Full Length Research Paper

Characterization of the Platelet-derived Growth FactorReceptor Alpha and c-kit Genes in the PufferfishFugu rubripes

HAWYS WILLIAMS, SYDNEY BRENNER and BYRAPPA VENKATESH*

Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore, Singapore 117609

(Received 11 March 2002)

The receptor tyrosine kinase type III subfamily includesthe PDGF receptors (a and b), c-kit and the colonystimulating factor 1 receptor (CSF1R). The similargenomic organization of the genes encoding thesereceptors and the observation that in mammals, thegenes for PDGFRa and PDGFRb are tandemly linkedwith the c-kit and CSF1R genes, respectively, suggest thatthese genes arose from an ancestral receptor tyrosinekinase gene. We have previously cloned two copies eachof the PDGFRb and CSF1R genes from the pufferfish,Fugu rubripes, and shown that they are tandemly linked.We have now cloned the Fugu PDGFRa and c-kit genesand show that they are also tandemly linked. Theintergenic region between the two genes in the Fugu(13 kb) and humans (350 kb) contains a conserved 59 bpelement, which may play a role in regulation. The FuguPDGFRa gene is transcribed from two promoters givingrise to alternate transcripts.

Keywords: Platelet-derived growth factor receptor (PDGFR); c-Kit;Fugu; CSF1R

Abbreviations: PCR, polymerase chain reaction; PDGFR, platelet-derived growth factor receptor; RACE, rapid amplification ofcDNA ends; RT-PCR, reverse transcriptase PCR

INTRODUCTION

The receptors for the platelet-derived growth factor(PDGFRa and PDGFRb), colony stimulating factor-1(CSF1R) and the stem cell factor (c-kit) belong totype III subfamily of receptor tyrosine kinase thatshare a common structure comprising five immuno-globulin-like domains in the extracellular region and

two tyrosine kinase domains separated by a kinaseinsert domain in the intracellular region.The PDGFRs mediate the proliferation anddifferentiation of glial and mesenchymal cells(Heldin and Westermark, 1999), whereas the CSF1Rmediates survival, growth and differentiation of cellsbelonging to the monocyte–macrophage lineage(Bourette and Rohrschneider, 2000). The proto-oncogene c-kit mediates the growth and differen-tiation of melanocytes, and hematopoietic and germcells (Rosnet and Birnbaum, 1993; Rawls andJohnson, 2000). The PDGFRa- and b receptorsdisplay differences in their binding affinity todifferent isoforms of PDGF (AA, AB and BB) anddiffer in the signals they transduce. Therefore, theeffect of PDGF on a cell type is determined by thetype and level of the PDGF receptor expressed on itssurface. Typically, fibroblasts and smooth musclecells express both PDGFRa- and b, with the latterbeing expressed at relatively higher levels. Somecells such as O-2A glial precursor cells, humanplatelets and rat endothelial cells express onlyPDGFRa, whereas other cell types such as mousecapillary endothelial cells express only PDGFRb(Heldin and Westermark, 1999). The CSF1R isexpressed exclusively in blood cells of mononuclearphagocyte lineage and in placental trophoblasts(Bourette and Rohrschneider, 2000), whereas the c-kitexpresses in diverse cell lineages including hemato-poietic stem cells, mast cells, megakaryocytes, earlyerythroid cells, B cell precursors, melanocytes and

ISSN 1042-5179 print/ISSN 1029-2365 online q 2002 Taylor & Francis Ltd

DOI: 10.1080/1042517021000011627

*Corresponding author. Tel.: þ65-6874-3633. Fax: þ65-6779-1117. E-mail: mcbbv@imcb.nus.edu.sg

DNA Sequence, 2002 Vol. 13 (5), pp. 263–270

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primordial germ cells (van der Greer et al., 1994;Wehrle-Haller and Weston, 1997). C-kit is alsoexpressed at high levels in the CNS of the developingand adult mouse (Qiu et al., 1988). Understanding thetranscriptional regulation of these genes is critical toanalyzing their function at various stages ofdevelopment and differentiation.

In humans and mouse, the genes encodingPDGFRa and PDGFRb are tandemly linked to thec-kit and CSF1R genes, respectively (Roberts et al.,1988; Gronwald et al., 1990; Eccles, 1991; Brunkowet al., 1995). Based on the structural similarity andgenomic organization of these four genes, it has beenproposed that they arose from an ancestral receptortyrosine kinase gene through two rounds ofduplication. We have previously cloned two copieseach of PDGFRb and CSF1R genes from thepufferfish, Fugu rubripes, and shown that they aretandemly linked like the mammalian genes(How et al., 1996; Williams et al., 2002). We havenow sequenced the Fugu PDGFRa and c-kit locus,and report here the genomic organization andexpression profile of the Fugu PDGFRa ( fPDGFRa )and c-kit ( fc-kit ) genes. The two genes are singlecopy in the Fugu genome and are tandemly linkedlike their mammalian homologs. The Fugu PDGFRagene gives rise to alternative transcripts generatedfrom two different promoters.

MATERIALS AND METHODS

Cloning and Sequencing of the Fugu PDGFRa andc-kit Genes

To isolate tyrosine kinase receptor genes from theFugu, we designed several pairs of degenerate PCRprimers corresponding to the consensus sequenceflanking the catalytic domains of mammalianreceptor tyrosine kinases. The gene fragments werePCR amplified from the Fugu genomic DNA, clonedinto a T vector and sequenced. The sequences weresearched for homology using the BLASTX program atthe National Center for Biotechnology Information(NCBI). One of the PCR products, TJ8, which wasamplified using the primers (50 TGYATHCAYGAY-MGNGT 30) and (50 CCRTARCTCCANACRTC 30)corresponding to residues CIHRDV and DVWSYG ofthe mammalian kinases, showed high sequencesimilarity to zebrafish PDGFRa. This clone was usedto probe a gridded Fugu cosmid library (Greg Elgar,Unpublished), and eight positive cosmids wereisolated. One of the cosmids, c31A19, was selectedfor sequencing by the shotgun method. About 5mg ofcosmid DNA was partially digested using Sau3A andfragments in the range of 1.6–2.0 kb were cloned intopBluescript and sequenced using the ABI Big dyeterminator chemistry (Perkin Elmer). The shotgun

sequences were assembled using the programSEQMAN II (DNAStar). The gaps and discrepanciesin the sequence were resolved by sequencing cosmidDNA using walking primers. A homology searchindicated that cosmid c31A19 contains the entirePDGFRa coding sequence and sequence for the first110 amino acids of the c-kit gene. In order to isolatethe entire c-kit gene, a Sau3A subclone containing the50 end of the c-kit gene was used to reprobe thecosmid library. Nine positive cosmids were isolatedand an overlapping cosmid, c17K21, was sequencedcompletely. The exon–intron structure and thetranscription start site of fPDGFRa and fc-kit geneswere determined by sequencing cDNA clonesgenerated by 50RACE and RT-PCR (SMART RACEcDNA Amplification kit, Clontech).

RNA Preparation and Reverse Transcription-PCR

Total RNA was extracted from various Fugu tissuesusing Trizol reagent (Gibco BRL). First strand cDNAwas synthesized using the SMARTe RACE cDNAamplification kit (Clontech). The following primerswere used to amplify fragments of fPDGFRa andfc-kit: fPDGFRa1F (50 CATGCACACCGGCTACT-ACAG 30); fPDGFRa2F (50 TGCCTTCTACGACGG-CAGATTGG 30); fPDGFRaR (50 GTATGTTTGGCC-GTCAGCTCAACG30); fC-KITF (50 GGTGACACCA-AGCCTACCATT 30); fC-KITR (50 GGATCTGTTGC-CAGGCAGGGA 30).

Primer pair fPDGFRa1F and fPDGFRaR wereused to amplify transcript 1 and primer pairfPDGFRa2F and fPDGFRa for transcript 2. Afragment of actin cDNA was amplified as an internalcontrol for the quality of mRNA using the primersfACTF (50 AACTGGGAYGACATGGAGAA 30) andfACTR (50 TTGAAGGTCTCAAACATGAT 30). Allthe primer pairs used span at least one intron.

Amplification of 50 Ends of cDNA

The transcription start sites were mapped byamplifying 50 ends of cDNA by RACE-PCR usinggene specific primers together with “Universalprimers” supplied with the RACE kit (Clontech).The RACE products were cloned and sequenced.

Sequence Comparison

ClustalX (Thompson et al., 1997) was used to createalignments of amino acid sequences. Pipmaker(Schwartz et al., 2000) was used to identify conservednon-coding sequences in the Fugu and humanloci. The human sequence was masked usingRepeatmasker prior to performing the alignment.The Advanced Pipmaker application was used withthe chaining option.

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Determining the Copy Number of Fugu PDGFRaGene

The amino acid sequences of all known vertebratePDGFRa including the Fugu PDGFRa isolatedin this study were aligned to identify conserved

regions. Degenerate PCR primers correspon-ding to the conserved sequences TEYCFYG(50ACNGARTAYTGYTTYTAYGG30) and DYM-DMKQ (50GYTTCATRTCCATRTARTC30) weredesigned and used to amplify PDGFRa gene

FIGURE 1 Schematic diagram of the (a) Fugu and (b) human PDGFRa and c-kit loci (GenBank accession number AF456419). Horizontalarrows represent genes and indicate the direction of transcription. Vertical arrows indicate the position of conserved putative regulatoryelement identified in this study. Note the different scales for the Fugu and human loci. The Fugu sequence was obtained from cosmidsc31A19 and c17K21 by sequencing both strands of DNA at least once. Human PDGFRa and c-kit data are from the draft human genomesequence (Ensembl browser; accession number AC009614). The deduced ordering of human BAC clones (RP11-231C18, RP11-24O10, RP11-545H22, RP11-33C2) containing the sequences for PDGFRa and c-kit is shown below the human sequence.

FIGURE 2 Alternative splicing of the Fugu PDGFRa gene. (a) Exon–intron structure of the Fugu PDGFRa genomic DNA and thealternative transcripts generated from two different promoters. Transcription start sites are shown by arrows. Coding exons arerepresented by open boxes and introns by lines. The 50 untranslated exons are shown by stippled box. Size of the exons (bp) are shownabove the exons and size of the introns (bp) are shown below the line. (b) Comparison of the alternative amino acid sequences encoded bythe Fugu PDGFRa transcripts 1 and 2, and the zebrafish PDGFRa sequence. Intron positions in the Fugu gene are shown by arrowheads.The conserved cysteine residues are boxed.

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FIGURE 3 ClustalX alignment of the Fugu and other vertebrate c-kit sequences. Arrowheads indicate the position of introns in the Fuguand human c-kit genes. Asterisks indicate identical residues and periods indicate conservative substitutions. The conserved cysteineresidues in the extracellular ligand-binding domain are boxed, and the consensus tyrosine residue in the catalytic domain is shown in bold.The transmembrane domain is also boxed and the kinase-insert domain that splits the intracellular kinase domain is underlined. Accessionnumbers of amino acid sequences used for generating the alignment: Fugu c-kit (fkit), this study; zebrafish c-kit (zkit), AF153446; chickenc-kit (ckit), D13225; human c-kit (hkit), AAC50969.

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fragment from the Fugu genomic DNA. The PCRconditions were 958C for 5 min followed by 35 cyclesof 958C for 30 s, 508C for 1 min and 728C for2 min followed by a final elongation at 728C for5 min. The PCR products were cloned andsequenced.

RESULTS AND DISCUSSION

Genomic Organization of Fugu PDGFRa and c-kitGenes

A total of 54 kb contiguous sequence was determinedfrom two overlapping cosmids, c31A19 and c17K21(submitted to GenBank under accession numberAF456419). This locus contains the completesequences for the fPDGFRa and fc-kit gene on thesame strand of DNA (Fig. 1). The genomic arrange-ment of these genes is similar to that of theirmammalian homologs (Gronwald et al., 1990;Brunkow et al., 1995) as well as that of themammalian and Fugu PDGFRb and CSF1R genes(Roberts et al., 1988; Eccles, 1991; Williams et al.,2002). The identical genome organization of thesegenes suggests that the duplication of the ancestraltyrosine kinase receptor gene that gave rise to

the four receptor kinase genes in vertebratesoccurred before the divergence of the Fugu andmammalian lineages about 450 million years ago,and that the linkage of PDGFRa gene with c-kit gene,and PDGFRb gene with CSF1R gene has beenconserved in teleosts and mammals during evol-ution. It is possible that the conservation of linkage isthe result of a selective pressure on some sequencesin this locus, which are essential for the functioningof both the linked genes.

The Fugu c-kit gene comprises 21 exons similar toits human ortholog (Andre et al., 1997). The trans-cription start site of fc-kit gene is located 303 bpupstream of the translation initiation codon. Theentire gene, from transcription start site to the poly Asignal, spans only 16.5 kb. This is in contrast to thelarge human c-kit gene, which occupies about 83 kb.The large size of the human gene is due to expandedintrons that are typical of human genes. The firstintron of the human c-kit alone is 37.4 kb (Andre et al.,1997); its Fugu counterpart is only 1.6 kb.

The Fugu PDGFRa gene has two promoters givingrise to alternate transcripts with different 50 exons(Fig. 2). We designate the transcript generated fromthe proximal promoter as transcript 1 and that fromthe distal promoter as transcript 2. Transcript 1

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consists of 21 coding exons and two 50 untranslatedexons, whereas transcript 2 contains 22 coding exonsand an untranslated exon at the 50 end (Fig. 2a). Thegenomic structure of the latter is identical tothe human PDGFRa gene (Kawagishi et al., 1995).The overall size of the fPDGFRa gene, including all

the alternate exons, is only 12 kb. Its human orthologis about five times larger (Kawagishi et al., 1995).The amino acid sequence encoded by the alternateexons of the fPDGFRa contain conserved cysteineresidues characteristic of the extracellular domain ofthis family of receptor tyrosine kinases (Fig. 2b). Nosuch alternate transcripts have been identified forPDGFRa gene either in mammals or in zebrafish,and thus it is not clear whether the PDGFRa gene inother vertebrates has alternate promoters. From thesequence comparison it appears that the alternateexons in the Fugu gene arose due to a tandemduplication of the 50 exons and the promotersequence. This could be a common mechanism thatgives rise to alternative transcripts transcribed fromdifferent promoters.

The intergenic distance between the Fugu PDGFRaand c-kit genes is 13 kb. The homologous region in thehumans is about 350 kb. We searched the humanintergenic sequence for the presence of othergenes, known or novel, by homology search and

FIGURE 4 ClustalX alignment of Fugu and other vertebrate PDGFRa sequences. Arrowheads indicate the position of introns in the Fuguand human PDGFRa genes. Asterisks indicate identical residues and periods indicate conservative substitutions. The conserved cysteineresidues in the extracellular ligand-binding domain are boxed, and the consensus tyrosine residues in the catalytic domain are shown inbold. The transmembrane domain is also boxed. The kinase-insert domain that splits the intracellular kinase domain is underlined.Accession numbers of amino acid sequences used for generating the alignment: Fugu PDGFRa, this study (only isoform 1 is shown);zebrafish PDGFRa, AAG43479; Xenopus PDGFRa, AAA49929; chicken PDGFRa, AAF01460; and human PDGFRa, M22734.

FIGURE 5 Expression patterns of Fugu PDGFRa and c-kit genesanalyzed by RT-PCR. PCR primers were designed such that theyspan at least one intron. Actin was amplified as a control for thequality of RNA.

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gene prediction programs, but did not identifyany genes. However, our analysis indicated that thisregion contains a large number of transposon-derived sequences such as Alus, MIRs, LINES, LTRelements and MERs. These elements account fornearly 45% of the non-coding sequence in the humangenome (IHGS consortium, 2001), and are mainlyresponsible for the large size of the human genome.The Fugu, not well understood at this stage, by somemechanism has managed to keep its genome compactby suppressing the spread of these elements.

We have previously isolated two copies each of theFugu PDGFRb and CSF1R genes and shown that theduplicate copies are the result of an additional roundof duplication specific to the teleost lineage (Williamset al., 2002). In order to check if the PDGFRa-ckit isalso duplicated in the Fugu genome, we investigatedthe number of copies of PDGFRa in the Fugu bydegenerate PCR. Our analysis identified only onecopy of the gene that was cloned by us in this study.We also BLAST searched the recently completed draftFugu genome sequence (http://www.fugu-sg.org)using amino acid sequences of the human, zebrafishand Fugu PDGFRa (isolated in this study) and ckit asquery sequences. Our search identified only onecopy each of PDGFRa and ckit genes in the Fugugenome, indicating that these genes are single copyin the Fugu. This suggests that the two copies ofPDGFRb and CSF1R genes in the Fugu could be theresult of a segmental duplication in the Fugu lineageinvolving the locus of these genes. Alternatively, it ispossible that if the teleost lineage did undergo anancient whole-genome duplication as proposed byAmores et al. (1998), the extra copies of the PDGFRaand ckit have been lost from the Fugu lineage.

Comparison of PDGFRa and c-kit ProteinSequences

The Fugu c-kit gene encodes a protein with 984amino acids which is 62% identical to the zebrafishc-kit and 43% identical to the human c-kit (Fig. 3).The Fugu c-kit contains all the motifs that arecharacteristic of the type III receptor tyrosine kinasefamily (Fig. 3). The fPDGFRa gene codes for twoisoforms of PDGFRa with 1064 and 1079 residues.These isoforms share 71 and 68% amino acid identitywith zebrafish PDGFRa, and 68 and 57% identitywith the human PDGFRa, respectively (Figs. 2band 4). Both isoforms contain all the functionallyimportant amino acid residues that are found in thePDGFRa from tetrapods and zebrafish (Fig. 4). Thus

we conclude that both isoforms are functionalproteins.

Expression Profile of the Fugu PDGFRa and c-kitGenes

Expression patterns of the fPDGFRa and the fc-kitgenes were analyzed by RT-PCR using gene specificprimers. The fc-kit gene showed detectable levels ofexpression in all the tissues investigated, except inblood where a very faint RT-PCR band was observed(Fig. 5). This is consistent with the expression patternof c-kit gene in mammals that shows expression indiverse cell types including those in the centralnervous system (Qiu et al., 1988; Wehrle-Haller andWeston, 1997). The Fugu PDGFRa transcripts 1 and 2show slightly different patterns of expression.Although both express in the brain, eye, heart,kidney, liver, muscle, ovary and skin, the levels oftranscript 2 in the eye, muscle, ovary and skin isconsiderably lower than those of transcript 1 (Fig. 5).This suggests that the two transcripts are under thecontrol of different regulatory elements. The physio-logical significance of the different expressionprofiles of the two transcripts is not clear. Interest-ingly, neither of the isoforms of PDGFRa showdetectable levels of expression in the Fugu blood. Wehave previously shown that the Fugu PDGFRb genesexpresses at relatively higher levels in the blood(Williams et al., 2002). This suggests that PDGFRb isthe predominant form of PDGF receptor expressedon the surface of blood cells in teleosts.

Conserved Non-coding Element in thePDGFRa-c-kit Locus

Comparison of the intergenic sequences in the Fuguand human loci identified an evolutionarily con-served element that is 76% identical across 59 bp(Fig. 6). It is located downstream of the PDGFRa polyA signal in both loci (Fig. 1). Since this element isconserved during a long evolutionary period, it islikely to be involved in the expression of eitherPDGFRa and/or c-kit gene. This element does notshow homology to any known transcription factorbinding sites and hence its function is not known.The compact promoters of the Fugu isolated by usare useful in understanding the tissue and develop-mental stage specific expression of these genes andalso serve as useful tools for labeling and targetingexpression to specific cell lineages.

FIGURE 6 Conserved putative regulatory element in the Fugu and human c-kit promoter region. The nucleotide positions are in relationto the first codon of the c-kit gene.

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Acknowledgements

The authors would like to thank Ms Diane Tan andMs Boon Hui Tay for the technical assistance, and theUK HGMP Resource Center for the Fugu cosmids.B.V. is an adjunct staff member of the Department ofPediatrics, National University of Singapore.

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