and B-chain mRNAs (10, 11).The coding region of B-chain mRNA from endothelial cells isidentical to that of tumor-derived cDNA (10). In contrast, weand others have shown (12, 13) that A-chain cDNA clonesderived from nontransformed cultured human endothelialcells lack an internal 69-base-pair (bp) region present in thereported glioma cDNA sequence. This region encodes anextremely basic C-terminal portion of the A-chain precursor,which appears to be required for efficient assembly of amitogenic A-A homodimer.

To define the nature of A-chain mRNA processing and tocompare the organization of A- and B-chain genes, we haveisolated and characterized the normal human PDGF A-chaingene.¶ The internal 69-bp region of the glioma-derivedcDNA is a single exon of the normal A-chain gene, confirm-ing the suggestion (5, 12) that the two forms of mRNA ariseby alternative splicing. Consistent with the homologousprimary structures of A and B chains, the exon-intronstructures of the two PDGF genes are similar. We havefurther localized the A-chain gene to the distal end of theshort arm of chromosome 7 by in situ hybridization.

MATERIALS AND METHODSCell Culture. Growth of human passaged umbilical vein

endothelial cells and RNA preparation have been described(11, 12). HepG2 cells were grown in Dulbecco's modifiedEagle's medium containing 10%o fetal bovine serum (GIBCO).

Isolation of Genomic Clones. A library of human peripheralblood DNA partially digested with Sau3A was constructedin the vector AEMBL3 (14). It was screened by plaquehybridization with 5' end [EcoRI-Mst II of clone D-1 (5), 290bp] and 3' end [Bal I-EcoRI of clone dT1.1 (12), 724 bp]cDNA probes.DNA Sequencing. Sequencing was performed mostly by

the Sanger method, using M13 and plasmid subclones ofisolated restriction fragments (15, 16). The nucleotide ana-logue 7-deazaguanosine trisphosphate (17) was helpful insome (G +C)-rich 5' regions, but others required use ofchemical degradation techniques (18).

Primer Extension. Oligonucleotides DB1 (5' CTGCGAGCT-GCGAGCCGCCCTG 3') and DB2 (5' CACACCGATCA-CCTGCCTGAAC 3') were labeled with [32PJATP and poly-nucleotide kinase to a specific activity 1-2 x 109 cpm/pug; 5x 105 cpm were hybridized to each RNA sample at 580C for1 hr in 12 Al1 of 100 mM KCI/10 mM MgCl2/25 mM Tris'HCI,pH 8.5. The reverse transcription reactions (40 ,ul) contained30 mM KCl, 8 mM MgCI2, 50 mM Tris HCl (pH 8.5), 500 .uM(each) dNTP, 50 ,ug of actinomycin D per ml, 4 units ofRNasin (Promega Biotec, Madison, WI), and 50 units of avianmyeloblastosis virus reverse transcriptase (Molecular GeneticResources, Tampa, FL) at 42°C for 30 min.

Filter Hybridization. Restriction fragments were labeledby random priming (19). A 69-bp probe specific for exon 6was made from overlapping complementary oligonucleotidesPDGF-1 (5' CTGGTTGGCTGCTTTAGGTGGGTTTTAAC-

Abbreviations: PDGF, platelet-derived growth factor; IVS, inter-vening sequence.tPresent address: Department of Paediatrics, Royal PostgraduateMedical School, Hammersmith Hospital, Du Cane Road, LondonW12 OHS, United Kingdom.§To whom reprint requests should be addressed.IThis sequence is being deposited in the EMBL/GenBank data base(Bolt, Beranek, and Newman Laboratories, Cambridge, MA, andEur. Mol. Biol. Lab., Heidelberg) (accession no. J03638).

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Proc. Nati. Acad. Sci. USA 85 (1988) 1493

CTTT 3') and PDGF-2 (5' GAAGGCCTAGGGAGTCAGG-TAAAAAACGGAAAAGAAAAAGGTTAAAACCC 3') asdescribed (20).Chromosomal in Situ Hybridization. The 1.75-kilobase (kb)

endothelial cell-derived cDNA clone dTl.1 (12) was labeledto a specific activity of 1.2 x 107 cpm/,ug by nick-translationwith all four tritiated nucleotides (New England Nuclear)(21). In situ hybridization to metaphase chromosome prep-arations of normal male peripheral blood lymphocytes wasperformed as described (21).

RESULTSStructure of the PDGF A-Chain Gene. The restriction map

of a 47-kb region containing the A-chain gene (Fig. 1) wasconstructed by analysis of overlapping bacteriophage clonesisolated from a human genomic library. The gene spans -24kb. Exons and their corresponding intron boundaries werelocated and sequenced (Fig. 2). Like the related B-chaingene (22), the A-chain gene contains seven exons, whichcorrelate with functional subdivisions of the growth factor.The unusually long 5' untranslated region and signal peptideconstitute exon 1. Exons 2 and 3 encode the N-terminalpropeptide; exons 4 and 5 encode the mature PDGF subunit.The long exon 7 consists almost entirely of 3' untranslatedsequences.The 69-bp region present in a glioma-derived cDNA clone

but missing in normal endothelial cell-derived cDNAs cor-responds to exon 6. This demonstrates that the two observedforms of A-chain cDNA are indeed the consequence ofalternative processing of transcripts of the normal A-chaingene. Hybridization of genomic Southern blots with a syn-thetic 69-bp probe corresponding to exon 6 established thatno gross rearrangement had occurred in the vicinity of thisexon (not shown).

All splice junction sequences conform to the GT/AG rule(23). Unusual features were seen near the splice donor sitesof intervening sequences 3 and 4 (IVS3 and IVS4). In theformer case, there is a perfect 41-bp repeat (underlined inFig. 2). IVS4 begins with an unbroken stretch of 100 pyrim-idine residues that includes multiple repeats of the sequenceCCCT.Assignment of the Transcriptional Start Site. To define the

transcription initiation site(s) of the A-chain gene, primerextension analysis was performed by using RNA from pas-saged human endothelial cells and from the hepatoblastomaline HepG2 (Fig. 3). The latter has been reported to secretea PDGF-like mitogen (24), and we have confirmed by RNAtransfer blotting (not shown) that it expresses a low level ofA- and B-chain mRNAs. Single bands, of 352 and 389nucleotides, were generated by using each of two oligonu-

cleotide primers, designated DB1 and DB2, respectively,spaced 39 bp apart on the mRNA. This placed the start site36 bp (+ 5 bp) downstream of a consensus TATAA (Gold-berg-Hogness box) sequence (Fig. 3). This result is consis-tent with the TATAA motif being a functional element of theA-chain promoter. No other TATAA sequences were iden-tified in 1 kb of upstream sequence (data not shown). Sinceno other extension products were seen in the 370 bp down-stream of this site, multiple transcriptional start sites down-stream from the consensus TATAA are unlikely to accountfor the heterogeneous size of A-chain mRNAs (approxi-mately 2.6, 2.3, and 1.6 kb).Exon 7: Poly(A) Sites. In view of the unique transcriptional

start site, mRNA size heterogeneity is likely to reflect use ofalternative poly(A) sites. Exon 7 extends at least as far as thearrowheads at position 3157 in Fig. 2, which represents theend of the cDNA clone dTM.1 (12) and corresponds to amRNA size of at least 2.0 kb. The shortest form of mRNApresumably results from cleavage and polyadenylylationfarther upstream, though the closest match to a consensusAATAAA signal upstream of this point is a number of copiesof AACAAA, which is not thought to be an efficient substi-tute (25). The short (750 bp) form of mouse dihydrofolatereductase mRNA is also derived from an alternative noncon-sensus poly(A) signal within the 3' untranslated region of thegene (26). Farther beyond the end of the endothelial cDNAclone, the only close matches to AATAAA in the genomicsequence are CATAAA at position 3201 and a perfect match,AATAAA, at position 3372. The latter predicts a mRNA of2.25 kb plus poly(A) tail, which may correspond to the largeor intermediate mRNA species seen on RNA transfer blots.An (A+ U)-rich sequence, including repeats of AUUUA,

is characteristic of the 3' untranslated regions of somerapidly degraded mRNAs for inflammatory mediators andoncogenes (27). Such a sequence is not present in theexisting A-chain exon 7 sequence (Fig. 2) but is present inthe 3' untranslated region of the B-chain mRNA (22). This isconsistent with the short half-life of the B-chain mRNA(35-40 min) compared to that of the A chain (>3 hr) incultured rat pup smooth muscle cells (28).Chromosomal Localization of the PDGF A-Chain Gene.

The genes for A and B chains of PDGF have been localizedto chromosomes 7 (5) and 22 (6), respectively. To map theA-chain gene more precisely, we performed in situ hybrid-ization to metaphase chromosomes. The results of analysisof 150 metaphase spreads are shown in Fig. 4. Of the 383silver grains recorded, 29 (7.6%) were located on or besidebands 7p21-p22 (most likely within 7p22). We note that 7p22is the site of one of a defined subset of mutagen-inducedfragile sites, many of which coincide with the locations ofknown oncogenes (29), and is also the site of one breakpoint

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FIG. 1. Map of the cloned region of the PDGF A-chain gene. The scale at the top is in kilobases. The restriction map was derived fromfour overlapping bacteriophage clones, as shown beneath. All sites are shown for the enzymes HindIII (H), Kpn I (K), EcoRI (R), Sal I (S),and Xba I (X). Exons are numbered 1-7.

Biochemistry: Bonthron et al.

1494 Biochemistry: Bonthron et al.

1IGAGGGAGGGGCGCGGAGCCCCGGCGCGGAGCCGGGCGCGGGGCTTTGATGGATTTAGCTGCTTGCGCGAGCGCGTGTGTGCTCCCTGCCGCAGCGGCGGCGCCCGGGCCCTGCCGGGTCC121 GCACGAACCCCGAGCGCTTCCAAGGTGCGGGTCCCAGGCCCGGAATCCGGGGGAGGCGGGGGGGGGGGGGCGGGGGCGGGGGCGGGGGAGGGGCGCGGCGGCGGCGT cCCTCTCC241 CCGCCGCCGGCCGGCTCCAC jSCGCGCGCCCTGCGGAGCCCGCCCAACTCCGGCGAGCCGGGCCTGCGCCTACTCCTCCTCCTCCTCTCCCGGCGGCGGCTGCGGCGGAGGCGCCGACTC360 GGCCTTGCGCCCGCCCTCAGGCCCGCGCGGGCGGCGCAGCGAGGCCCCGGGCGGCGGGTGGTGGCTGCCAGGCGGCTCGGCCGCGGGCGCTGCCCGGCCCCGGCGAGCGGAGGGCGGAGC480 GCGGCGCCGGAGCCGAGGGCGCGCCGCGGAGGGGGTGCTGGGCCq:GCTGTGCCCGGCCGGGCGGCGGCTGCAAGAGGAGGcCGGAGGCGAGCGCGGGGCCGGCGGTGGGCGCGCAGGGC600 GGCTCGCAGCTCGCAGCCGGGGCCGGGCCAGGCGTTCAGGCAGGTwATCGGTGTGGCGGCGGCGGCGGCGGCGGCCCCAGACTCCCTCCGGAGTTCTTCTTGGGGCTGATGTCCGCAAAT720 ATGCAGAATTACCGGCCGGGTCGCTCCTGAAGCCAGCGCGGGGAGCGAGCGCGGCGGCGGCCAGCACCGGGAACGCACCGAGGAAGAAGCCCAGCCCCCGCCCTCCGCCCCTTCCGTCCC840 CACCCCCTACCCGGCGGCCCAGGAGGCTCCCCGCGCTGCGGGCGCGCACTCCCTGTTTCTCCTCCTCCTGGCTGGCGCTGCCTGCCTCTCCGCACTCACTGCTCGCGCCGGGCGCGCTCC960 GCCAGCTCCGTGCTCCCCGCGCCACCCTCCTCCGGGCCGCGCTCCCTAAGGGATGGTACTGAATTTCGCCGCCACAGGAGACCGGCTGGAGCGCCCGCCCCGCGGCCTCGCCTCTCCTCC

1080 GAGCAGCCAGCGCCTCGGGACGCGATGAGGACCTTGGCTTGCCTGCTGCTCCTCGGCTGCGGATACCTCGCCCATGTTCTGGCCGAG GTTGGTGCCGCCCCCGCGCCCCGTCCCTGCGCMetArgThrLeuAlaCysLeuLeuLeuLeuGlyCysGlyTyrLeuAlaHisValLeuAlaGluj

1199 CGGCTCCTCCG.......... IVS 1 (1.5 kb) .........GGCCGCGGGCGCTGACCGTGTGGCCTCTGCTTGCAG rGAAGCCGAGATCCCCCGCGAGGTGATCGAGAGGCTGGCCLGluAlaGluIleProArgGluValIleGluArgLeuAla

1285 CGCAGTCAGATCCACAGCATCCGGGACCTCCAGCGACTCCTGGAGATAGACTCCGTAG1 GTAAATCGCGCCCCTTCCCTCCGCGCGCGGGGAGGGCGCGGGC.... IVS 2 (5.8 kb)ArgSerGlnIleHisSerIleArgAspLeuGlnArgLeuLeuGluIleAspSerValGj

1386. GCGGGAGCTGTGAGGGGCTGTGCCGCCAGGTGCCTGTTCCCCAGTGGCTCCCAAAGCTGGTCTGTGGGAAGTGCGGCTGGACAGGCCCAGGGCACAGCGCACGGGCCTGGGGCA1500 TTCACGGTGTTCTCCTTCCGCCTGCAG rGGAGTGAGGATTCTTTGGACACCAGCCTGAGAGCTCACGGGGTCCACGCCACTAAGCATGTGCCCGAGAAGCGGCCCCTGCCCATTCGGAGG

LlySerGluAspSerLeuAspThrSerLeuArgAlaHisGlyValHisAlaThrLysHisvalProGluLysArgProLeuProIleArgArg1619 AAGAGAAGCATCG1 GTGAGTCCAGGAGGCCGCGATGGGCAGGGCAGGGCCGGQTCGGGGTGAGTCCAGGAGGCCGCGATGGGCAGGGCAGGGCCGGGTGGGGAGGAGGTGCCC .......

LysArgSer IleGJ1731 . . IVS 3 (1.4 kb) ... GAGGCCGGTCCCCGCTCACTGTGCCCCCGCCGTTGCAG FAGGAAGCTGTCCCCGCTGTCTGCAAGACCAGGACGGTCATTTACGAGATTCCTCGGAGTCAG

LluGluAlaValProAlaValCysLysThrArgThrVal IleTyrGlu IleProArgSerGln18 31 GTCGACCCCACGTCCGCCAACTTCCTGATCTGGCCCCCGTGCGTGGAGGTGAAACGCTGCACCGGCTGCTGCAACACGAGCAGTGTCAAGTGCCAGCCCTCCCGCGTCCACCACCGCAGC

ValAspProThrSerAla~snPheLeuIleTrpProProCysValGluValLysArgCysThrGlyCysCysAsnThrSerSerValLysCysGlnProSerArgVal isHiSArgSer1951 GTCAAG GTGAGCCCTCCCCTCCCCTCCCCT... IVS 4 (10.0 kb) .. .CCGAAGCTCCATGCAGGCATTCATGGCCGGGCTCTGTTCTCTCTGGCAG GTGGCCAAGGTGGAATAC

ValLysj ValAlaLy sValGluTyr2048 GTCAGGAAGAAGCCAAAATTAAAAGAAGTCCAGGTGAGGTTAGAGGAGCATTTGGAGTGCGCCTGCGCGACCACAAGCCTGAATCCGGATTATCGGGAAGAGGACACG GTGAGTGGCT

ValArgLysLysProLysLeuLysGluvalGlnvalArgLeuGluGluH isLeuGluCysAlaCysAlaTh rThrSerLeuAsnProAspTyrA rgGluGl uAspThrG2167 GCCTTCGTCGGCATCGGTGTTGGAGAACAGGTCTTCAGAGCCTTGCTTTTGGGGTGTTAGGTGGCCCCCTT...IvS 5 (0.5 kb) ... GCCGTAGGTATTTGTTGCTTCAGTTCTTC2267 ACCCCGACGGCCGTCCCTCGGCCCACTCACCGCCCTGCCCTTTTGTTAACAG jAAGGCCTAGGGAGTCAGGTAAAAAACGGAAAAGAAAAAGGTTAAAACCCACCTAAAGCAGCCAACC

LlyArgProArgGluSerGlyLysLysArgLysArgLysArgLeuLysProThr***2386 AG] GTAGGACTGTCTGCCGGACACACTGAGTCCTGCAGGGCGGGGAGGTGCAACTGACTGATCACACCAGCAAATCACGCTTCCTGTCTCGCC... IVS 6 (1.9 kb) .. .TTTTCCT2485 CCTGTGCAGCGATGTGTGTGCTGGTGGTTCACTTGGTCGGTGGTCTCAGTGTCTGTCTTTCCCTTCTCCCTGCAG fATGTGAGGTGAGGATGAGCCGCAGCCCTTTCCTGGGACATGGAT

LspValArg* * *26C0 4 GTACATGGCGTGTTACATTCCTGAACCTACTATGTACGGTGCTTTATTGCCAGTGTGCGGTCTTTGTTCTCCTCCGTGAAAAACTGTGTCCGAGAACACTCGGGAGAACAAAGAGACAGT27 24 GCACATTTGTTTAATGTGACATCAAAGCAAGTATTGTAGCACTCGGTGAAGCAGTAAGAAGCTTCCTTGTCAAAAAGAGAGAGAGAGAGAGAGAGAGAGAAAACAAAACCACAAATGACA2844 AAAACAAAACGGACTCACAAAAATATCTAAACTCGATGAGATGGAGGGTCGCCCCGTGGGATGGAAGTGCAGAGGTCTCAGCAGACTGGATTTCTGTCCGGGTGGTCACAGGTGCTTTTT2964 TGCCGAGGATGCAGAGCCTGCTTTGGGAMACGACTCCAGAGGGGTGCTGGTGGGCTCTGCAGGGGCCCGCAGGAAGCAGGAATGTCTTGGAAACCGCCACGCGAACTTTAGAAACCACACC3084 TCCTCGCTGTAGTATTTAAGCCCATACAGAAACCTTCCTGAGAGCCTTAAGTGGTTTTTTTTTTTTGTTTTTGqrTTGTTTTTTTTTTTTTTGTTTTTTTTl'-TTTTTTTTTTTACACCAl'3204 AAAGTGATTATTAAGCTTTCCTTTTTACTCTTTGGCTAGCTTTTTTTTTTTTTTTTTTTAATTATCTCTTGGATGACATTTACACCGATAACACACAGGCTGCTGTAACTGTCAGGACAG3324 TGCGACGGTATTTTTCCTAGCAAGATGCAAACTAATGAGATGTATT TGGTATACCTACCTATGCATCATTICCTAAATGTTTCTGGCTTTGTGTTTCTCCCTTACCCTGC3444 TTTATTTGTTAATTTAAGCCATTTTGAAAGAACTATGCGTCAACCAATCGTACGCCGTCCCTGCGGCACCTGCCCCAGAGCCCGTTTGTGGCTGAGTGACAACTTGTTCCCCGCAGTGCA3564 CACCTAGAATGCTGTGTICCCACGCGGCACGTGAGATGCATTGCCGCTTCTGTCTGTGTTGTTGGTGTGCCCTGGTGCCGTGGTGGCGGTCACTCCCTCTGCTGCCAGTGTTTGGACAGA3684 ACCCAAATTCTTTATTTTTGGTAAGATATTGTGCTTTACCTGTATTAACAGAAATGTGTGTGTGTGGTTTrGTTTTTTTT 3762

FIG. 2. Nucleotide sequence of the PDGF A-chain gene. Approximate sizes of introns are indicated. The 5' and 3' limits of the cDNA clonedT1.1 are shown by upturned arrowheads. The three ATG codons upstream of the A-chain coding region are underlined. The TATAA andAATAAA sequences are boxed; exons are bracketed; the indicated transcriptional start site is only approximate, due to the large size of theprimer extension products. A 41-bp direct repeat at the start of IVS3 is indicated by long arrows.

of a deletion (7pll.2-p22) seen in myelodysplastic syndrome(29). Monosomy for 7p22 appears to result in a syndrome ofcraniosynostosis and other dysmorphic features (30).

DISCUSSION

Consistent with their presumed common evolutionary ori-gin, the intron-exon structures of the PDGF A- and B-chain

DB-1 DB-2 DB-1 DB-2M Tb A b pM M '; b b -n MFabca bcab cM

||WUNIX-H :: .* :: - 389

E~~~~~~~~~~~~~l :f`352

FIG. 3. Mapping of the PDGF A-chain cap site. Primer exten-sion products were run for 2 hr (Left) or 4.5 hr (Right) on a 6%acrylamide/8 M urea gel. Markers: end-labeled pBR322 digestedwith Hinfl (lanes M) or Hpa II (lanes M'). Two micrograms ofpoly(A)+ RNA from passaged human umbilical vein endothelialcells (lanes a), 4 ,ug of poly(A) + RNA from HepG2 cells (lanes b), or10 jig of tRNA (lanes c) was used with each of the oligonucleotides,DB1 or DB2. Exposure: 14 days at - 70 C with intensifying screen.Nucleotide positions are indicated on the right.

genes are very similar (see refs. 22 and 31). In regions ofsubstantial amino acid homology-for example, exons 1 and2-the sequences and splice junctions can be preciselyaligned (Fig. 5). Exons 4 and 5 are also well conserved.Exons 3 and 6, in contrast, have few residues in commonbetween A and B chains. Nonetheless, exon 6 of A and Bchains share the feature of encoding a very basic C-terminaldomain [predicted net charge + 11 (A chain), + 9 (B chain)].Exon 6 is the exon "skipped" during splicing of the endo-thelial-type A-chain mRNA; we have provided evidence thatan active A-chain homodimer can be readily assembled onlyfrom a precursor containing the exon 6 basic domain (12).Whether the exon 6 domain of the B-chain precursor servesa similar function and is required for the assembly of B-Bhomodimers and B-small A heterodimers is undefined.There is currently no evidence for alternative splicing ofexon 6 of the PDGF B chain, but it will be important to assayfor this event specifically, by nuclease protection.The 5' end of the A-chain gene is strikingly (G +C)-rich

(78.6% over the first 1209 bp of Fig. 2) and fits the criteria fora hypomethylated "HTF island" (32). The primer extensionexperiment suggests that the TATAA sequence within thisregion is likely to be a functional component of the A-chainpromoter. A- and B-chain genes also contain more than onecopy of the consensus binding sequence for transcriptionfactor Spl (GGGCGG) upstream and downstream of the capsite. This feature is often associated with multiple transcrip-tional start sites [e.g., epidermal growth factor receptor (33),hydroxymethylglutaryl CoA reductase (34)]. The A- andB-chain promoters may resemble the (G +C)-rich promoterregion of the c-myc gene, in which each of two start sites isdefined by a TATAA element (35, 36). The TATAA, but notthe upstream GGGCGG, sequences of the B-chain promoterare required for promoter activity in transient expressionexperiments (D.T.B., T.C., and S.H.O., unpublished).

Proc. Natl. Acad. Sci. USA 85 (1988)

Proc. Natl. Acad. Sci. USA 85 (1988) 1495

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FIG. 4. In situ hybridization of PDGF A-chain cDNA to metaphase chromosomes. The histogram displays data for all grains counted in150 metaphases. The ideogram of chromosome 7 shows the distribution of grains on that chromosome and the assignment of PDGF A chainto 7p2l-p22.

An unusual feature conserved between A- and B-chainmRNAs is the presence of three ATG triplets, upstream ofthe authentic initiator codon, within a long 5' untranslatedregion. In the A chain, the first two of these are closetogether in the same reading frame and are followed shortlyby a stop codon. The third heads an open reading frame(ORF) that could encode a 31-residue peptide; the terminatorfor this reading frame overlaps the authentic ATG initiatorcodon in such a way that if this ORF were translated (37),reinitiation would have to follow a backward "jump" of onenucleotide. Backward movement of the ribosome is believedto explain a -1 frame shift in the translation of a retroviralmRNA (38). The 5' untranslated regions of A- and B-chainmRNAs would be expected to have substantial secondary

structure due to their high G-C content; this and the presenceof upstream ATGs would be expected to decrease transla-tional efficiency (39). The possibility that the upstreamATGs are involved in translational control of PDGF expres-sion (as demonstrated for the yeast GCN4 product) (40)requires analysis by site-directed mutagenesis of these re-

gions.Since alternative splicing can generate functionally dis-

tinct PDGF A-chain mRNAs, developmental and cell-type-specific regulation of this process may have important con-sequences for physiological and pathological processes,such as embryogenesis and atherosclerosis. At present, theonly direct evidence that exon 6 is utilized remains theisolation of the original glioma clone D-1 of Betsholtz et al.

A MRTLACLLLLGCGYLAHVLAE EAEIPREVIERLARSQIHSIR... DLQRLLEIDSV GSEDSLDTSLRAHGVHATKHVPEKRPLPIRRKRSIB MNRCWALFLSLCCYLRLVSAE GDPIPEELYEMLSD ...HSIRSFDDLQRLLHGDPG .EEDGAELDLNMTRS .... HSGGELESLARGRRSL

L.* ** *** * ** ** ****,*******J ** * * **,*L4

A ......EEAVPAVCKTRTVIYEI PRSQVDPTSANFLIWPPCVEVKRCTGCCNTSSVKCNPSRVHHRSVKB GSLTIAEPAIIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQ

.5 6 -7A VAKVEYVRKKPKLKEVQVRLEEHLECACATT.SLNPDYREEDT.................GRPRESGKKRKRKRLKPT........I..DVRB VRK IEIVRKKPIFKKATVTLEDHLACKCETVAAARPVTRSPGGSQEQ AKTPQTRVTIRTVRVRRPPKGKHRKFPHTHDKTALKETLGA ...

A CHAIN 1.5 5.2 1.4 10.0 0.5 1.9

B CHAIN 8.8 2.0 1.5 1.5 6.8 0.3

FIG. 5. Intron-exon structures of PDGF A and B chains. (Upper) The alignment is similar to that of Betsholtz et al. (5) but shows that thenonhomologous regions of A and B chains are largely confined to exons 3 and 6. Dots represent gaps introduced to improve alignment. TheN-terminal propeptide cleavage sites are shown by arrows toward the right-hand end of exon 3, and each exon is numbered above. Asterisksindicate matching residues. (Lower) The diagram compares the splicing patterns ofA and B chains. Sizes of exons are to scale; those of introns(from ref. 22 in the case of the B chain) are indicated (in kb).

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Biochemistry: Bonthron et al.

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1496 Biochemistry: Bonthron et al.

(5). However, several tumor cell lines appear to secreteA-chain homodimers. If, as implied by our cDNA expressionexperiments (12), the A-chain precursor lacking exon 6cannot efficiently form such homodimers, use of this exonmay not be rare in malignant cells. Cells cultured fromhuman atheromatous plaques may likewise produce aPDGF-like growth factor composed of A but not B chains(41). Therefore, in a variety of circumstances, alteration ofthe splicing pattern to generate the longer form of A-chainprecursor, and consequent assembly of an A-A homodimer,may provide the cell with an autocrine growth advantage.

Steady-state mRNA levels of A and B chains of PDGFvary in an inducible fashion in some cell lines (42, 43).Together with the structural data discussed above, thissuggests that regulation of transcription, alternative mRNAsplicing, differing stabilities of A- and B-chain mRNAs, andtranslational control could all be important determinants of acomplex pattern of PDGF expression.

We thank Michael Gimbrone for human endothelial cell cultures,Christer Betsholtz for supplying the cDNA clone D-1, and RobinBrown for help with labeling the PDGF 1 and 2 oligonucleotides.This work was partly supported by grants from the NationalInstitutes of Health (to T.C., C.C.M., and S.H.O.). T.C. is a PewScholar in the Biomedical Sciences. S.H.O. is an Investigator of theHoward Hughes Medical Institute.

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2. Greenberg, M. E. & Ziff, E. B. (1984) Nature (London) 311,433-438.

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