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The Uroguanylin Gene (Guca1b) Is Linked to Guanylin (Guca2) on Mouse Chromosome 4
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The Uroguanylin Gene (Guca1b) Is Linked to Guanylin (Guca2) on Mouse Chromosome 4 Teresa L. Whitaker,* Kris A. Steinbrecher,* Neal G. Copeland,† Debra J. Gilbert,† Nancy A. Jenkins,† and Mitchell B. Cohen*,1 *Division of Pediatric Gastroenterology and Nutrition, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229; and †Mammalian Genetics Laboratory, ABL–Basic Research Program, NCI–Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received May 5, 1997; accepted August 4, 1997
Uroguanylin is an endogenous ligand of the intestinal receptor guanylate cyclase-C (GC-C). Both uroguanylin and the related peptide ligand guanylin bind to GC-C and stimulate an increase in cyclic GMP, inducing chloride secretion via the cystic fibrosis transmembrane conductance regulator. We describe the cloning of the complete mouse uroguanylin gene (Guca1b) and show that Guca1b is tightly linked to the mouse guanylin gene on chromosome 4. The two genes are structurally similar, being composed of three short exons; the uroguanylin gene spans 2.4 kb and the guanylin gene spans 1.7 kb. Uroguanylin mRNA is most prominent in proximal small intestine, whereas guanylin mRNA is predominantly expressed in distal small intestine and colon. The upstream promoter sequence of the mouse uroguanylin gene contains a canonical TATA element at the site of transcription initiation and consensus binding sites for several known transcription factors, including HNF-1 and Sp1 within the first 1 kb. Although the gene structure and coding sequences of uroguanylin and guanylin are similar, the 5* flanking sequences and patterns of expression of these two genes in the intestine are different. It is likely that uroguanylin and guanylin represent gene duplications that have evolved to allow overlapping and complementary patterns of expression in the intestine. q 1997 Academic Press
INTRODUCTION
Binding of the bacterial heat-stable enterotoxin (STa) to the intestinal receptor guanylate cyclase-C (GC-C) triggers a signal transduction cascade that results in secretory diarrhea. Activation of GC-C leads to Sequence data from this article have been deposited with the Mouse Genome Database under Accession No. MGD-JNUM-41432 and with the GenBank Data Library under Accession No. U95182. 1 To whom correspondence should be addressed. Telephone: (513) 636-4415. Fax: (513) 636-7805. E-mail: [email protected].
0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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increased intracellular levels of guanosine 3*,5-cyclic monophosphate (cGMP); this activates a cGMP-dependent protein kinase which phosphorylates the cystic fibrosis transmembrane conductance regulator, resulting in net chloride (Cl0) and water secretion (Schulz et al., 1990; Pfeifer et al., 1996; Cuthbert et al., 1994; Chao et al., 1994). Guanylin and uroguanylin are two endogenous mammalian ligands for GC-C (Hamra et al., 1993; Currie et al., 1992). They are structurally related to STa but are less potent activators of this signal transduction pathway (Hamra et al., 1993). Guanylin was originally isolated from rat jejunum and has subsequently been shown to be expressed highly in the intestine (Currie et al., 1992; Lewis et al., 1993). Although uroguanylin was originally identified in opossum and human urine (Hamra et al., 1993; Kita et al., 1994), similar to guanylin, uroguanylin mRNA is predominantly expressed in the intestine (Miyazato et al., 1996; Fan et al., 1996). Uroguanylin mRNA expression has also been reported in opossum kidney and heart (Fan et al., 1996) and in rat lung, pancreas, and kidney (Miyazato et al., 1996). We report here the genomic sequence and tissue distribution of mouse uroguanylin. We demonstrate that the mouse uroguanylin gene is closely linked to the mouse guanylin gene on chromosome 4. The gene structure and coding sequences of uroguanylin and guanylin are similar; however, the 5* flanking sequences of these two genes and their patterns of expression in the intestine differ. This suggests that uroguanylin and guanylin are examples of a gene duplication that has evolved to allow these ligands to cooperatively regulate salt and water metabolism in the intestine through interactions with GC-C. MATERIALS AND METHODS Genomic cloning and sequencing. The full-length human uroguanylin cDNA (gift from Oliver Hill) was used to probe a Lambda DASH II Sau3A partial 129/Sv mouse genomic library (gift from Marcia Shull). Briefly, phage aliquots were adsorbed to LE392 bacteria and plated at a density of 10,000 plaque-forming units on 90-mm plates. Plaques
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SEQUENCING AND MAPPING OF THE MOUSE UROGUANYLIN GENE were transferred to a nylon membrane (Gibco BRL, Gaithersburg, MD), denatured, neutralized, and crosslinked by ultraviolet illumination (Stratalinker) as previously described (Sciaky et al., 1994). Membranes were hybridized to an [a-32P]dATP random-labeled human uroguanylin cDNA and washed to a final stringency of 0.11 SSC, 1% SDS. Two positive plaques, l6 and l12, were isolated to purity as previously described (Sciaky et al., 1994) from the 5 1 105 plaques screened. Overlapping restriction fragments from BamHI, PstI, EcoRI, and XhoI digests of l12, the isolate containing the largest genomic insert, were subcloned in pBluescript II SK (/) (Stratagene, La Jolla, CA) and sequenced using Sequenase (US Biochemicals, Cleveland, OH). Southern blot analysis was used to generate a physical map of the genomic fragment. The sequence was analyzed using MacVector analysis software (Kodak) and TFSEARCH Version 1.3 (Yutaka Akiyama, Kyoto University, Kyoto, Japan). Oligonucleotides complementary to the mouse sequence were designed to amplify a 98-bp cDNA fragment extending from nucleotide 288 to 386 and a near full-length cDNA product extending from nucleotide 38 to 480 by RT-PCR. Primer extension. To determine the nucleotides that encode the 5* end of the uroguanylin transcript, primer extension analysis was performed with a 32P-end-labeled oligonucleotide, PEXT1 (5*-CCTCCAACTCATTCAGCTTCTTCACTGATTC-3*), complementary to RNA encoded within the second exon. The oligonucleotide was annealed to total RNA isolated from adult mouse duodenum and distal jejunum and extended with SuperScript RNase H0 reverse transcriptase. The primer extension products were electrophoresed through a 6% denaturing acrylamide sequencing gel with a sequencing ladder. The gels were dried and visualized using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). Interspecific mouse backcross mapping. Interspecific backcross progeny were generated by mating (C57BL/6J 1 Mus spretus)F1 females and C57BL/6J males as previously described (Copeland and Jenkins, 1991). A total of 205 F2 mice were used to map the Guca1b locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed as described (Jenkins et al., 1982). All blots were prepared with Hybond-N/ nylon membrane (Amersham, Arlington Heights, IL). The probe, a 4.0-kb XhoI–XhoI mouse genomic DNA fragment, was labeled with [a-32P]dCTP using a nick-translation labeling kit (Boehringer Mannheim, Mannheim, Germany); washing was performed to a final stringency of 0.81 SSCP, 0.1% SDS, 657C. A fragment of 9.6 kb was detected in BglI-digested C57BL/6J DNA, and a fragment of 14.0 kb was detected in BglI-digested M. spretus DNA. The presence or absence of the 14.0-kb BglI M. spretus-specific fragment was followed in backcross mice. A description of the probes and restriction fragment length polymorphisms (RFLPs) for the loci linked to Guca1b, including Jun, Guca2, and Gja4, has been reported previously (Ceci et al., 1989; Sciaky et al., 1995; Haefliger et al., 1992). Recombination distances were calculated using Map Manager, Version 2.6.5. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. Northern analysis. Total RNA was isolated from a panel of FVB mouse tissues using acid guanidine isothiocyanate–phenol–chloroform extraction (Chomczynski and Sacchi, 1987). Mouse tissues were harvested under an IACUC-approved protocol. Agarose gel electrophoresis and Northern blotting were performed as previously described (Laney et al., 1992). A 98-bp RT-PCR product extending from nucleotide 288 to 386 of the uroguanylin cDNA was random labeled with [a-32P]dATP and hybridized to total RNA extracted from mouse tissues. Blots were reprobed with an end-labeled oligonucleotide complementary to 18S rRNA to normalize for loading (Mann and Lingrel, 1991). Image analysis was performed on a PhosphorImager system using ImageQuant software (Molecular Dynamics).
RESULTS
Genomic Cloning and Sequencing of Guca1b The insert from l12, approximately 18 kb, was digested with restriction enzymes, subcloned, and se-
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quenced. The sequence of the entire uroguanylin gene is present in a single 4-kb XhoI–XhoI fragment (Genbank Accession No. U95182; Fig. 1). By comparing the mouse genomic uroguanylin sequence with the mouse cDNA sequence generated by RT-PCR, we determined that Guca1b is composed of three exons ranging from 165 to 226 nucleotides and spans 2.4 kb including intervening sequences. The exon/intron junctions follow consensus donor/acceptor splice sites (Jackson, 1991). Similar to guanylin, the sequence of the 15-amino-acid mature peptide for uroguanylin is encoded in the third exon. Through the use of a parallel sequencing ladder as a molecular weight marker for the primer extension products, the transcription start site was localized 85 to 95 nucleotides 5* of the translation start site located in exon 1 (Fig. 2). The polyadenylation signal sequence is located 165 nucleotides downstream of the translation stop site. The size of the mouse uroguanylin mRNA including 5* and 3* untranslated sequences is 582 bp. Computer analysis of 1 kb of 5* flanking sequence of the uroguanylin gene was performed to identify possible regulatory elements by homology to known consensus binding sites. This analysis revealed a 13/15-bp (underlined) match (5*-GTTAATAAGTGACCA-3*) with a consensus binding site for HNF-1 (5*-GTTAATG/TAA/TTNACCA-3*) and an 8/10-bp (underlined) match (5*-GGGGTGGGGT-3*) for Sp1 binding (5*-GA/GGCA/GGGGA/T-3*), both within 60 nucleotides of the transcription start site (Fig. 1). In addition, there is a consensus TATA site at nucleotide /1 to /5. Guca1b is Located on Mouse Chromosome 4 The mouse chromosomal location of Guca1b was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J 1 M. spretus)F1 1 C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2300 loci that are well distributed among all the autosomes as well as the X chromosome (Copeland and Jenkins, 1991). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using a mouse genomic Guca1b probe. The 14.0-kb BglI M. spretus RFLP was used to follow the segregation of the Guca1b locus in backcross mice. The mapping results indicated that Guca1b is located in the central region of mouse chromosome 4, linked to Jun, Guca2, and Gja4. Although 85 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 3), up to 149 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios for the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are centromere–Jun–14/110–Guca2–0/88–Guca1b–6/149–Gja4. The recombination frequencies (expressed as genetic
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FIG. 1. Sequence of the mouse uroguanylin gene and 5* and 3* flanking regions. The transcription start site located at the TATA box is denoted by /1, and the polyadenylation signal is underlined. Putative HNF-1 and Sp1 binding sites within the 5* flanking region are indicated above the sequence with asterisks. The amino acid sequence of the uroguanylin precursor encoded by exons 1–3 is indicated below the sequence. The stop codon (TGA) is double underlined. The antisense oligonucleotide PEXT1 used in primer extension analysis is indicated with a dotted line above the sequence in exon 2.
distances in centimorgans { the standard error) are Jun–12.7 { 3.2–[Guca2, Guca1b]–4.0 { 1.6–Gja4. No recombinants were detected between Guca2 and Guca1b in 88 animals typed in common, suggesting that the two loci are within 4.1 cM of each other (upper 95%
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confidence limit). This close linkage suggests that the two genes may have arisen by tandem duplication. In fact, sequencing information showed that the l12 insert contained the complete genomic sequence for both uroguanylin (Guca1b) and guanylin (Guca2) (Fig. 4).
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and Guca1b suggests that GUCA1B will map to 1p35– p34, as well. We have compared our interspecific map of chromosome 4 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (provided by Mouse Genome Database, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Guca1b mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). Recently, the human uroguanylin gene was isolated and sequenced (Maegert et al., 1996). The mouse and human gene structures for uroguanylin are very similar, each being composed of three short exons spanning FIG. 2. Determination of the 5* end of the uroguanylin transcript. Primer extension analysis with oligonucleotide PEXT1 using total RNA extracted from mouse jejunum. The size of the primer extension product was determined by electrophoresis of the product against a known sequencing ladder. The PEXT1 reaction resulted in five products; when compared to the corresponding 5* flanking sequence indicated on the left, the primary product was located at the start of the TATA box (star), and four minor products (asterisks) were located within 4 nucleotides of the TATA box.
By physical mapping of the l12 insert, the complete guanylin gene is located approximately 11 kb upstream of the uroguanylin gene, confirming the tight linkage between Guca1b and Guca2. Tissue Distribution A 98-bp fragment extending from nucleotide 288 to 386 was used as a probe for Northern blot analysis to localize mouse uroguanylin mRNA. A single predominant mRNA species of approximately 600 bp was recognized with this probe. Signal was present in all intestinal tissues examined, with the highest concentration observed in jejunum (Fig. 5). Expression was also seen in the kidney (Fig. 5). No signal was detected by Northern analysis of total RNA from ova/testes, diaphragm, heart, lung, spleen, liver, thymus, pancreas, or stomach (Fig. 5). DISCUSSION
We have isolated and sequenced the gene for mouse uroguanylin. Tight gene linkage between Guca1b and Guca2 was demonstrated by interspecific backcross analysis, which placed these two genes very close together on mouse chromosome 4. This confirmed that in the clone that we isolated, the location of Guca1b, 11 kb downstream of the mouse guanylin gene start site, was not an artifact of library construction. The central region of mouse chromosome 4 shares a region of homology with human chromosome 1 (summarized in Fig. 4). In particular, GUCA2 has been mapped to human 1p35–p34. The tight linkage between mouse Guca2
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FIG. 3. Guca1b maps to the central region of mouse chromosome 4. Guca1b was placed on mouse chromosome 4 by interspecific backcross analysis. The segregation patterns of Guca1b and flanking genes in 85 backcross animals that were typed for all loci are shown at the top. For individual pairs of loci, more than 85 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J 1 M. spretus)F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 4 linkage map showing the location of Guca1b in relation to linked genes is shown at the bottom. Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).
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FIG. 4. Structure of the insert of genomic clone l12. Exons 1–3 of mouse guanylin and mouse uroguanylin are indicated by hatched boxes. Numbering is indicated relative to the start site of the uroguanylin gene (/1). Restriction sites are shown for XhoI (X) to define the 4.0-kb XhoI–XhoI genomic fragment containing the entire uroguanylin gene. The position of a GA repeat sequence in intron 1 of the uroguanylin gene is shown by an open box. The locations of sequences encoding the mature peptides for guanylin (G) and uroguanylin (U) are denoted by arrows. Consensus elements for HNF-1, AP1, and Sp1 are shown in their relative positions. The start codon (ATG) is indicated for each gene.
approximately 2.4 kb. The mouse guanylin gene is also composed of three short exons although this gene spans only 1.7 kb (Sciaky et al., 1994). The mouse and human uroguanylin genes are approximately 70% identical in the coding regions while the mouse guanylin and uroguanylin genes are 51% identical in the coding regions. This high degree of structural similarity and coding sequence homology between uroguanylin and guanylin genes is suggestive of a gene duplication event. Uroguanylin mRNA is expressed in mouse intestine and kidney. Similar to that of the mouse, uroguanylin is expressed in intestine and kidney in human (Hill et al., 1995), rat (Miyazato et al., 1996), and opossum (Hamra et al., 1993). However, it is also expressed in opossum heart (Hamra et al., 1993) and in rat lung and pancreas (Miyazato et al., 1996), tissues in which we did not see expression by Northern analysis in mouse. Two short regions of the mouse uroguanylin gene promoter, at nucleotides 0109 to /18 and 0348 to
0310, share homology with regions in the human uroguanylin gene promoter (Maegert et al., 1996). In the region from 0109 to /18 there is a TATA box at the site of transcription initiation as determined by primer extension; a putative binding site for Sp1 is present at position 052 to 043. There is also a consensus binding site for HNF-1, at position 033 to 019. HNF-1 has been shown to regulate the transcriptional activation of a number of genes expressed in the intestine including guanylin (Hochman et al., 1997), sucrase isomaltase (Wu et al., 1994), a-fetoprotein (Tyner et al., 1990), villin (Robine et al., 1993), and a1-antitrypsin (Perlmutter et al., 1989). A Cdx-2 consensus binding site, which along with HNF-1 is important for transcriptional activation of sucrase isomaltase (Suh et al., 1994), is not present in the uroguanylin promoter. Although there is conservation of an HNF-1 consensus sequence in the uroguanylin and guanylin promoters, there is little sequence homology between the 5* flank-
FIG. 5. Tissue distribution of mouse uroguanylin mRNA. Total RNA was separated and the blot was probed as described under Materials and Methods. The positions of 28S and 18S rRNA are indicated along the side of the blot. Uroguanylin mRNA was present throughout the intestine. Signal was highest in small intestine with very little signal in the cecum and colon. Uroguanylin signal was also seen in the kidney but not in ova/testes, diaphragm, heart, lung, spleen, liver, thymus, pancreas, or stomach. For quantitation purposes the blot was reprobed with an oligonucleotide that recognizes 18S rRNA (Mann and Lingrel, 1991), and the results are shown at the bottom.
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ing sequences of these genes. Furthermore, uroguanylin and guanylin have distinct patterns of mRNA expression; uroguanylin mRNA is primarily expressed in the proximal small intestine, whereas guanylin mRNA is predominantly expressed in the distal small intestine and colon (Whitaker et al., 1997). Evolutionary divergence is also supported by the demonstration of robust uroguanylin mRNA but not guanylin mRNA expression in mouse kidney. Both uroguanylin and guanylin have been shown to activate Cl0 secretion in the intestine and to have potent natriuretic activity (Forte et al., 1996). Although these ligands activate the same signal transduction pathway, they do not necessarily have the same physiologic functions. For example, uroguanylin is much more active at an acidic pH, in contrast to guanylin, which has a more basic pH optima (Hamra et al., 1997). It is possible that differences in their patterns of gene expression have evolved to allow these two ligands to cooperatively regulate salt and water metabolism in the intestine and kidney. ACKNOWLEDGMENTS We thank Mary Barnstead for excellent technical assistance. This work was supported in part by a grant from the National Institutes of Health, DK 47318, and by the National Cancer Institute, DHHS, under contract with ABL.
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The Uroguanylin Gene (Guca1b) Is Linked to Guanylin (Guca2) on Mouse Chromosome 4 Teresa L. Whitaker,* Kris A. Steinbrecher,* Neal G. Copeland,† Debra J. Gilbert,† Nancy A. Jenkins,† and Mitchell B. Cohen*,1 *Division of Pediatric Gastroenterology and Nutrition, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229; and †Mammalian Genetics Laboratory, ABL–Basic Research Program, NCI–Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received May 5, 1997; accepted August 4, 1997
Uroguanylin is an endogenous ligand of the intestinal receptor guanylate cyclase-C (GC-C). Both uroguanylin and the related peptide ligand guanylin bind to GC-C and stimulate an increase in cyclic GMP, inducing chloride secretion via the cystic fibrosis transmembrane conductance regulator. We describe the cloning of the complete mouse uroguanylin gene (Guca1b) and show that Guca1b is tightly linked to the mouse guanylin gene on chromosome 4. The two genes are structurally similar, being composed of three short exons; the uroguanylin gene spans 2.4 kb and the guanylin gene spans 1.7 kb. Uroguanylin mRNA is most prominent in proximal small intestine, whereas guanylin mRNA is predominantly expressed in distal small intestine and colon. The upstream promoter sequence of the mouse uroguanylin gene contains a canonical TATA element at the site of transcription initiation and consensus binding sites for several known transcription factors, including HNF-1 and Sp1 within the first 1 kb. Although the gene structure and coding sequences of uroguanylin and guanylin are similar, the 5* flanking sequences and patterns of expression of these two genes in the intestine are different. It is likely that uroguanylin and guanylin represent gene duplications that have evolved to allow overlapping and complementary patterns of expression in the intestine. q 1997 Academic Press
INTRODUCTION
Binding of the bacterial heat-stable enterotoxin (STa) to the intestinal receptor guanylate cyclase-C (GC-C) triggers a signal transduction cascade that results in secretory diarrhea. Activation of GC-C leads to Sequence data from this article have been deposited with the Mouse Genome Database under Accession No. MGD-JNUM-41432 and with the GenBank Data Library under Accession No. U95182. 1 To whom correspondence should be addressed. Telephone: (513) 636-4415. Fax: (513) 636-7805. E-mail: [email protected].
0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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increased intracellular levels of guanosine 3*,5-cyclic monophosphate (cGMP); this activates a cGMP-dependent protein kinase which phosphorylates the cystic fibrosis transmembrane conductance regulator, resulting in net chloride (Cl0) and water secretion (Schulz et al., 1990; Pfeifer et al., 1996; Cuthbert et al., 1994; Chao et al., 1994). Guanylin and uroguanylin are two endogenous mammalian ligands for GC-C (Hamra et al., 1993; Currie et al., 1992). They are structurally related to STa but are less potent activators of this signal transduction pathway (Hamra et al., 1993). Guanylin was originally isolated from rat jejunum and has subsequently been shown to be expressed highly in the intestine (Currie et al., 1992; Lewis et al., 1993). Although uroguanylin was originally identified in opossum and human urine (Hamra et al., 1993; Kita et al., 1994), similar to guanylin, uroguanylin mRNA is predominantly expressed in the intestine (Miyazato et al., 1996; Fan et al., 1996). Uroguanylin mRNA expression has also been reported in opossum kidney and heart (Fan et al., 1996) and in rat lung, pancreas, and kidney (Miyazato et al., 1996). We report here the genomic sequence and tissue distribution of mouse uroguanylin. We demonstrate that the mouse uroguanylin gene is closely linked to the mouse guanylin gene on chromosome 4. The gene structure and coding sequences of uroguanylin and guanylin are similar; however, the 5* flanking sequences of these two genes and their patterns of expression in the intestine differ. This suggests that uroguanylin and guanylin are examples of a gene duplication that has evolved to allow these ligands to cooperatively regulate salt and water metabolism in the intestine through interactions with GC-C. MATERIALS AND METHODS Genomic cloning and sequencing. The full-length human uroguanylin cDNA (gift from Oliver Hill) was used to probe a Lambda DASH II Sau3A partial 129/Sv mouse genomic library (gift from Marcia Shull). Briefly, phage aliquots were adsorbed to LE392 bacteria and plated at a density of 10,000 plaque-forming units on 90-mm plates. Plaques
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SEQUENCING AND MAPPING OF THE MOUSE UROGUANYLIN GENE were transferred to a nylon membrane (Gibco BRL, Gaithersburg, MD), denatured, neutralized, and crosslinked by ultraviolet illumination (Stratalinker) as previously described (Sciaky et al., 1994). Membranes were hybridized to an [a-32P]dATP random-labeled human uroguanylin cDNA and washed to a final stringency of 0.11 SSC, 1% SDS. Two positive plaques, l6 and l12, were isolated to purity as previously described (Sciaky et al., 1994) from the 5 1 105 plaques screened. Overlapping restriction fragments from BamHI, PstI, EcoRI, and XhoI digests of l12, the isolate containing the largest genomic insert, were subcloned in pBluescript II SK (/) (Stratagene, La Jolla, CA) and sequenced using Sequenase (US Biochemicals, Cleveland, OH). Southern blot analysis was used to generate a physical map of the genomic fragment. The sequence was analyzed using MacVector analysis software (Kodak) and TFSEARCH Version 1.3 (Yutaka Akiyama, Kyoto University, Kyoto, Japan). Oligonucleotides complementary to the mouse sequence were designed to amplify a 98-bp cDNA fragment extending from nucleotide 288 to 386 and a near full-length cDNA product extending from nucleotide 38 to 480 by RT-PCR. Primer extension. To determine the nucleotides that encode the 5* end of the uroguanylin transcript, primer extension analysis was performed with a 32P-end-labeled oligonucleotide, PEXT1 (5*-CCTCCAACTCATTCAGCTTCTTCACTGATTC-3*), complementary to RNA encoded within the second exon. The oligonucleotide was annealed to total RNA isolated from adult mouse duodenum and distal jejunum and extended with SuperScript RNase H0 reverse transcriptase. The primer extension products were electrophoresed through a 6% denaturing acrylamide sequencing gel with a sequencing ladder. The gels were dried and visualized using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). Interspecific mouse backcross mapping. Interspecific backcross progeny were generated by mating (C57BL/6J 1 Mus spretus)F1 females and C57BL/6J males as previously described (Copeland and Jenkins, 1991). A total of 205 F2 mice were used to map the Guca1b locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed as described (Jenkins et al., 1982). All blots were prepared with Hybond-N/ nylon membrane (Amersham, Arlington Heights, IL). The probe, a 4.0-kb XhoI–XhoI mouse genomic DNA fragment, was labeled with [a-32P]dCTP using a nick-translation labeling kit (Boehringer Mannheim, Mannheim, Germany); washing was performed to a final stringency of 0.81 SSCP, 0.1% SDS, 657C. A fragment of 9.6 kb was detected in BglI-digested C57BL/6J DNA, and a fragment of 14.0 kb was detected in BglI-digested M. spretus DNA. The presence or absence of the 14.0-kb BglI M. spretus-specific fragment was followed in backcross mice. A description of the probes and restriction fragment length polymorphisms (RFLPs) for the loci linked to Guca1b, including Jun, Guca2, and Gja4, has been reported previously (Ceci et al., 1989; Sciaky et al., 1995; Haefliger et al., 1992). Recombination distances were calculated using Map Manager, Version 2.6.5. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. Northern analysis. Total RNA was isolated from a panel of FVB mouse tissues using acid guanidine isothiocyanate–phenol–chloroform extraction (Chomczynski and Sacchi, 1987). Mouse tissues were harvested under an IACUC-approved protocol. Agarose gel electrophoresis and Northern blotting were performed as previously described (Laney et al., 1992). A 98-bp RT-PCR product extending from nucleotide 288 to 386 of the uroguanylin cDNA was random labeled with [a-32P]dATP and hybridized to total RNA extracted from mouse tissues. Blots were reprobed with an end-labeled oligonucleotide complementary to 18S rRNA to normalize for loading (Mann and Lingrel, 1991). Image analysis was performed on a PhosphorImager system using ImageQuant software (Molecular Dynamics).
RESULTS
Genomic Cloning and Sequencing of Guca1b The insert from l12, approximately 18 kb, was digested with restriction enzymes, subcloned, and se-
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quenced. The sequence of the entire uroguanylin gene is present in a single 4-kb XhoI–XhoI fragment (Genbank Accession No. U95182; Fig. 1). By comparing the mouse genomic uroguanylin sequence with the mouse cDNA sequence generated by RT-PCR, we determined that Guca1b is composed of three exons ranging from 165 to 226 nucleotides and spans 2.4 kb including intervening sequences. The exon/intron junctions follow consensus donor/acceptor splice sites (Jackson, 1991). Similar to guanylin, the sequence of the 15-amino-acid mature peptide for uroguanylin is encoded in the third exon. Through the use of a parallel sequencing ladder as a molecular weight marker for the primer extension products, the transcription start site was localized 85 to 95 nucleotides 5* of the translation start site located in exon 1 (Fig. 2). The polyadenylation signal sequence is located 165 nucleotides downstream of the translation stop site. The size of the mouse uroguanylin mRNA including 5* and 3* untranslated sequences is 582 bp. Computer analysis of 1 kb of 5* flanking sequence of the uroguanylin gene was performed to identify possible regulatory elements by homology to known consensus binding sites. This analysis revealed a 13/15-bp (underlined) match (5*-GTTAATAAGTGACCA-3*) with a consensus binding site for HNF-1 (5*-GTTAATG/TAA/TTNACCA-3*) and an 8/10-bp (underlined) match (5*-GGGGTGGGGT-3*) for Sp1 binding (5*-GA/GGCA/GGGGA/T-3*), both within 60 nucleotides of the transcription start site (Fig. 1). In addition, there is a consensus TATA site at nucleotide /1 to /5. Guca1b is Located on Mouse Chromosome 4 The mouse chromosomal location of Guca1b was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J 1 M. spretus)F1 1 C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2300 loci that are well distributed among all the autosomes as well as the X chromosome (Copeland and Jenkins, 1991). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using a mouse genomic Guca1b probe. The 14.0-kb BglI M. spretus RFLP was used to follow the segregation of the Guca1b locus in backcross mice. The mapping results indicated that Guca1b is located in the central region of mouse chromosome 4, linked to Jun, Guca2, and Gja4. Although 85 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 3), up to 149 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios for the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are centromere–Jun–14/110–Guca2–0/88–Guca1b–6/149–Gja4. The recombination frequencies (expressed as genetic
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FIG. 1. Sequence of the mouse uroguanylin gene and 5* and 3* flanking regions. The transcription start site located at the TATA box is denoted by /1, and the polyadenylation signal is underlined. Putative HNF-1 and Sp1 binding sites within the 5* flanking region are indicated above the sequence with asterisks. The amino acid sequence of the uroguanylin precursor encoded by exons 1–3 is indicated below the sequence. The stop codon (TGA) is double underlined. The antisense oligonucleotide PEXT1 used in primer extension analysis is indicated with a dotted line above the sequence in exon 2.
distances in centimorgans { the standard error) are Jun–12.7 { 3.2–[Guca2, Guca1b]–4.0 { 1.6–Gja4. No recombinants were detected between Guca2 and Guca1b in 88 animals typed in common, suggesting that the two loci are within 4.1 cM of each other (upper 95%
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confidence limit). This close linkage suggests that the two genes may have arisen by tandem duplication. In fact, sequencing information showed that the l12 insert contained the complete genomic sequence for both uroguanylin (Guca1b) and guanylin (Guca2) (Fig. 4).
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and Guca1b suggests that GUCA1B will map to 1p35– p34, as well. We have compared our interspecific map of chromosome 4 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (provided by Mouse Genome Database, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Guca1b mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). Recently, the human uroguanylin gene was isolated and sequenced (Maegert et al., 1996). The mouse and human gene structures for uroguanylin are very similar, each being composed of three short exons spanning FIG. 2. Determination of the 5* end of the uroguanylin transcript. Primer extension analysis with oligonucleotide PEXT1 using total RNA extracted from mouse jejunum. The size of the primer extension product was determined by electrophoresis of the product against a known sequencing ladder. The PEXT1 reaction resulted in five products; when compared to the corresponding 5* flanking sequence indicated on the left, the primary product was located at the start of the TATA box (star), and four minor products (asterisks) were located within 4 nucleotides of the TATA box.
By physical mapping of the l12 insert, the complete guanylin gene is located approximately 11 kb upstream of the uroguanylin gene, confirming the tight linkage between Guca1b and Guca2. Tissue Distribution A 98-bp fragment extending from nucleotide 288 to 386 was used as a probe for Northern blot analysis to localize mouse uroguanylin mRNA. A single predominant mRNA species of approximately 600 bp was recognized with this probe. Signal was present in all intestinal tissues examined, with the highest concentration observed in jejunum (Fig. 5). Expression was also seen in the kidney (Fig. 5). No signal was detected by Northern analysis of total RNA from ova/testes, diaphragm, heart, lung, spleen, liver, thymus, pancreas, or stomach (Fig. 5). DISCUSSION
We have isolated and sequenced the gene for mouse uroguanylin. Tight gene linkage between Guca1b and Guca2 was demonstrated by interspecific backcross analysis, which placed these two genes very close together on mouse chromosome 4. This confirmed that in the clone that we isolated, the location of Guca1b, 11 kb downstream of the mouse guanylin gene start site, was not an artifact of library construction. The central region of mouse chromosome 4 shares a region of homology with human chromosome 1 (summarized in Fig. 4). In particular, GUCA2 has been mapped to human 1p35–p34. The tight linkage between mouse Guca2
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FIG. 3. Guca1b maps to the central region of mouse chromosome 4. Guca1b was placed on mouse chromosome 4 by interspecific backcross analysis. The segregation patterns of Guca1b and flanking genes in 85 backcross animals that were typed for all loci are shown at the top. For individual pairs of loci, more than 85 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J 1 M. spretus)F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 4 linkage map showing the location of Guca1b in relation to linked genes is shown at the bottom. Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).
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FIG. 4. Structure of the insert of genomic clone l12. Exons 1–3 of mouse guanylin and mouse uroguanylin are indicated by hatched boxes. Numbering is indicated relative to the start site of the uroguanylin gene (/1). Restriction sites are shown for XhoI (X) to define the 4.0-kb XhoI–XhoI genomic fragment containing the entire uroguanylin gene. The position of a GA repeat sequence in intron 1 of the uroguanylin gene is shown by an open box. The locations of sequences encoding the mature peptides for guanylin (G) and uroguanylin (U) are denoted by arrows. Consensus elements for HNF-1, AP1, and Sp1 are shown in their relative positions. The start codon (ATG) is indicated for each gene.
approximately 2.4 kb. The mouse guanylin gene is also composed of three short exons although this gene spans only 1.7 kb (Sciaky et al., 1994). The mouse and human uroguanylin genes are approximately 70% identical in the coding regions while the mouse guanylin and uroguanylin genes are 51% identical in the coding regions. This high degree of structural similarity and coding sequence homology between uroguanylin and guanylin genes is suggestive of a gene duplication event. Uroguanylin mRNA is expressed in mouse intestine and kidney. Similar to that of the mouse, uroguanylin is expressed in intestine and kidney in human (Hill et al., 1995), rat (Miyazato et al., 1996), and opossum (Hamra et al., 1993). However, it is also expressed in opossum heart (Hamra et al., 1993) and in rat lung and pancreas (Miyazato et al., 1996), tissues in which we did not see expression by Northern analysis in mouse. Two short regions of the mouse uroguanylin gene promoter, at nucleotides 0109 to /18 and 0348 to
0310, share homology with regions in the human uroguanylin gene promoter (Maegert et al., 1996). In the region from 0109 to /18 there is a TATA box at the site of transcription initiation as determined by primer extension; a putative binding site for Sp1 is present at position 052 to 043. There is also a consensus binding site for HNF-1, at position 033 to 019. HNF-1 has been shown to regulate the transcriptional activation of a number of genes expressed in the intestine including guanylin (Hochman et al., 1997), sucrase isomaltase (Wu et al., 1994), a-fetoprotein (Tyner et al., 1990), villin (Robine et al., 1993), and a1-antitrypsin (Perlmutter et al., 1989). A Cdx-2 consensus binding site, which along with HNF-1 is important for transcriptional activation of sucrase isomaltase (Suh et al., 1994), is not present in the uroguanylin promoter. Although there is conservation of an HNF-1 consensus sequence in the uroguanylin and guanylin promoters, there is little sequence homology between the 5* flank-
FIG. 5. Tissue distribution of mouse uroguanylin mRNA. Total RNA was separated and the blot was probed as described under Materials and Methods. The positions of 28S and 18S rRNA are indicated along the side of the blot. Uroguanylin mRNA was present throughout the intestine. Signal was highest in small intestine with very little signal in the cecum and colon. Uroguanylin signal was also seen in the kidney but not in ova/testes, diaphragm, heart, lung, spleen, liver, thymus, pancreas, or stomach. For quantitation purposes the blot was reprobed with an oligonucleotide that recognizes 18S rRNA (Mann and Lingrel, 1991), and the results are shown at the bottom.
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ing sequences of these genes. Furthermore, uroguanylin and guanylin have distinct patterns of mRNA expression; uroguanylin mRNA is primarily expressed in the proximal small intestine, whereas guanylin mRNA is predominantly expressed in the distal small intestine and colon (Whitaker et al., 1997). Evolutionary divergence is also supported by the demonstration of robust uroguanylin mRNA but not guanylin mRNA expression in mouse kidney. Both uroguanylin and guanylin have been shown to activate Cl0 secretion in the intestine and to have potent natriuretic activity (Forte et al., 1996). Although these ligands activate the same signal transduction pathway, they do not necessarily have the same physiologic functions. For example, uroguanylin is much more active at an acidic pH, in contrast to guanylin, which has a more basic pH optima (Hamra et al., 1997). It is possible that differences in their patterns of gene expression have evolved to allow these two ligands to cooperatively regulate salt and water metabolism in the intestine and kidney. ACKNOWLEDGMENTS We thank Mary Barnstead for excellent technical assistance. This work was supported in part by a grant from the National Institutes of Health, DK 47318, and by the National Cancer Institute, DHHS, under contract with ABL.
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