Proc. Nati. Acad. Sci. USA Vol. 88, pp. 5335-5339, June 1991 Biochemistry The gene: Evolutionary history, alternative splicing, and developmental regulation (intestine/preprohormone/polymerase chain reaction) ALAN S. KOPIN*t, MICHAEL B. WHEELER*, JUNKO NISHITANI*, EDWARD W. MCBRIDE*, TA-MIN CHANGt, WILLIAM Y. CHEYt, AND ANDREW B. LEITER* *Division of Gastroenterology, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111; and tIsaac Gordon Center for Digestive Diseases and Nutrition, The Genesee Hospital, University of Rochester School of Medicine and Dentistry, Rochester, NY 14609 Communicated by Viktor Mutt, March 26, 1991 (receivedfor review January 8, 1991)

ABSTRACT The gene encoding the hormone secretin has gene differs in structure from other genes of the - been isolated and structurally characterized. The transcrip- secretin family. Similarities within the gene family appear to tional unit is divided into four exons spanning 813 nucleotides. be limited to the exons that encode the biologically active Comparison ofthe rat secretin gene to the other members ofthe . glucagon-secretin gene family reveals that similarities are We have reported (1) the tissue distribution of secretin restricted to the exons encoding the biologically active peptides. mRNA within the rat . Unexpectedly, Analysis of RNA from porcine intestine indicates that at least the highest levels of secretin mRNA were found in the ileum, two transcripts are generated from the porcine secretin gene as distant from the regions of the intestine that are exposed to a result of differential splicing. The longer and more abundant the highest concentrations of gastric acid and fat, the major transcript appears to be identical to a previously isolated stimuli for secretin release (4-6). To further investigate the cDNA, which encodes a precursor that includes a 72-amino acid relationship between secretin gene expression and the pres- C-terminal extension . The shorter transcript does not ence of its enteral secretagogues, we have examined the contain the third exon and, as a result, encodes only 44 residues ontogeny of this hormone in developing rats. The develop- beyond the C terminus of secretin. The amino acid sequence mental studies reported here demonstrate that intestinal deduced from the shorter transcript is identical to a precursor secretin mRNA levels are highest before birth, antedating the form of secretin recently isolated from porcine duodenum onset of gastric acid production and feeding. These obser- [Gafvelin, G., Jornvall, H. & Mutt, V. (1990) Proc. Natl. Acad. vations suggest that the secretin gene is developmentally Sci. USA 87, 67814785]. Developmental studies reveal that regulated by factors other than its established enteral secre- both secretin mRNA and peptide levels in the intestine are tagogues. highest just before birth, prior to the onset of gastric acid secretion and feeding. This observation implies that secretin biosynthesis in developing animals is controlled independently MATERIALS AND METHODS of the principal factors known to regulate secretin release in Isolation of Recombinant Bacteriophage Containing the Rat adult animals. Secretin Gene. A library constructed from a partial Hae III digest ofrat genomic DNA and propagated in A Charon 4A (7) We have described (1) the isolation of cDNAs encoding was screened with a 424-base rat secretin cDNA probe porcine and rat secretin precursors. The deduced structures labeled by priming with random hexamers (8). Of 500,000 of the rat and porcine secretin precursors are similar, con- plaques examined, 4 positive recombinants were identified. sisting ofa signal peptide, an N-terminal peptide, secretin, an A 2.6-kilobase EcoRI restriction fragment was isolated from amidation-cleavage sequence, and a 72-amino acid C-termi- plaque-purified phage DNA, subcloned into the plasmid nal peptide. A precursor form of porcine secretin extending pUC19, and sequenced by the chain-termination method (9) only 44 amino acids beyond secretin has recently been using modified T7 DNA polymerase (United States Biochem- purified from intestinal extracts and sequenced (2). Within ical). Computer analysis of nucleotide and deduced peptide the C-terminal peptide of the shorter precursor, a single sequences utilized the University of Wisconsin Genetics arginine residue replaced 32 amino acids predicted from the Computer Group Software version 6.3 (10). cDNA. The remaining 40 amino acids at the C terminus ofthe Determination of the Transcriptional Initiation Site. The short precursor were identical to the sequence derived from transcriptional initiation site was mapped using a 32P-end- the cDNA. We now report the identification of differentially labeled (1 x 107 cpm/pmol) 24-base oligonucleotide probe, spliced secretin transcripts in porcine intestine that account 5'-GTGCAGGAAGCACGAAAGAACTTG-3'. The primer for both precursor forms. was annealed to poly(A)+ RNA (5.8 ,ug) isolated from rat Secretin is structurally related to several other regulatory small intestinal mucosa for 2 hr at 60°C in 250 mM KCl/10 peptides including glucagon, vasoactive intestinal peptide mM Tris Cl, pH 7.5/1 mM EDTA. Transcripts were extended (VIP), gastric inhibitory polypeptide (GIP), - for 1 hr at 37°C using Moloney murine leukemia virus reverse releasing hormone (GHRH), and pituitary adenylate cyclase- transcriptase. The size of the extended product was deter- activating protein, based on the occurrence of common mined by electrophoresis on a denaturing 8% polyacrylamide N-terminal amino acids (3). To further understand the evo- gel. lutionary relationship of secretin with other members of its Northern Blot Hybridization Assays. RNA from intestinal peptide family, we have isolated and structurally character- tissue of fetal and postnatal rats (Taconic Farms) was pre- ized the rat secretin gene.§ We found that the rat secretin Abbreviations: VIP, vasoactive intestinal peptide; GIP, gastric in- hibitory polypeptide; GHRH, growth hormone-releasing hormone. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M64033). 5335 Downloaded by guest on September 28, 2021 5336 Biochemistry: Kopin et al. Proc. Natl. Acad. Sci. USA 88 (1991) pared by extraction in acid phenol (11). For Northern blot encoding porcine secretin and the end of its C-terminal analyses, equal amounts of RNA were separated on 1.3% peptide. After heating at 980C for 5 min, the reaction mixture agarose/0.66 M formaldehyde gels and transferred to Nytran was cooled to 720C, and 2.5 units of Thermus aquaticus (Taq) membranes by capillary blotting. Blots were hybridized at DNA polymerase (Perkin-Elmer/Cetus) was added. DNA high stringency with the same secretin cDNA probe used to was amplified through 30 cycles of denaturation at 940C for screen the genomic library. To assure that equal amounts of 60 sec followed by a one-step annealing/extension at 680C for RNA were loaded into each lane, the RNA was quantitated 60 sec. A 133-bp Apa I-Sst I fragment ofa 201-bp product was by the absorbance at 260 nM, and the blots were reprobed for subcloned and sequenced. 13-actin. Selected blots were rehybridized with a rat chole- Determination of Secretin-Like Immunoreactivity in Tis- cystokinin cDNA probe at high stringency (12). The relative sues. Intestinal tissue was rapidly removed from rats of abundance of secretin mRNA on the autoradiographs was selected ages, frozen in liquid nitrogen, and stored at -70°C. determined by scanning densitometry. Radioimmunoassay for secretin-like immunoreactivity in in- Ribonuclease Protection Assays. Porcine or rat intestinal testinal extracts was performed as described (14). RNA samples were hybridized overnight at 45°C (13) with 2-10 fmol of antisense RNA probes (105 cpm/fmol). The probes consisted of a sequence complementary to a 366- RESULTS nucleotide Sal I-Nco I restriction fragment ofthe rat secretin Isolation of the Rat Secretin Gene. Screening -500,000 cDNA plus 42 nucleotides of pIBI31 vector and a 229-base- plaques in a rat genomic library with a rat secretin cDNA pair (bp) Nco I-BamHI restriction fragment of the porcine probe identified 4 identical clones. Southern blot analysis of secretin cDNA plus 37 bp of pGEM-5 vector. The hybrids the phage DNA revealed that the rat secretin cDNA probe were digested with RNase A (30 ,g/ml) and RNase T1 (2 hybridized to a single 2.6-kilobase EcoRI restriction frag- ,ug/ml) at 25°C for 30 min and the size of the protected ment. The same fragment hybridized to oligonucleotide fragments was determined by electrophoresis on denaturing probes specific for the 5' and 3' untranslated regions of the 5% polyacrylamide gels. mRNA, suggesting that the entire transcription unit was cDNA Amplification. Oligo(dT)-primed first-strand cDNA isolated. was reverse-transcribed from poly(A)+ porcine duodenal Nucleotide Sequence Analysis of the Cloned Rat Secretin mucosal RNA for use as template in the polymerase chain Gene. The rat secretin gene spans 813 nucleotides from the reaction as described (1). Amplification of 100 ng oftemplate transcriptional start site to the polyadenylylation addition was primed with two oligonucleotides, 5'-GACACTCG- site. The gene includes four exons, 119, 102, 105, and 197 bp GACGGGACGTTCACGA-3' and 5'-ACCCCTCCGCAG- long, separated by three introns, 81, 105, and 104 nucleotides CAGGGATGGC-3', corresponding to regions of the mRNA long (Fig. 1). The first exon encodes the 5' untranslated

tgtccatttcgctacctggtagc AP-2/SP1 actacccctctacttcctcccqccccagccagttgaggggcgccaacacggcggtagggacaggaggaggccggacgacag AP-2/SP1 AP-2/SP1 ctqqqqqqqccccctgaccttcccgcaatcgatggggtgcggccggccgcgcggaqccqqqqcqqtgccggagcccccc AP-2 I gcctqcqqgtcgggcagctataaagcggtggcgcggggctcaggctgAAGGTGCAGCATTTATCACACCCAGAACCCGAC Exon 1 [------Signal Peptide------][------NTP----- MetGluProLeuLeuProThrProProLeuLeuLeuLeuLeuLeuLeuLeuLeuSerSerSerPheValLeuProAlaP CATGGAGCCTCTACTGCCCACGCCGCCGCTACTGCTGCTGCTGCTGCTGCTGCTCTCAAGTTCTTTCGTGCTTCCTGCAC

roProAr CTCCCAGgtgagcggggtcgggaattggtttcatcccggccacgcccacccctcggtttccgactcttaaggctccaccc

_ ] [ ------Secretin- gThrProArgHisSerAspGlyThrPheThrSerGluLeuSerArgLeuGlnAspSerAlaArgLeuGlnAr ctccccagGACCCCAAGACACTCGGACGGGACGTTCACCAGCGAGCTCAGCCGCTTGCAGGACAGTGCCAGGCTGCAGCG Exon 2

gLeuLeuGinGlyLeuValGlyLysArgSe CCTGCTGCAGGGTCTGGTGGGGAAGCGCAGgtgagccagcactaggtagaattttccagacctgctggtatgggacaggc

rGluGluAspThrGluAsnIlePro ctgtcacatcccagtacaccactaattctactttgggcttgggggtggggggcagCGAGGAGGACACAGAAAATATTCCA Exon 3 ------C-Tenninal Peptide------GluAsnSerValAlaArgProLysProLeuGluAspGlnLeuCysLeuLeuTrpSerAsnThrGlnAlaLeuGlnAspTr GAGAACAGCGTGGCCCGTCCCAAGCCATTAGAGGACCAACTCTGCTTGCTGTGGTCGAACACTCAGGCCCTACAGGATTG gtgagcactccatcctcagctggattcctaagtccctagccatgtgggtgcctgcactctggtggccagtcactagcccc

pLeuLeuProArgLeuSerLeuAspGlySerLeuSerLeuTrpLeuProProGlyP ctcaccagctatctccctccacagGCTTCTGCCCAGGCTGTCCCTGGATGGGTCCCTGTCTCTCTGGCTGCCTCCTGGAC Exon 4

roArgProAlaValAspHisSerGluTrpThrGluThrThrArgGlnProArg CAAGGCCTGCTGTCGACCATTCAGAGTGGACTGAAACAACCAGGCAGCCCAGATGAGGGAGGAAGGGGAGTCTCCAGGAG CCTGACTGGAGTAGGGATTGGTTGTCCTTGGCATCAAXAAGAAGGAATTTAGACCCTGGTcttttagctcatggtgtgt gatatctgataaaatcaaggcagtctctgctccgtgaacctggcaggcttggacggatccatccatctcattcagtgact aggaccacacccagttgttgccctttgaacctggaaagccagctagggcattttttatctctgacatcagctgaacccct cttttcctagc FIG. 1. Nucleotide sequence of the rat secretin gene. Exons are shown in capital letters; introns and flanking sequences are shown in lowercase letters. The amino acid sequence of the translated regions is indicated above the nucleotide sequence. Peptide structures within the precursor are identified above their respective amino acid sequences. The Goldberg-Hogness promoter (TATAAA), the polyadenylylation signal (AATAAA), and a potential "CAAT" box are italicized and single-underlined. The transcriptional initiation site is indicated by an arrow. Potential binding sites for AP-2 and Spl are double underlined. NTP, N-terminal peptide. Downloaded by guest on September 28, 2021 Biochemistry: Kopin et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5337

1 2 A 1 2 3 B 1 2 3 4 149 145

266- *@l 113 -* --113112 408- 1--i09 200-s -- 82

-*4

4 - 69

FIG. 2. Determination ofthe transcription initiation site ofthe rat secretin gene by primer extension. A 3 P-end-labeled 24-base syn- FIG. 3. Analysis of secretin transcripts in intestinal RNA. (A) thetic oligonucleotide probe was annealed to poly(A)+ rat small Analysis of rat secretin transcripts by RNase protection assay. intestinal RNA (lane 1) or to yeast tRNA (lane 2) and then extended Lanes: 1, 20 Ag of RNA from small intestine; 2 and 3, undigested with Moloney murine leukemia virus reverse transcriptase. The probe, 0.003 fmol and 0.012 fmol, respectively. The protected extension products were separated on a-denaturing polyacrylamide fragment is indicated by a heavy arrow. Size of the undigested gel. The size of the major product (in bp) is shown on the left and is fragment is shown on the left in nucleotides. The gel was exposed to indicated by a bold arrow; an incompletely extended product is Kodak XAR film with two intensifying screens for 76 hr at -85°C. indicated by a light arrow. The positions of DNA size markers are (B) RNase protection assay of porcine RNA. Lanes: 1, 3 ,ug of shown on the right in bp. poly(A)+ RNA from duodenal mucosa; 2, 0.016 fmol of undigested porcine probe; 3, undigested probe hybridized to 10 ,ug of porcine region, the signal peptide, and 8 amino acids of the N-ter- duodenal RNA; 4, probe hybridized to 30 ,g of yeast tRNA and Major protected fragment is indicated by a heavy arrow, minal peptide. The second exon encodes the remaining 2 digested. and the predominant minor transcript is indicated by a light arrow. amino acids of the N-terminal peptide, secretin, the amida- Size markers in nucleotides are indicated on the left. The gel was tion cleavage sequence Gly-Lys-Arg, and 2 nucleotides ofthe exposed with two intensifying screens for 16 hr at -85°C. first serine residue ofthe C-terminal peptide. Exon 3 encodes the next 35 amino acids of the C-terminal peptide plus 2 Analysis of Transcripts Generated from the Secretin Gene. nucleotides of the tryptophan codon that follows. The re- RNA samples from both rat and porcine small intestine were maining 36 amino acids of the C-terminal peptide and the 3' examined by RNase protection assays to determine the untranslated region of the mRNA are encoded by the final number and relative abundance of secretin transcripts gen- exon. erated from the rat and porcine genes. Probes specific for the The transcriptional initiation site was determined by prim- rat and porcine secretin genes were utilized to determine the er-extension analysis using a 24-base synthetic oligonucleo- presence of alternately spliced transcripts. Analysis of RNA tide probe (Fig. 2). The major extension product was 113 from rat small intestine revealed a major transcript consistent nucleotides long, localizing the transcription initiation site to with the full-length cDNA reported previously (1) (Fig. 3A). 29 bases 3' of the first thymidine of a consensus TATA box. Examination of RNA from porcine duodenum revealed that A second site, 6 nucleotides downstream of the major site, the major protected fragment was -230 nucleotides long, the may represent either an incompletely extended product or an size expected for a full-length transcript. In addition, we alternate transcriptional initiation site. observed a predominant minor protected fragment, 90-100 Analysis of the 5' flanking sequences for potential regula- nucleotides shorter than the major porcine transcript (Fig. tory sequences reveals the consensus sequence for transcrip- 3B). tion factor AP-2 (15, 16) at positions -47, -72, -125, and To determine the sequence of the shorter porcine secretin -189 (Fig. 1). Potential Spl binding sites are found at transcript, the corresponding cDNA was isolated by ampli- positions -67, -121, and -183 (17). Characteristic GT/AG fying the first-strand cDNA template with primers specific for splice donor/acceptor sequences flank all introns in the rat the second and fourth exons. The primers were selected to secretin gene. The nucleotide sequences of the four exons of enable amplification of all known precursor forms of secretin the cloned gene were identical to the cDNA sequence de- (1, 2). The cDNA corresponding to the shorter transcript was scribed previously (1). identified by DNA blot hybridization, subcloned, and se-

<------SECRETIN------GlyLysArgAr gMet ------39aa------> 1. <--CAGCGGCTGCTGCAGGGCCTGGTGGGGAAGCGCAG GATGCCCATGAAGCCCCCAGTGGATCAGGCCTGGTCTCCC--> 111111111111111111111111111111111 11111111111111111 111111111111111111111 2. <--CAGCGGCTGCTGCAGGGCCTGGTGGGGAAGCGCAGC<--9Obp-->TGGATGCCCATGAAGCCCCCAGTGGATCAGGCCTGGTCTCCC--> <------SECRETIN------GlyLysArgSer<--30aa-->TrpMet3------39aa------> _-- cvrvi o -[ Cew q F-f EN,.y~

FIG. 4. Nucleotide sequence alignment of two secretin transcripts present in porcine duodenum. Sequence 1 corresponds to a shorter transcript isolated by cDNA amplification. Sequence 2 corresponds to a previously reported cDNA sequence (1). Identical nucleotide sequences are indicated by vertical lines. Amino acid sequence is indicated above and below the respective nucleotide sequence. aa, Amino acids. The nucleotide sequence identity of the two cDNAs extends 34 and 30 bp from the 5' and 3' ends of the sequence illustrated. Downloaded by guest on September 28, 2021 5338 Biochemistry: Kopin et al. Proc. Natl. Acad. Sci. USA 88 (1991)

A FETAL--1 POSTNATALL third exon of the rat gene. Since the exon/intron boundaries of other polypeptide hormone genes have been well con- DAY 16 17 18 19 20 21 1 3 7 10 14 21 28 35 A served in most mammalian species, these observations sug- gest that the shorter precursor form of porcine secretin, . | t a.: :.:.: *4 lacking the sequences encoded by the third exon of the ii SECRETIN porcine gene, occurs by differential splicing of a single mRNA precursor. Ontogeny of Secretin Gene Expression. To determine whether secretin gene expression is developmentally regu- a ,- *I ipi I* e a CCK lated, we have compared the relative abundance of secretin mRNA in developing intestinal tract of fetal, neonatal, and adult rats by Northern blot hybridization analysis. Secretin

.. .. :11:11 ..Wru.'.As-1 &-i 3ACTIN transcripts were first detectable in duodenum on day 17 of gestation, reached peak abundance 3 days later, and gradu- ally fell -10-fold after birth to adult levels (Fig. 5). To B demonstrate that mRNA abundance reflects the synthesis of 140 peptide, we have shown that secretin immunoreactivity par- 12,- (F20) allels transcript levels during development. The temporal pattern ofsecretin gene expression in developingjejunum and 10.1 TI. -120 * ileum was similar to the duodenum (data not shown). In contrast to secretin, mRNA encoding was -100 5 present at low levels late in gestation and did not increase 0 M significantlys until after birth (Fig. 5A). 0. z w m E DISCUSSION E Structural analysis of the rat secretin gene reveals both differences when compared to the in C)L-u similarities and significant w MU genes encoding glucagon, VIP, GIP, and GHRH (Fig. 6). U, UM Like glucagon and VIP, the entire secretin sequence is encoded by a single exon (18-20). In contrast, the sequences for GIP and GHRH are each encoded on their respective 20 genes by two exons (21, 22). The secretin gene appears to AGE (DAYS) encode only a single copy of its biologically active peptide, whereas glucagon and VIP are duplicated within their re- FIG. 5. Ontogeny of secretin and cholecystokinin in the rat spective genes as glucagon-like peptides 1 and 2 and peptide duodenum. (A) Northern blot analysis of secretin and cholecystoki- histidine-isoleucine. The secretin gene is the only member of nin mRNA in the developing rat. Each lane was loaded with 7.5 ug this family that does not have an intron separating the of RNA and probed sequentially for secretin, cholecystokinin, and transcriptional and translational start sites. ,f-actin. Blots were exposed to Kodak XAR film with two intensi- Alignment of the amino acid sequences of the hormone fying screens at -85TC as follows: 2 days for secretin, 3 days for cholecystokinin, and 12 hr for /3-actin. A, adult; CCK, cholecysto- coding exons for the members of the secretin-glucagon kinin. (B) Ontogeny of secretin immunoreactivity (IR) and mRNA in family demonstrates considerable similarity near the N ter- developing rat duodenum. Secretin immunoreactivity was measured minus ofeach hormone (Fig. 7). The amino acids encoded by in tissue extracts in triplicate and data are reported as mean ± SEM. the remainder of each hormone-coding exon exhibit consid- Secretin mRNA levels were quantitated from the autoradiographs of erable sequence divergence. The exon boundaries relative to Northern blots by scanning densitometry and are shown as relative the biologically active peptides have not been highly con- densitometry units (RDU). An arrow indicates birth. F17 and F20, served. No clear relationship could be found among the days 17 and 20 of fetal gestation, respectively; AD, adult animals. amino acid sequences of the other functional domains of the precursors. Similarly, comparison of the translated nucleo- quenced (Fig. 4). The sequence of the 133-bp amplified tide sequence of the entire rat secretin gene with the exon fragment was identical to the reported porcine cDNA except sequences in the other genes revealed no significant similar- for the exclusion of sequence corresponding to the entire ities beyond those exons encoding the biologically active

...... SECRETIN / ...... I...... 4 / -_` .. I I

m...... - ... GIP //...... -//...... "I... .1 ...... / /...... :.. .. H

GHRH 1:1 ~~~~~~...... : .11:l| Z1 ...

GLUCAGON E}i / E ...= LiZ].// -11/l|t||| /l"'I_ro fl //1

VIP I E...... // "**"'1"1LZI mii go

KKE.

FIG. 6. Structural organization of the genes encoding members of the secretin-glucagon family. Exons encoding similar functional domains are aligned and are shown as boxes drawn to scale for the glucagon (rat), GHRH (rat), VIP (human), and GIP (human) genes. Introns are depicted as broken lines. Untranslated regions are indicated by open boxes. Cross-hatched boxes denote signal peptides. Biologically active peptides are shown as solid boxes. N- and C-terminal peptides are indicated by shaded boxes. Downloaded by guest on September 28, 2021 Biochemistry: Kopin et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5339 SECRE-TIN TPRBSDGTFTSZLSRLQDSARLQRLLQGLV-GKR GiHIRH VR IMAIYRRILCQLYARK ZIUNRQQ9 GIP ALPSLPVGSBAKVSSPQPR e YAF IFY&AMOKI QPDFVNWifAQifRAN VIP SSISZDPVPVKRJV DNYTJRKQMAVKXYJSILN K PHI NA#JAISVTS Y ISARRYi&Sl- I GLUCAC3ON 5sFPASQTIZPLZIDP DQINZ D 3Q:T Y& YLERE DFVERPURTEN GILP1 NNIAKRRDZFZEAZ -TFTS VESYLIGQAAKZFIAWJKE! iRGRR GILP2 FPZIVAIAZhLGRFJAAHSESDEJNTILJELATRDFINWEIQTJITD FIG. 7. Alignment ofthe amino acid sequences encoded by the hormone-coding exons ofthe secretin-glucagon family ofgenes. Amino acids identical to the corresponding exon in the secretin gene (top line) are enclosed in a box. Sequences shown are encoded by the respective rat genes with the exception of GIP (human). PHI, peptide histidine-isoleucine; GLP-1 and GLP-2, glucagon-like peptides 1 and 2, respectively. peptides. A relationship between sequences in the secretin 1. Kopin, A. S., Wheeler, M. B. & Leiter, A. B. (1990) Proc. gene and the introns of the glucagon, VIP, GIP, and GHRH Natl. Acad. Sci. USA 87, 2299-2303. cannot be ruled out until the 2. Gafvelin, G., Jornvall, H. & Mutt, V. (1990) Proc. Natl. Acad. genes complete intervening Sci. USA 87, 6781-6785. sequences for the latter genes are available. However, the 3. Bell, G. I. (1986) Peptides 7, 27-36. likelihood of extensive sequence conservation within introns 4. Meyer, J. H., Way, L. W. & Grossman, M. I. (1970) Am. J. is small. Our findings suggest that the secretin-glucagon gene Physiol. 219, 964-970. family has diverged extensively since the duplication of a 5. Meyer, J. H., Way, L. W. & Grossman, M. I. (1970) Am. J. common ancestral gene. Physiol. 219, 971-977. 6. Watanabe, S., Chey, W. Y., Lee, K. Y. & Chang, T. M. (1986) We have described (1) the sequence of a porcine secretin Gastroenterology 90, 1008-1017. precursor, deduced from the nucleotide sequence ofa cDNA, 7. Sargent, T. D., Wu, J. R., Sala-Trepst, T. M., Wallace, R. G., which contains an unusually long 72-amino acid C-terminal Reyes, A. A. & Bonner, J. (1979) Proc. Natl. Acad. Sci. USA peptide. A shorter precursor has recently been isolated from 76, 3256-3276. porcine intestinal extracts in which 32 residues of the C-ter- 8. Benton, W. D. & Davis, R. (1977) Science 196, 180-182. 9. Tabor, S. & Richardson, C. (1987) Proc. Natl. Acad. Sci. USA minal peptide were replaced by a single arginine residue (2). 84, 4767-4771. In the present study we confirm the existence of another 10. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids less-abundant transcript of the porcine secretin gene that Res. 12, 387-395. encodes the shorter precursor and does not contain any ofthe 11. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, sequences encoded by the third exon. Thus we dif- 156-159. report 12. Deschenes, R. J., Haun, R. S., Funckes, C. L. & Dixon, J. E. ferential splicing for a member of the secretin-glucagon (1985) J. Biol. Chem. 260, 1280-1286. family of peptides. 13. Zinn, K., DiMaio, D. & Maniatis, T. (1983) Cell 34, 865-872. In the present study, we have shown that the rat secretin 14. Chey, W. Y., Chang, T. M., Park, H. J., Lee, K. Y. & Es- gene is developmentally regulated. Several earlier develop- coffery, R. (1983) Endocrinology 113, 651-656. mental studies in neonatal animals have shown that secretin 15. Imagawa, M., Chiu, R. & Karin, M. (1987) Cell 51, 251-260. 16. Mitchell, P. J., Wang, C. & Tjian, R. (1987) Cell 50, 847-861. immunoreactivity in the intestine initially falls after birth 17. Dynan, W. S. & Tjian, R. (1985) Nature (London) 316, 774- (23-25). Here we report the ontogeny of secretin from the 778. fetal period to adulthood. Secretin gene expression clearly 18. Heinrich, G., Gros, P. & Habener, J. F. (1984) J. Biol. Chem. antedates the production of gastric acid by at least several 259, 14082-14087. days (26). These results suggest that the secretin gene is 19. Tsukada, T., Horovitch, S., Montminy, M., Mandel, G. & developmentally regulated by mechanisms independent of Goodman, R. H. (1985) DNA 4, 293-300. the established stimuli for secretin release in adult animals. 20. Linder, S., Barkhem, T., Norberg, A., Persson, H., Schalling, M., Hokfelt, T. & Magnusson, G. (1987) Proc. Natl. Acad. Sci. The time course of secretin gene expression in developing USA 84, 605-609. animals contrasts with cholecystokinin, another hormone 21. Inagaki, N., Seino, Y., Takeda, J., Yano, H., Yamada, Y., known to regulate pancreatic secretion. Unlike secretin, Bell, G. I., Eddy, R. L., Fukushima, Y., Byers, M. G., Shows, cholecystokinin is expressed at low levels until feeding begins T. B. & Imura, H. (1989) Mol. Endocrinol. 3, 1014-1021. after birth (27). 22. Mayo, K. E., Cerelli, G. M., Rosenfeld, M. G. & Evans, R. M. The function of secretin in developing rats is unknown. (1985) Nature (London) 314, 464-467. Administration of secretin to experimental animals has been 23. Larsson, L. I., Sundler, F., Alumets, J., Hakanson, R., Schaf- falitzky de Muckadell, 0. B. & Fahrenkrug, J. (1977) Cell shown to stimulate pancreatic growth (28). The trophic Tissue Res. 181, 361-368. effects of secretin appear to be additive with the growth- 24. Paquette, T. L., Shulman, D. F., Alpers, D. H. & Jaffe, B. M. promoting effects ofcholecystokinin and (29, 30). The (1982) Am. J. Physiol. 243, G511-G517. expression of secretin in fetal animals may indicate a poten- 25. Ichihara, K., Eng, J. & Yalow, R. S. (1983) Biochem. Biophys. tial role for this hormone in promoting the rapid pancreatic Res. Commun. 112, 891-898. growth that occurs late in gestation (31). At the present time, 26. Takeuchi, K., Peitsch, W. & Johnson, L. R. (1981) Am. J. however, a direct cause and effect relationship between high Physiol. 240, G163-G169. 27. Brand, S. J. & Fuller, P. J. (1988) J. Biol. Chem. 263, 5341- levels of secretin in the fetus and pancreatic growth has yet 5347. to be established. 28. Dembinski, A. B. & Johnson, L. R. (1980) Endocrinology 106, 323-328. We thank Jack Dixon for providing the rat cholecystokinin cDNA 29. Solomon, T. E., Vanier, M. & Morisset, J. (1983) Am. J. probe. This work was supported by National Institutes of Health Physiol. 245, G99-G105. Grants P30 DK39428, from the Center for Gastroenterology Re- 30. Solomon, T. E., Morisset, J., Wood, J. G. & Bussjaeger, L. J. search on Absorptive and Secretory Processes, and DK01934. J.N. (1987) Gastroenterology 92, 429-435. was supported by National Institutes of Health Fellowship F32 DK 31. Han, J. H., Rall, L. & Rutter, W. J. (1986) Proc. Natl. Acad. 08556. Sci. USA 83, 110-114. Downloaded by guest on September 28, 2021