Structure, Chromosomal Localization, and Expression of Mouse reg ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 268, No. 21, Issue of July 25. pp. 15974-15982, 1993 Printed in U.S. A .

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure, Chromosomal Localization, and Expressionof Mouse reg Genes, reg I and reg I1 A NOVELTYPE

OF reg GENE, reg 11, EXISTS INTHEMOUSEGENOME* (Received for publication, July 23, 1992, and in revised form, April 8, 1993)

Michiaki UnnoS, Hideto Yonekura, Kan-ichi Nakagawara, Takuo Watanabe, Hikari Miyashita, Shigeki Moriizumi, and HiroshiOkamotos From the Departmentof Biochemistry, Tohoku University Schoolof Medicine, Sendai 980, Miyagi, Japan

Takako Itoh and Hiroshi Teraoka From Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Osaka, Japan

We previously identified a gene, reg (i.e. regenerat- is expressed in regenerating islets and normal exocrine paning gene), in the screening of a regenerating isletcreas, but not in the normal islets (3,4). The rat reg cDNA derived cDNA library of rat (Terazono, K., Yamamoto, had a single open reading frame that encoded a 165-amino H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, acid protein with a 21-amino acid signal peptide. ImmunocyY., and Okamoto, H. (1988)J. Biol. Chem. 263,2111- tochemical analyses of the rat pancreas showed that the Reg 21 14),and isolated a human reg cDNA and gene (Watanabe, T., Yonekura, H., Terazono, K., Yamamoto, protein was expressed in normal acinar cells and in islets in H., and Okamoto, H. (1990)J.Biol. Chem. 265,7432- the regenerating state to a similar extent as in acinar cells, 7439);the rat and human cDNAs encode 165- and 166- and that the Reg protein was co-localized with insulin in pamino acid proteins, respectively. Until now, it was cell secretarygranules of regeneratingislets (5). We also thought that there is a single locus for Reg protein in isolated the humanreg cDNA (3),which encoded a 166-amino the mammalian genome. In this study, we isolated two acid protein witha 22-amino acidsignal peptide, and the distinct cDNAs and genes, one of which was a mouse human reg gene (6),which spans about3 kbp’ and iscomposed homologue to rat andhuman reg gene, the othera novel of six exons andfive introns. Recently, itwas concluded that type of reg gene. We designated them reg I and reg 11, human Reg protein, pancreatic stone protein(PSP) and panrespectively. The two proteinsencoded by these genes creatic thread protein (PTP) are just different names for a share 76% aminoacidsequenceidentitywitheach single protein existing in several molecular forms but derived other. Both genes span about3 kilobase pairs, and the from a singlereg gene, since the functionalreg gene is a single genomic organization of six exons and five intronsis copy gene and the primary amino acid sequence of PSP and conserved between them. Chromosomal mapping stud-PTP is identical with that of theprotein encodedin the ies indicate that the reg I gene is localized on mouse human chromosome 12, whereas thereg11 gene is localized on human reg gene (6). PSP has beenisolatedfrom pancreatic stone and juice and has been reported to inhibit chromosome 3. By Northern blot analysis, both reg I and regI1 mRNAs are detected inthe normal pancreas formation and precipitation of crystals of CaC03 in vitro, and hyperplastic islets of aurothioglucose-treated suggesting that it has a role in pancreatic lithogenesis (7, 8). mice, but not in the normalislets. It is remarkable that PTP has been found to exhibit pH-dependent globule-fibril transformation (9). More recently, Rouquier et al. (10)rein the gallbladderr e g I is expressed, but reg I1 is not. ported that a complete sequence identity was observed bereg mRNA. It was tween the rat PSP mRNA and the rat therefore concluded that there is a single reg gene for Reg In 1984, we found that administrationof poly(ADP-ribose) protein/PSP/PTP in mammalian tissues. synthetase inhibitors such as nicotinamide to 90% depancrea-Inthepresentstudy, we haveisolated two distinct reg tized rats induces regeneration of pancreatic islets (1, 2). In cDNAs and genes of the mouse, determined their structures screeningtheregenerating islet-derived cDNAlibrary, we and chromosomallocalization, and examined theirexpression came across a novel gene, reg (i.e. regenerating gene), which in various mouse tissues. ~

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* This work was supported in partby a Grant-in-Aid for Scientific Research from the Ministryof Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of pagecharges. Thisarticlemustthereforebe hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. T h e nucleotide sequence(s) reportedin thispaperhas been submitted totheGenBankTMfEMBLDataBankwith accession number(s) Dl4010 and 014011. $ Portions of this work were submitted as partial fulfillment of the degree of Doctor of Medical Science at Tohoku University. § To whom all correspondence should be addressed Dept. of Biochemistry, Tohoku UniversitySchool of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980, Miyagi, Japan. Tel.: 81-22-274-1111(ext. 2211); Fax: 81-22-272-7273.

EXPERIMENTALPROCEDURES

Materials-Male C57BL/6J mice weighing 25-30 g were used for all experiments except induction and isolation of hyperplastic islets. Male NON mice (11), a strain derivedfrom ICR mice, received intraperitoneal injections of aurothioglucose a t a dose of 0.5 g/kg of body weight on the 42nd and 56th day after birth. Six months after ___._______~_____~ __ _ _ ~ The abbreviations used are; kbp, kilobase pair(s); bp, base pair(s); PSP, pancreaticstoneprotein; PTP, pancreaticthreadprotein; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; PIPES, piperazine-N,N’-bis(2-ethanesulfonicacid); FITC, fluorescein 5-isothiocyanate; PI, propidium iodide; DAPI, 4’,6-diamidino-2-phenylindole;PAP, pancreatit.is-associated protein; HIP, hepatocellular carcinoma, intestine and pancreas.

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and 7-deaza-deoxyguanosine triphosphate aspreviously described (17, the aurothioglucose treatment, hyperplastic islets were isolated as 18). described (1$. Determination of Transcription Initiation Sites by Primer ExtenDeoxvcvtidine 5'-[a-3'Pltri~hos~hate(la-32PldCTP) (3000 c i / sion Analyses-Forty-base synthetic oligodeoxyribonucleotides commmol), adenosine 5':[y-32P]trfiphosphate ([Y-~'P]ATP) (>5000 Ci/ mmol), Megaprime'" labeling system andHybond-N'" were purchased plementary to the sequence of 21-25 plus 303-337 of the mouse reg I from Amersham Corp., UK. X ZAP 11, pBluescript, T 3 RNA polym- gene (see Fig. 3C) and complementary to thesequence of 324-363 of erase, T7 RNA polymerase, and Prime-Erase'" push column were the mouse reg I1 gene (see Fig. 3 0 ) were labeled at the 5' end with T4 kinase. 32P-Labeledoligodeoxyrifrom Stratagene, La Jolla,CA. X GEM 11 was from Promega, Madi- [ Y - ~ ~ P ] A T P a n dpolynucleotide bonucleotides (1 X lo5cpm) and 20 pg of RNA from mouse pancreas son,WI.Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, terminaldeoxynucleotidyltransferase,S1 nuclease and reverse were hybridized and extended by reverse transcriptase as described (19) and electrophoresed on6% polyacrylamide, 8.3 M urea gels. transcriptase were from Takara Shuzo, Kyoto, Japan. Sequenase'" Determination of TranscriptionInitiationSites by SI Nuclease was from U. S. Biochemical Corp. Aurothioglucose was from Sigma. Mapping-Eighty-base synthetic oligodeoxyribonucleotides compleNick translationkitandbiotin16-dUTP were fromBoehringer, Mannheim, Germany. FITC-avidin and biotinylated goat anti-avidin mentary to the sequence of -20 to 25 plus 303-337 of the mouse reg I gene (see Fig. 3C) and complementary to thesequence of -20 to 25 antibody were from Vector Laboratories, Burlingame,CA. Oligodeoxyribonucleotides were synthesized witha model 380B automated DNA plus 313-347 of the mouse reg I1 gene (see Fig. 32)) were labeled at the5'end with (Y-~'P]ATPandT4 polynucleotidekinase. :'*Psynthesizer (Applied Biosystems). Labeled oligodeoxyribonucleotides (1 X lo5cpm) and 20 pg of RNA Complementary DNA Cloning-Mouse reg I and reg I1 cDNAs were amplified using the RACE protocol (anchoring PCR) (13).A (dT)17- from mouse pancreas were hybridized for 16 h a t 60 "C in10 p1 of 0.4 M NaC1, 5.5 mM PIPES (pH 6.4), and 1 mM EDTA. Hybrids were adaptor primer (5'-GACTCGAGTCGACATCGATTTTTTTTTTT digested with 100 units of S1 nuclease and electrophoresed on 6% T T T T T T - 3 ' ) was annealed to 1 pg of mouse total RNA from pancreasandextendedwith reverse transcriptase,and was then polyacrylamide, 8.3 M urea gels. Chromosomal Mapping of Mouse reg I and reg I I Genes by Fluoresamplified successively with rat reg cDNA sense primer, RAT4 (5'GGCCAGGAGGCTGAAGAAGA-3'; nucleotides 61-80, see Ref. 3), cence in Situ Hybridization-The fluorescence in situ hybridization plus an adaptor primer lacking the poly(dT) sequence (5'- procedure was carried outaccording to the methodof Trask (20) with GACTCGAGTCGACATCG-3'). 35 cycles of amplification in 50 mM some modifications as follows. Mouse reg I and reg I1 cDNA clones KCl, 2.5 mM MgC12,10 mM Tris, pH 8.3,0.1%gelatin were performed were labeled with biotin 16-dUTP using a nick translation kit. Metusing a step cycle file in a Perkin-Elmer Cetus Instruments thermal aphase chromosomes prepared from bone marrow cells of C57BL/6J cycler with denaturation a t 94 "C for 30 s, annealing a t 55 "C for 1 mice were hybridized with the DNA probe for 16 h in asolution min,andextensionat 72 "C for2 min.Reactionproducts were containing 2 X SSC, 50% formamide, 1 X Denhardt's solution, 10% digested with restriction enzymes whose sites were contained within dextran sulfate, and5-25 pg/ml sonicated and denaturedmouse liver primers, isolated through a gel of 1% low melting temperature agarose, DNA. Slides were washed once in 2 X SSC, 50% formamide, once in and subcloned into pBluescript for further analysis. For isolation of 2 X SSC without formamide,once in 1 X SSC, and once in 0.1 X cDNAs containing the 5' endof mouse reg I and reg 11, the 5'-RACE SSC. All the washes were carried out a t 37 "C for 30 min.Slides procedure (13) was followed. An antisense primer complementary to hybridized to the biotinylated probe were treated a t room temperature as follows: 1)washed in 4 x SSC for 5 min, 2) preblocked in 4 x SSC, both reg I and reg I1 cDNAs (nucleotides 1446-1464 and nucleotides 1449-1467, respectively; see Fig. 3, C and D)was annealed to 1 pg of 0.1% bovine serumalbumin for5 min,3)incubatedin 2.5 pg/ml mouse pancreaticRNAandextendedwith reverse transcriptase. FITC-avidinin 4 X SSC, 0.1%bovine serumalbumin for 1 h,4) Excess primer was removed using a Prime-Erase'" push column. The washed in 4 X SSC, 4 X SSC with 0.1% Triton X-100, 4 X SSC, and cDNAs were tailed using dATP and terminal deoxynucleotidyltrans- P N (0.1 M phosphate buffer at pH 8.0 containing 0.1% Nonidet P40) for 5 min, 5) preblocked in PNM (PN containing 5% nonfat dry ferase and amplified successively with a nested antisense primer, MRlPEX3A, 5'-TCTGCATCAGCCCAAGTTAAACGG-3', comple- milk and 0.01% sodium azide, spun for 15 min a t 200 X g to remove mentaryto reg I cDNA (nucleotides 1025-1048, see Fig. 3C), or milk solids) for 5 min, 6) incubated in5 pg/ml biotinylated goat antiMR2PEX3A, 5'-GTCTTTTTCAGCCAAGGGGAAGTC-3', comple- avidin antibody in PNM for 1 h, 7) washed three times in P N for 10 mentary toreg I1 cDNA (nucleotides 999-1022, see Fig. 3 0 ) , a (dT),,min each, 8) preblocked in PNM for 5 min, 9) incubated in 2.5 pg/ adaptor primer, plus adaptor primer under the conditions as described ml FITC-avidin in PNM for 1 h, 10) stained with actinomycin D for above. DNA was digested, subcloned, andsequenced. 3 h, and 11)washed three times in P N for 10 min each. An antifade Genomic Southern Blot Analyses of Mouse reg I and reg IIsolution containing 0.1 pg/ml DAPI and P I was applied to all slides Genomic DNA was extracted from the liver of male C57BL/6J mice before viewing. as previously described (14). Twentymicrogram of genomic DNA was Northern Blot Analyses of RNAs from Various Mouse Tissuesdigested with nine restriction enzymes as depicted in Fig. 2, electro- RNA was extractedfromhyperplasticislets of aurothioglucosephoresed on 0.8% agarose gel, and transferred to nylon filters (15). treated mice and various tissues of normal mice by the methods of The filters were hybridized as previouslydescribed (16) with 32P- Chirgwin et al. (21) using cesium trifluoacetate. RNAs were electrolabeled mouse reg I or reg I1 cDNA as probe, washed to a stringency phoresed and transferred tonylon filters. The filterswere prehybridof 2 X SSPE and 0.1% SDS at 65 "C, and autoradiographed. After ized for 4h a t 50 'C in 50% (v/v) formamide,5 X SSPE, 5 X exposure to the first probe, the filter was boiled in 0.1 X SSPE and Denhardt's solution, 200 pg/ml herring sperm DNA, and 0.5% SDS, 0.1% SDS prior to hybridization with the second probe. and then hybridized for 16 h a t 50 "C in the same buffer using "PIsolation of Mouse reg I Gene-Mouse DNA wasdigested with labeled mouse reg I cDNA or reg I1 cDNA as a probe. The filters were HincII, ligated with EcoRI adaptor (5'-AATTCGGCACGAG-3' and washed to a stringency of 0.1 X SSPE and 0.1% SDS a t 65 "C for 30 5'-CTCGTGCCG-3')andphosphorylatedwithT4 polynucleotide min and autoradiographed. The cDNA fragments of reg I and reg I1 kinase. This productwas fractionated by agarose gel electrophoresis. were subcloned into pBluescript,which carries bacteriophage T3 and DNA was recovered from a gel piece containing 4-8-kbp fragments T7 promoters, and were then transcribed into the sense orientation and ligated with X ZAP I1 arms. Ligated DNA was packaged in uitro by T 3 or T7 RNA polymerase. The transcripts (synthetic reg I RNA and plated on a lawn of Escherichia coli XL1-Blue. The library was and reg I1 RNA) were used for positive and negative control. The screened using 32P-labeled mousereg I cDNA as probe. Recombinant filters were re-hybridized a t 42 "C in the same buffer as described phage DNAs in the hybridization-positive clones were excised and above with 32P-labeledmouse pancreatic a-amylase (nucleotides 511recircularized in uiuo, and the resulting Bluescript plasmids were 906,seeRef.22) orelastase I1 (nucleotides 163-519, see Ref. 23) isolated. cDNA, which was obtained by PCR, and washed to a stringency of Isolation of Mouse reg I I Gene-Mouse DNA was digested with 0.1 X SSPE and 0.1% SDS at65 "C. BamHI and fractionated by agarose gel electrophoresis. DNA was Quantification of Mouse reg I and reg II mRNA by Slot Blot recovered from agel piece containing 9-20-kbp fragments andligated Analysis-The synthetic reg I RNA and reg I1 RNA described above with X GEM-11 arms. Ligated DNA was packaged in uitro and plated were used for the quantitative standard. The concentration of the on a lawn of E. coli KW231. The library was screened using 32P- synthetic RNAs was determined by measuring the absorption at 260 labeled mouse reg I1 cDNA as probe. From the positively hybridized nmafterdenaturation at 65 "Cand calculatingfrom the molar clones,DNA inserts were isolatedand subcloned intopBSand extinction coefficient and base composition of the RNAs. The integpBluescript vectors. rity of the synthetic RNA preparationwas evaluated by electrophoNucleotide Sequencing-Thenucleotidesequences were deter- resis through denaturing agarose gel. These RNAs were denatured mined by the dideoxy chain termination method using Sequenase'" and spotted in sequential dilutions onto nitrocellulose filters (800, ~~

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400,200,100,50,25, 12.5, and 6.25 pg for each slot). Total RNA from reg cDNA and a genome clone in order toproduce transgenic hyperplastic islets (200, 100, and 50 ng), normal islets (1000,500, andmice. T o isolate a mouse reg cDNA clone, we used the 3‘ 250 ng) and normal pancreas (200,100, and 50 ng) were also spotted onto the filters. The filters were hybridized and washed as described RACE method with mouse pancreatic RNA and an oligonuabove. The radioactivity was measured using a bioimage analyzer, cleotide primer, RAT4,based on the ratreg cDNA sequence. BAS 2000 (Fuji Photo Co., Ltd., Tokyo, Japan) and the amount of Unexpectedly, two DNA fragments were amplified by PCR. reg I or reg I1 mRNA/pg RNA was obtained by least-square regression We then obtained the 5‘ portion of the full-length cDNAs by analysis of the results. the 5’ RACE method usingoligonucleotide primers, MRlP3A

and MR2P3A, respectively, and determined their nucleotide sequences (Fig. l),which corresponded to thecoding regions Complementary DNA Cloning and Amino Acid Sequences of of the genes described later (see Fig. 3). In comparing the Reg Proteins-Our original objective was to isolate a mouse nucleotide sequences, 76% identity between the two distinct reg homologues was found. We designated thetwo mouse reg MR1P3A homologues reg I and reg 11. reg I cDNA is 753 bp in length adaptor and has one open reading frame encodes that 165 aminoacids, b 4 RAT 4 adaptor and reg I1 cDNA is692 bp in length and has one open reading D frame that encodes 173 amino acids. The ATG start codon in P E I I each cDNA is assigned according to the similarity to the reg I 4 eukaryotic consensus start sequence as described by Kozak (24). TheNH2-terminal 21- and 22-aminoacidsequences (Reg I and Reg 11, respectively) are hydrophobic and likely represent a signal peptide (25). The predicted mature Reg I and Reg I1 proteins have molecular weights of 16,240 and 16,855, respectively. A comparison of theamino acid sequences showed that there was 76% identity between Reg I and Reg I1 proteinsandthat all7cysteineresidues are conserved in Reg I and Reg I1 proteins, suggesting that the proteins may have similar overall conformations. Reg I1 protein has a unique sequence of 7 amino acids in the aminoterminal region (amino acid residues 29-35, see Fig. 8) and 4 @ RAT4 its signal peptide is 1 amino acid longer than that of Reg I adaptor (see Fig. 3, panels C and D, and Fig. 8). The amino acid 0 4 S sequence of the rat Reg protein (3) is more similar to thatof reg 11 mouse Reg I (87% identical) than that of Reg I1 protein (73% identical). c Genomic Southern Blot Analysis-To ascertain whetherreg t c I and reg I1 mRNA areencoded by twodistinct genes, genomic DNA frommouseliver was digested with BamHI, HincII, EcoRI, PstI, SpeI, XbaI, BglII, Sad, and DraI and analyzed u by Southern blot hybridization using mouse reg I cDNA or 1OObp reg I1 cDNA as a probe (Fig. 2). The blot hybridized to reg I FIG. 1. PCR and sequencing strategies for mouse reg I and cDNA was clearlydifferent from that toreg I1 cDNA. BamHI, reg I1 cDNAs. The heavy lines indicate the open reading frames. HincII, XbaI, and SacI generatedsingle restriction fragments Arrowheads indicate the primers for 5’-RACE, or 3’-RACE as described under “Experimental Procedures.”Arrows indicate the direc- that hybridized to reg I cDNA, while BamHI, EcoRI, SpeI, tion and extent of sequence determination. Restriction enzymes: P, XbaI, and BglII generated single restriction fragments that hybridized to reg I1 cDNA. The sizes of the restriction fragPstI; E, EcoRI; K , KpnI; S,SacI. RESULTS

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A 1 2 3 4 15 26 37 48 59 6 7 8 9 FIG. 2. Southern blot analysis of mouse reg I and reg I1 genes. Twenty

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microgram of genomic DNA mouse from liver was digested with various restrictionenzymesandelectrophoresedon 0.8% agarose gel: Lane I , BamHI; lane 2, HincII; lane 3, EcoRI; lane 4, PstI; lane Fi, SpeI; lane 6, XbaI; lane 7, BglII; lane 8, S a d ; lane 9, DraI. A, hybridization with radiolabeled reg I cDNA; B, hybridization with radiolabeled reg I1 cDNA. Positions of size markers are presented on the left.

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FIG. 3. Restriction maps of reg I gene (A) and reg I1 gene ( B ) and nucleotide sequences of reg I gene (C)and reg I1 gene ( D ) .A and B , restriction sites are shown at the top. The six exons are indicated as boxes; filled boxes and open bones indicate protein coding regions and untranslated regions, respectively. Arrows indicate the direction and extent of sequence determination. Restriction enzymes: H , HindIII; B , BamH1; X , XbaI; Hc, HincII; P, PstI; Bg, BglII; C, ClaI; K , KpnI; E, EcoRI; S, SacI. C and D,capital letters indicate exons and lowercase letters are used for introns and 5'- and 3"flanking sequences. Numbering of nucleotides begins at thetranscription initiation site; negative numbers indicate 5"flanking sequence. The Goldberg-Hogness promoter sequence (TATAAA) is underlined, as are the polyadenylor its ation recognition signals (AATAAA). Sequences similar to the consensus sequence (5'-G(T/A)CACCTGT(G/C)CTTTTCCCTG-3' complementary sequence) of pancreatic exocrine enhancer (27) are indicated in the 5"flanking region by underlined with a dashed line.

ments detected with the reg I or reg I1 probe correspond to the restriction map of reg I or reg I1 (Fig. 3). Isolation and Nucleotide Sequence Determination of Mouse reg I and reg I I Genes-To isolate the mouse reg I and reg 11 genes, genomic DNA libraries were constructed and screened with mouse reg I or reg I1 cDNA as a probe. The positively hybridized clones were plaque-purified. The reg I gene was isolated in a fragment of 6.5 kbp from the HincII-digested library, and the reg I1 gene was isolated in a fragment of 12 kbp from the BamHI-digested library. We determined the complete nucleotide sequences of the genes including 5'- and 3"flanking regions on both strands (Fig. 3, A and B ) . The genes spanned about 3 kbp inlength. Comparison of the

genomic sequences with the cDNA sequences revealed that the coding regions of both reg I and reg I1 were divided into six exons separated by five introns and that all exon-intron junctions conformed to the GT/AG rule (26). In both reg I and reg I1 genes, exon 1 encodes the 5"untranslated region; exon 2 encodes the remainder of the 5"untranslated region andthe putative signal sequences; exons 3-6 encode the remainder of Reg I or Reg I1 protein. The 3"untranslated regions and a consensus polyadenylation signal (AATAAA) are located in exon 6. The reg 11 gene has a 21-bp insertion that encoded 7 unique amino acids in exon 3. A comparison of the nucleotide and deduced amino acid sequences between the corresponding regions of mouse reg I

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FIG. 4. Localization of the transcription initiation site of mouse reg I gene and reg I1 gene by primer extension analysis (A and B ) and by S1 nuclease mapping (C and D ) . A and E , RNA from mouse pancreas was hybridized to 5'-end-labeled 40-base oligodeoxyribonucleotidesas described under "Experimental Procedures." The reverse product of reg I (&ne1 ) or reg I1 (&ne2)was analyzed by electrophoresis on 6% acrylamide-urea gel. The major extended band is indicated by an arrowhead. Lanes A, C, G,and T display size markers of DNA which were obtained from the dideoxy sequencing using the sameprimers and same sequence of mouse reg I or reg I1 gene. C and D,RNA from mouse pancreas was hybridized to 5'-end-labeled 80-base oligodeoxyribonucleotides as described under "Experimental Procedures." The hybrid with reg I probe ( l a n e 1 ) or reg I1 probe ( l a n e 2) was digested with S1 nuclease, and protected fragments were analyzed by electrophoresis on 6% acrylamide-urea gel. The protected fragment corresponding to the major extended band of primer extensionanalysis (Fig.4, A or B ) is indicated by an arrowhead. Lanes A, C, G,and T display size markers of DNA, which were obtained from the dideoxy sequencing using the same sequence of mouse reg I or reg I1 gene and 92P-labeled primer the 5'-end of which corresponded to the 5'-end of the S1 probe. The nucleotide sequence of the region encompassing the transcription initiation site was marked by asterisk.

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FIG.5. Chromosomal mapping of mouse reg I ( A and B ) and reg I1 (C and D ) genes by fluorescence in situ hybridization and distributionof the signals. A , PI staining. B, DAPI staining.Arrows indicate that thesymmetrical twin fluorescent spots are localized near the middle of chromosome 12. C, PI staining. D,DAPI staining.Arrows indicate that thefluorescent spots are localized near the middle of chromosome 3. E, histogram of the signals, which are hybridized to reg I probe, located on mice chromosomes. F, histogram of the signals, which hybridized to reg I1 probe, located on mice chromosomes.

reg/PSP Gene Family

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and reg I1 reveals a high degree of homology in the coding regions but no significant homology in the intervening sequences. Exon 1 of reg I and reg I1 genes are 25 bp in length and display no significant homology. Exon 2 of the reg I and reg I1 genes are 107 and 110 bp in length, respectively, 18s- ’ displaying 72% homology. Exon 3 of reg I1 has a 21-bp insertion, and the homology to that of reg I is 79%, not counting the 21-bp insertion. Exons 4 and 5 of reg I, which are 138 and 112 bp long, respectively, are exactly the samein length as reg I1 exons 4 and 5, displaying 88 and 83% homology. Exon 6 of reg I and reg I1 are very different in length; Exon 6 of reg I and reg I1 are 268 and 167 bp long, respectively. The reg I introns 1, 2, 3, 4, and 5 are 277, 521, 293, 651, and 173 bp long, whereas the reg I1 introns 1, 2, 3, 4, and 5 are B 287,559, 224, 991, and 190 bp long, respectively. 1 2435 6 .“7 8 9 . Transcription Initiation Sites and 5”Upstream Regions of , t --Mouse reg I and reg II Genes-The transcription initiation 28s sites of mouse reg I and reg I1 genes were determined by S1 nucleasemappings.One primer extension analyses and 18s . major band and some minor bands were extended by reverse transcriptase (Fig. 4, A and B ) . The major extended band of reg I corresponded to the majorprotected band of the S1 4s nuclease mapping of reg I (Fig. 4C). The major extended band of reg I1 corresponded to oneof the protected bands of the S1 nuclease mapping of reg I1 (Fig. 4 0 ) . From these results we concluded that the adenineresidues labeled I in Fig. 3 (C and 1 2 3 C D )were the major transcription initiation sites of reg I and reg I1 genes, respectively. TATA box sequences were found at the same position (-30 to -25). There are no typical CAAT 28 S boxes in the 5”upstream region of either gene. 18s Chromosomal Mapping of Mouse reg I and reg II Genes by Fluorescence in Situ Hybridization-We have examined 100 metaphase chromosomes showing typical Q bands for both reg I and reg I1 probes. When mouse reg I cDNA or reg I1 4scDNA labeled withbiotin was hybridized with the mouse chromosomes in situ, major signals of the reg I gene were recognized on chromosome 12 and the fluorescent spotswere 1 2 3 localized near themiddle of chromosome 12 (Fig. 5, A and B ) . D In contrast,those of the reg I1 gene were recognized on 3 and the fluorescent spots were localized near chromosome 28 S the middle of chromosome 3 (Fig. 5, C and D).The same 18Sresults were obtained using themouse reg I or reg I1 genomic As shown in Fig. 5E, the distribution clones (data not shown). of the signals hybridized with the reg I probe was as follows; 10 (10%)of these chromosomes exhibited complete symmet4srical twin spots on chromosome 12, and 22 (22%)had single spotsononechromatid.Theremainingsignals appeared randomly throughout the rest of the genome. As shown in E Fig. 5F, the distributionof the signalshybridized with the reg 1 2 3 I1 probe was as follows; 2 (2%)of these chromosomes exhib28 S ited complete symmetrical twin spots onchromosome 3, and 15 (15%)had single spots on one chromatid. Theremaining 18s signals appeared randomly throughout the of rest the genome. Northern Blot Analysesof RNAs from Hyperplastic Islets of FIG. 6. Northern blot analysis of RNAs isolated from nor- Aurothioglucose-treated Mice and Normal Mouse Tisswsmal islets, hyperplastic islets, and pancreas using mouse reg We examinedreg I and reg I1 expression in hyperplasticislets, I or reg I1 cDNA. Lane 1 , RNA from islets from untreated ICR normal islets, and pancreas by Northern blot analysis using mice ( 2 pg); lune 2, RNA from hyperplastic islets of aurothioglucosetreated NON mice ( 2 pg); lune 3, RNA from mouse pancreas (2 pg). mouse reg I or reg I1 cDNA as a probe (Fig. 6, A and B ) . We Lane 4-6, mouse reg 1 RNA (0.1, 1.0, and 10.0 ng/lane, respectively) confirmed that cross-hybridization between reg I and reg I1 prepared as described under “Experimental Procedures.” Lane 7-9, did not occur under thehybridization condition of high strinmouse reg I1 RNA (0.1, 1.0, and 10.0 ng/lane, respectively) prepared gency (Fig. 6, A and B, lanes 4-9). Both reg I and reg I1 as described under “Experimental Procedures.”A, hybridization with mRNAs of 0.9 kilobases were detected in the hyperplastic mouse reg 1 cDNA; B, hybridization with mouse reg I1 cDNA. C, islets and normal pancreas but not in normal islets. The same hybridization with mouse n-amylase cDNA. D, hybridization with mouse elastase I1 cDNA.Positions of 28, 18, and 4 S RNAs are results were obtained using antisenseoligonucleotide specific presented on the left. E , 28 and 18 S ribosomal RNAs stained by 33 to mouse reg I or reg I1 as a probe (data not shown). There pg/ml acridine orange are indicated below as an internal control. was no expressionof a-amylase nor elastaseI1 in normalislets

A

1 2435 6 7 8 9

--

-

-

s

15980

reg/PSP Gene Family

A I

2 3 84765

910111213

15 14

28S-

4

18 S-

FIG. 7. Northern blot analysis of RNAs from various mouse tissues using mouse reg I or reg I1 cDNA. Lune 1, pancreas (0.5pg of RNA/lane); lane 2, liver; lane 3, kidney; lane 4, brain; lane 5, thyroid gland; lane 6, stomach; lane 7,lung; lane 8, heart; lane 9,testis; lane IO, spleen; lane 11, rectum; lane 12, gallbladder; lane 13, adrenal gland, (lane 2-13, 5 pg of RNA/lane). Lane 14 and 15, mouse reg I RNA (0.1 and 1.0 ng/ lane, respectively) prepared as described under “Experimental Procedures.” Lane 15 and 16, mouse reg I1 RNA (0.1 and 1.0 ng/lane, respectively) prepared as described under “Experimental Procedures.” A, hybridization with mouse reg I cDNA, B, hybridization withmouse reg I1 cDNA. Positions of 28, 18, and 4 S RNAs are presented on the left. C, 28 and 18 S ribosomal RNAs stained by 33 pg/ml acridine orange are indicated below as an internal control.

4

1)

s-

B

(I)

--

10111213 3984765

21

1617

1415 1617 7

28S18 S-

4

s-

C 1

and hyperplastic islets (Fig. 6, C and D), indicating that our preparation of islets had little contamination of pancreatic exocrine cells. We also examined the expression of mouse reg I and reg I1 in normal mouse tissues using mouse reg I or reg I1 cDNA as a probe. As shown in Fig. 7, reg I is expressed strongly in pancreas, moderately in gallbladder, and weakly in liver. reg I1 is expressed strongly in pancreas, weakly in liver, but not a t all in gallbladder. Neither reg I nor reg I1 was detected inkidney, brain, thyroid grand, stomach, lung, heart, testis, spleen,colon, or adrenal glandby Northern blot analysis. Byslot blot analysis (see “Experimental Procedures”), amounts of reg I mRNA andreg I1 mRNA in the hyperplastic islets were estimated as 1.57 and 1.17 ng/pg RNA, respectively. By the same analysis, the amounts of reg I mRNA and reg I1 mRNA in normal isletswere determined to beless than 0.06 ng/pg RNA. reg I mRNA and reg I1 mRNA in normal pancreas were 1.30 and 0.41 ng/pg RNA, respectively.

2 3 4 5 6 7 8 910111213

six exons andfive introns (Fig. 3). This genomic organization is conserved among the two mouse reg genes, the human reg gene (7) and the rat reg gene.’ Mouse reg I gene and rat reg gene are more closely related than are mouse reg I gene and reg I1 gene, suggesting that the ancestral gene for reg may have been duplicated prior to divergence of mouse and rat. The 5”flanking sequence of mouse reg I gene has anoverall homology of 56% when aligned with the corresponding region of mouse reg I1 gene. The TATA box sequence is present 30 bp upstream from the transcription initiation site of both the genes, but there are no typical CAAT box sequences in either gene. As shown in Fig. 3 (Cand D),sequences similar to the motif of 5’-G(T/A)CACCTGT(G/C)CTTTTCCCTG-3’ or its complementary sequence (which was proposed as a pancreatic exocrine consensussequence by Bouletet al. (27)) are present a t a similar distance upstream from the transcription initiation sites in thetwo genes. This sequence contains 5’-CA(C/ G)CTG(T/C)-3‘ AP4 binding motif (28), which appears in DISCUSSION the rat insulinI “Nir box” (29), and thismotif is reported to We have found two related yet distinctgenes, reg I and reg be bound by several trans-acting factors: Pan-1, Pan-2 (30), 11, that encode Reg proteins differing in amino acid sequence. and AP-4 (31). These sequences in the reg genes may play a Based on the results of Southern blot analysis (Fig. 2), reg I role in specific transcription in pancreaticcells, although the and reg I1 mRNA are unequivocally encoded by two separate expression in other tissues, such as in gallbladder, might be genes, which are present ina single copy in the haploidset of regulated by other sequences of 5”flanking regions. the mouse genome. In addition, as determinedby fluorescence As shown in Fig. 8, mouse Reg I protein exhibits higher in situ hybridization (Fig. 5), these genes are locatedon homology to rat and humanReg protein and is thought to be different chromosomes; reg I gene is located on chromosome a mouse homologue to rat and human Reg protein. A novel 12, and reg I1 gene is located on chromosome 3. Both reg I * Y. Suzuki, H. Yonekura, and H. Okamoto, unpublished results. and reg I1 genes span about 3 kbp, and they arecomposed of

reg/PSP Gene Family Type 11

Type I

-

mouse reg 11 mouse reg I rat reg humanreg/PSP/PTP rat PAP

1 :

1 1 1 1 1 1 1

: : : : : : :

mouse reg I1 mouse reg I rat reg human reg/PSP/PTP rat PAP rat peptide 23 human HIP bovine PTP "Drickamer"

47 39 39 40 44 18 44 44

: :

mouse reg 1 1 mouse reg I rat reg human reg/PSP/PTP rat PAP human HIP bovine PTP "Drickamer"

97 89 89 90 94 94 94

:

mouse reg I1 mouse reg I rat reg human reg/PSP/PTP rat PAP human HIP bovine PTP "Drickamer"

142 134 134 135 144 144 144

: : : : : : :

bovine PTP 'Drickamer"

15981

: :

: :

: :

: : : : : :

FIG. 8. Alignment of amino acid sequences of the Reg/PSP family: mouse Reg I1 protein, mouse Reg I protein, rat Reg protein (3), human Reg protein/PSP/PTP (6), rat PAP (32), peptide 23 (33, 34), human HIP (35), and bovine PTP (36). Sequence gaps resulting from optimization of alignment are indicated by dashes. Residues identical to those of mouse Reg I protein are indicated by dots. The designation "Drickamer" indicates the amino acid residues found by Drickamer (37, 38) to he almost invariant in members of calcium-dependent lectin. Residues are indicated by single-letter amino acid code. X indicates undetermined residue.

type of Reg protein, Reg 11, has a unique sequence of 7 amino family." Fig. 8 shows that Reg and Reg-related proteinshave acids in the amino-terminal region (amino acid residues 29a high degree of homology with a consensus motif reported 35). During the courseof this study, several reg-related genes by Drickamer and colleagues (37, 38) to be almost invariant and proteins named pancreatitis-associated protein (PAP) inmembers of calcium-dependent(C-type)animal lectin. (32), peptide 23 (33, 34), and HIP ( 3 5 ) , were reported. PAP Lasserre et al. (35) have suggested that Reg and Reg-related is an additional proteinwhich appears in rat pancreaticjuice proteins belong to a fifth class of C-type animal lectin,more after the induction of pancreatic inflammation, and the cDNAclosely related to the invertebrate lectins than to other types encodes 175 amino acid residues (32). Peptide 23 has been of C-type animal lectins. Some C-type animal lectins such as reported to be released from rat anterior pituitary cells (33), Sarcophagalectin play animportant role inanautocrine and its expression has also been found in pancreatic islets manner in a specific stage of development and differentiation (34).3 Since the determined partial amino acid sequence of of the imaginaldiscs (39). In addition, plant and some animal peptide 23 is identical to residues 27 to 50 of PAP (see Fig. lectins possess the mitogenic activity on lymphocytes (40), 8), PAP and peptide23 may be the same protein. HIP cDNA and a growth factor from an avian sarcoma virus transformed was isolated from a human hepatocellular carcinoma cDNA rat NRK cell line has been reported to be identical to a 14library by differential screening. HIP mRNA is expressed at kDa P-galactoside-binding protein (41). a high level not only in some hepatocellular carcinomas but We have reported thataurothioglucose induces the islets to also in the normal pancreas and small intestine (35). The become hyperplastic in NON mice, and immunocytochemical amino acid sequences of rat PAP and human HIP protein examination has shown that the hyperplastic islets consist have significant homology to those of Reg proteins (ie. 44 predominantly of P-cells (3). By Northernblot analyses using and 47% identical to ratReg, respectively) and are even more mouse reg I or reg I1 cDNA as a probe, both reg I and reg I1 similar to that of the protein isolated as bovine PTP (36). genes are expressed in hyperplastic islets but not in normal Rat PAP, human HIP, and bovine PTP have a unique 5islets (Fig. 6). Miyaura et al. (42) noted the possibility that amino acid insertion in the same position of the carboxyl the expression of reg genes in regenerating islets is due to a terminus (amino acid residues 110-114) and display a high degree of homology with each other (65-70%). It is therefore contamination of exocrine cells. However,in thepresent study, this appears unlikely because a-amylase and elastase apparent that mouse Reg I1 protein is distinguishable from rat PAP/peptide 23, human HIP, and bovine PTP. As sum- I1 are not expressed in hyperplastic islets, whereas reg I and marized in Fig. 8, therefore, it appears that there areat least reg I1 are expressed (Fig. 6). In contrast, human reg mRNA three types of Reg and Reg-related proteins in mammalians, is expressed in colon and rectal cancers and may be related to tumorigenesis (6). In addition, HIP mRNA has also been e.g. (i) type I: mouse Reg I, rat Reg, and human Reg/PSP/ ( 3 5 ) . These PTP; (ii) type 11: mouse Reg 11; and (iii) type 111: rat PAP/ expressed in human hepatocellular carcinomas peptide 23, human HIP, and bovine PTP, and that the pro- results suggest that expression of members of the reg/PSP teins are derived from a multigene family, "the reg/PSP gene gene family are important in cell proliferation and/or differentiation, whereas thepossibility that reg/PSPgene would be Mouse reg I and reg I1 mRNAs were not detected in the pituitary activated during dedifferentiation,which may occur in regengland (M. Unno and H. Okamoto, unpublished results). erating or tumor tissues, assuggested by Rouquier et al. (lo), ~~

reg/PSP Gene Family

15982

cannot be excluded. In normal mouse tissues, both reg I and reg I1 genes arestronglyexpressedinpancreas (Fig. 7), suggesting that their expression in exocrine cells may maintain adequateexocrine pancreatic function (7-9). In the present study, we found that only reg I gene is expressed in the gallbladder. As for the expression of mouse reg I mRNA in the gallbladder, it is possible that the reg I protein may play a role in the inhibitionof CaC03 crystalgrowth, as suggested by De Caro et al. (7,8), in the bile juice. The tissue expression patterns for mouse reg I and reg I1 appear to be different from those of rat reg and human reg. The reg I and reg I1 of mouse aredetectedin liver (Fig. 7), whereasthehuman reg is detected in kidney and gastric mucosa (6). It is possible that these genes are expressed in ductal epithelium (gallbladder, biliary canalicular cells in liver, gastric mucosa, renal collecting duct, etc.) and an increased concentration of calcium or decreased pH might increase the tendency for calcification. New islets appear to generate outof pancreatic ductal tissue (43). We have suggested that the activation of reg may participate in @-cellregeneration (2-5). Recently, Francis et al. (44) pointed out a clear association between reg gene expression and isletcell replication in vitro using isolated islets from rat pancreas. We are attempting to determine whether the Reg protein has a growth-promoting activity for islet @-cells; we are exposing freshly isolated rat islets to recombinant rat Reg protein in an in vitro culture, and are investigating theeffect of Reg protein on islet cell replication (45). More recently,we have obtained evidence for such an e f f e ~ t Further .~ studies usinganimal models suchastransgenic mice seem tobe needed todeterminethe role of Reg proteinin islet cell replication. The physiological reasons for the maintenanceof two functional reg genesencoding proteins of different amino acid sequencein mice arestillunclear.Theexistence of two distinct reg genes in mouse genome may suggest the existence of non-allelic reg genes in rat and human genome. The cloning of other non-allelic reg genes of the rat and human and production of transgenic mice, especially mice carrying a mutant reg gene inactivated by gene targeting, could provide additional clues. Acknowledgments-We are indebted to Hideo Kumagai and Reiko Torigoe for skillful technical assistance and to Brent Bell for reading the manuscript. REFERENCES 1. Yonemura, Y., Takashima, T., Miwa, K., Miyazaki, I., Yamamoto, H., and Okamoto, H. (1984) Diabetes 33,401-404 2. Okamoto, H. (1990) in Molecular Bzology of the Islets of Langerhans (Okamoto, H., ed)pp. 209-231, CambridgeUniversityPress,Cambridge,

United Kingdom

T. Watanabe, Y. Yonemura, and H. Okamoto, unpublished results.

3. Terazono, K., Yamamoto,H.,Takasawa, S., Shiga,K.,Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J . Biol. Chem. 263,2111-2114 4. Terazono, K., Watanabe, T., and Yonemura,Y. (1990) in Molecular Biology of the Islets of Langerhans (Okamoto, H., ed) pp. 301-313, Cambridge

University Press, Cambridge, United Kingdom 5. Terazono, K., Uchiyama, Y., Watanabe, T., Yonekura, H., Yamamoto, H., and Okamoto, H.(1990) Diabetologia 33,250-252 6. Watanabe, T., Yonekura, H., Terazono, K., Yamamoto, H., and Okamoto, H. (1990) J . Biol. Chem. 265,7432-7439 7. De Caro, A. M., Bonicel, J. J., Rouimi, P., De Caro, J. D., Sarles, H., and Rovery, M. (1987) Eur. J . Biochem. 168,201-207 8. De Caro, A. M., Adrich, Z., Fournet, B., Capon, C., Bonicel, J. J., De Caro, J. D., and Rovery, M. (1989) Biochim. Biophys. Acta 994,281-284 9. Gross, J., Carlson, R. I., Brauer, A. W., Margolies, M. N., Warshaw, A. L., and Wands, J. R. (1985) J . Clin. Inuest. 7 6 , 2115-2126 10. Rouquier, S., Verdier, J. M., Iovanna, J., Dagorn, J. C., and Giorgi, D. (1991) J . Biol. Chem. 2 6 6 , 786-791 S. (1983) in Diabetic Microangiopathy 11. Tochino, Y., Kanaya, T., and Makino, (Abe, H., and Hoshi, M., eds) pp, 423-431, University of Tokyo Press,

Tokyo 12. Okamoto, H. (1981) Mol. Cell. Biochem. 37,43-61 13. Frohman. M. A,. Dush. M. K.. and Martin. G. R. (1988) . . Proc. Natl. Acad. Sci. U.'S. A . 8'5, 8998-9002 14. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning: A

LaboratoryManual. 2nd Ed., Cold Surine: HarborLaboratory, Cold Spring Harbor, NY Southern, E. M. (1975) J. Mol. Biol. 9 8 , 503-517 Yonekura, H., Nata, K., Watanabe, T., Kurashina, Y., Yamamoto, H., and Okamoto, H. (1988) J. Biol. Chem. 2 6 3 , 2990-2997 Hattori, M., and Sakaki, Y. (1986) Anal. Biochem. 152,232-238 Mizusawa, S., Nishimura, S., and Seela, F. (1986) Nuclerc Actds Res. 1 4 , ~I

15. 16. 17. 18.

1714-1WA lvly

"ll

19. Takasawa, S., Tohgo, A,, Unno, M., Yonekura, H., and Okamoto, (1992) H. FEES Lett. 307,318-323 20. Trask, B. J. (1992) Methods Cell Biol. 3 5 , 3-35 21. Chirewin. J. M.. Przvbvla, A. E.,MacDonald, R. J., and Rutter, W. J. (1979) biochemistrj 18,'5294-5299 22. Hagenbuchle, O., Bovey, R., and Young, R. A. (1980) Cell 21,179-187 23. Stevenson,B. J., Hagenbuchle, O., andWellauer, P. K. (1986) Nucleic Acids Res. 14,8307-8330 24. Kozak, M. (1987) Nucleic Acids Res. 15,8125-8148 25. Blobel, G., and Dobberstein, B. (1975) J. Cell B i d . 6 7 , 852-862 26. Padgett, R. A,, Grabowski, P. J., Konarska, M. M., Seiler, S., and Sharp, P. A. (1986) Annu. Reu. Biochem. 5 5 , 1119-I150 27. Boulet, A. M., Erwin, C. R., and Rutter, W. J. (1986) Proc. Natl. Acad. Sci. U. S. A . 8 3 , 3599-3603 28. Mermod, N., Wiliams, T. J., and Tjian, R. (1988) Nature 3 3 2 , 557-561 29. Nir, U., Walker, M. D., and Rutter, W. J. (1986) Proc.Natl.Acad.Sci. U. S. A . 8 3 , 3180-3184 30. Nelson, C., Shen, L., Meister, A., Fodor, E., and Rutter, W. J. (1990) Genes & Deu. 4 , 1035-1043 31. Fodor, E., Weinrich, S. L., Meister, A,, Mermod, N., and Rutter, W. J. (1991) Biochemistry 30,8102-8108 32. Iovanna, J., Orelle, B., Keim, V., and Dagorn, J. C. (1991) J. Biol. Chem. 266,24664-24669 33. Tachibana, K., Marquardt, H., Yokoya, S., and Friesen, H. G. (1988) Mol. Endocrinol. 2,973-978 34. Yamamoto, T., Katsumata, N., Tachibana, K., Friesen, H. G., and Nagy, J. I. (1992) J. Histochem. Cytochem. 4 0 , 221-229 35. Lasserre, C., Cbrista, L., Simon, M. T., Vernier, P., and Brechot, C. (1992) Cancer Res. 52,5089-5095 36. de la Monte, S. M.. Ozturk, M.. and Wands, J. R. (1990) J . Clin. Inuest. 86,1004-ioi3 37. Drickamer, K., and McCreary, V. (1987) J . Biol. Chem. 262,2582-2589 38. Drickamer, K. (1988) J. Biol. Chem. 263,9557-9560 39. Kawaguchi, N., Komano, H., and Natori, S. (1991) Deu. Biol. 144,86-93 40. Sharon, N., and Lis, H. (1989) Lectins, Chapman and Hall,Inc., New York 41. Yamaoka, K., Ohno, S., Kawasaki,H.,andSuzuki, K. (1991) Biochem. Biophys. Res. Commun. 1 7 9 , 272-279 42. Miyaura, C., Chen, L., Appel, M., Alam, T., Inman, L., Hughes, S. D., Milburn, J. L., Unger, R. H., and Newgard, C. B. (1991) Mol. Endocrinol. 5 , 226-234 43. Rosenberg, L., Brown, R. A,, and Duguid, W. P. (1983) J . Surg. Res. 3 5 , 63-72

J., Southgate, J. L., Wilkin, T. J., andBone, A. J. (1992) Diabetologia 3 5 , 238-242 45. Unno, M., Itoh, T., Watanabe, T., Miyashita, H., Moriizumi,S . , Teraoka, H., Yonekura,H., and Okamoto,H. (1992) in Pancreatic Islet Cell Regeneration and Growth (Vinik, A. I., ed) pp. 61-69, Plenum Press, New York

44. Francis,P.