Title: Structure of the Rat V1a Vasopressin Receptor Gene and Characterization of Its Promoter Region and Complete cDNA Sequence of the 3′-End
Abstract: The gene encoding the rat V1a arginine vasopressin (AVP) receptor was isolated, and its structural organization and 5′-flanking region were characterized. In addition, the complete cDNA sequence of the major transcript of the rat V1a receptor gene was determined. Southern blots demonstrated a single copy of the V1a receptor gene in the rat genome, spanning a region of 3.8 kilobases (kb) and consisting of two exons and one intron (1.8 kb). The location of the intron was unique among G protein-coupled receptor genes in that the first exon encodes six of the seven transmembrane regions, the seventh region being encoded by the second exon. Primer extension, RNase protection, and rapid amplification of the 5′-end of the cDNA identified three transcriptional initiation sites (−405, −243, and −237), the major transcription initiation sites being mapped to positions −243 and −237 base pairs (bp) upstream of the ATG initiation codon (+1 bp). This portion of the 5′-flanking region has neither a TATA nor a CCAAT box, is GC-rich but has no GC box motif, and has features of promoters seen in housekeeping genes. Chimeras containing 2.2 kb of the 5′-flanking region and deletion analyses using the chloramphenicol acetyltransferase gene indicated that a "minimal" region, exhibiting promoter activity and tissue specificity, is located between nucleotides −296 and −221, when transfected into vascular smooth muscle cells. Gel mobility shift assay and Southwestern blotting suggested that ∼30- and ∼28-kDa nuclear proteins specifically bind to this region. Rapid amplification of the 3′-end of the cDNA showed that the major transcript terminates 442 bp downstream of the stop codon, in agreement with the mRNA size (2.1 kb). This study demonstrated a distinctive feature in the structural organization of the AVP-oxytocin receptor family genes, and characterization of the 5′-flanking region reported here will lead to a better understanding of the mechanism of transcriptional regulation of the rat V1a AVP receptor gene. The gene encoding the rat V1a arginine vasopressin (AVP) receptor was isolated, and its structural organization and 5′-flanking region were characterized. In addition, the complete cDNA sequence of the major transcript of the rat V1a receptor gene was determined. Southern blots demonstrated a single copy of the V1a receptor gene in the rat genome, spanning a region of 3.8 kilobases (kb) and consisting of two exons and one intron (1.8 kb). The location of the intron was unique among G protein-coupled receptor genes in that the first exon encodes six of the seven transmembrane regions, the seventh region being encoded by the second exon. Primer extension, RNase protection, and rapid amplification of the 5′-end of the cDNA identified three transcriptional initiation sites (−405, −243, and −237), the major transcription initiation sites being mapped to positions −243 and −237 base pairs (bp) upstream of the ATG initiation codon (+1 bp). This portion of the 5′-flanking region has neither a TATA nor a CCAAT box, is GC-rich but has no GC box motif, and has features of promoters seen in housekeeping genes. Chimeras containing 2.2 kb of the 5′-flanking region and deletion analyses using the chloramphenicol acetyltransferase gene indicated that a "minimal" region, exhibiting promoter activity and tissue specificity, is located between nucleotides −296 and −221, when transfected into vascular smooth muscle cells. Gel mobility shift assay and Southwestern blotting suggested that ∼30- and ∼28-kDa nuclear proteins specifically bind to this region. Rapid amplification of the 3′-end of the cDNA showed that the major transcript terminates 442 bp downstream of the stop codon, in agreement with the mRNA size (2.1 kb). This study demonstrated a distinctive feature in the structural organization of the AVP-oxytocin receptor family genes, and characterization of the 5′-flanking region reported here will lead to a better understanding of the mechanism of transcriptional regulation of the rat V1a AVP receptor gene. INTRODUCTIONArginine vasopressin (AVP) 1The abbreviations used are: AVParginine vasopressinkbkilobase(s)bpbase pair(s)ntnucleotide(s)PCRpolymerase chain reactionCATchloramphenicol acetyltransferasePAGEpolyacrylamide gel electrophoresisRACErapid amplification of cDNA ends. has diverse actions, including the contraction of smooth muscle, stimulation of liver glycogenolysis, modulation of adrenocorticotropic hormone release from the pituitary, and the inhibition of diuresis(1Michell R.H. Kirk C.J. Billah M.M. Biochem. Soc. Trans. 1979; 7: 861-865Crossref PubMed Scopus (366) Google Scholar). These physiological effects are mediated through binding to specific membrane receptors of target cells. AVP receptors are coupled to G proteins and have been divided into at least three types, V1a, V1b, and V2. The V1a (vascular/hepatic) and V1b (anterior pituitary) receptors act through phosphatidylinositol hydrolysis to mobilize intracellular Ca2+(2Jard S. Gaillard R.C. Guillon G. Marie J. Schoenenberg P. Muller A.F. Manning M. Sawyer W.H. Mol. Pharmacol. 1986; 30: 171-177PubMed Google Scholar), while the V1a receptor mediates physiological effects such as cell contraction and proliferation, platelet aggregation, coagulation factor release, and glycogenolysis. The V1b receptor is expressed in the anterior pituitary where it stimulates adrenocorticotropic hormone release. V2 receptors are found mainly in the kidney and are linked to adenylate cyclase and the production of cyclic adenosine monophosphate associated with antidiuresis(3Thibonnier M. Kidney. Int. 1988; 34: S52-S56Google Scholar).Morel et al.(4Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (458) Google Scholar) recently reported the cloning of a cDNA encoding the rat V1a receptor; a 1354-nucleotide cDNA encodes a 394-amino-acid protein with seven membrane-spanning domains similar to bacterial rhodopsin and other G protein-coupled receptors. Moreover, Lolait et al.(5Lolait S.J. O'Carroll A.M. McBride O.W. Koning M. Morel A. Brownstein M.J. Nature. 1992; 357: 336-339Crossref PubMed Scopus (477) Google Scholar) and Birnbaumer et al.(6Birnbaumer M. Seibold A. Gilbert S. Ishido M. Barberis C. Anataramian A. Brabet P. Rosenthal W. Nature. 1992; 357: 333-335Crossref PubMed Scopus (484) Google Scholar) described the molecular cloning of human and rat V2 receptors, respectively. Simultaneously, Kimura et al.(7Kimura T. Tanizawa O. Mori K. Brownstein M.J. Okayama H. Nature. 1992; 356: 526-529Crossref PubMed Scopus (561) Google Scholar) reported the structure of a cDNA encoding the human oxytocin receptor and showed that its amino acid sequence has a high degree of similarity to those of V1 and V2 receptors. Thibonnier et al.(8Thibonnier M. Auzan C. Madhun Z. Wilkins P. Berti-Mattera L. Clauser E. J. Biol. Chem. 1994; 269: 3304-3310Abstract Full Text PDF PubMed Google Scholar) demonstrated that the cDNA for human V1a receptor has 72% amino acid sequence identity to the rat V1a receptor. These findings suggested that the AVP-oxytocin receptors belong to a subgroup of the family of G protein-linked receptors. With respect to genomic cloning of AVP-oxytocin receptor family genes, Seibold et al.(9Seibold A. Brabet P. Rosenthal W. Birnbaumer M. Am. J. Hum. Genet. 1992; 51: 1078-1083PubMed Google Scholar) have described the structure of the gene encoding the human V2 receptor, which showed two very small intervening sequences in the coding region. The sizes and location of these introns are unique among the G protein-coupled receptor genes in that the second exon encodes only six of the seven transmembrane regions, the seventh such region being encoded by exon 3. Very recently, Rosen et al.(10Rosen F. Russo C. Banville D. Zingg H.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 200-204Crossref PubMed Scopus (197) Google Scholar) reported that the rat oxytocin receptor gene contains two introns, the second of which interrupts the coding region between the sixth and seventh transmembrane domains. However, the structures of the V1 type receptor genes and analyses of the promoter regions of these genes remain to be determined. In addition, the cDNA sequence of the rat V1a receptor reported by Morel et al.(4Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (458) Google Scholar) lacked a 3′-noncoding region. In the present report, we describe the structural organization of the rat V1a receptor gene, the complete cDNA sequence, and also the DNA sequence of the promoter region. The V1a receptor gene spans 3.8 kb and consists of two exons and one intron. The 3′-end of the cDNA showed that the major transcript terminates 442 bp downstream of the stop codon, in agreement with the reported mRNA size. The promoter region has features common to promoters of housekeeping genes, and the promoter activity is specifically expressed in vascular smooth muscle cells, but not in NIH3T3 or skeletal muscle cells. We characterized the minimal region from nucleotides −296 and −222 which confers promoter activity and tissue specificity on the V1a receptor gene and found that 30- and 28-kDa nuclear proteins bind to this region.EXPERIMENTAL PROCEDURESLibrary Screening and DNA SequencingMethods of restriction analysis, screening recombinant genomic libraries, Southern blot transfer, hybridization to DNA on filters, subcloning into plasmid vectors, agarose, and polyacrylamide electrophoresis were performed as described(11Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocol in Molecular Biology. Greene Publishing Associates, New York1983Google Scholar). A Lambda DASH™ II rat genomic library prepared from Sprague-Dawley male testes (Stratagene) was screened using a rat V1a receptor cDNA fragment corresponding to nucleotides −91 to +1343 of a V1a receptor cDNA clone (4Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (458) Google Scholar) relative to the ATG initiation codon as a probe. The cDNA fragment was obtained by reverse transcriptase and polymerase chain reaction (PCR) as follows. Adult rat liver total RNA (1 μg) was transcribed with random hexamers (2.5 μM), dNTPs (1 mM), and RNase inhibitors (1 unit/μl) using murine Moloney leukemia virus reverse transcriptase (2.5 units/μl) for 45 min at 42°C as previously reported(12Matsubara H. Kanasaki M. Murasawa S. Tsukaguchi Y. Nio Y. Inada M. J. Clin. Invest. 1994; 93: 1592-1601Crossref PubMed Scopus (149) Google Scholar, 13Matsubara H. Suzuki J. Inada M. J. Clin. Invest. 1993; 92: 1659-1666Crossref PubMed Scopus (82) Google Scholar). The resultant single strand cDNA was amplified with the forward primer in the 5′-noncoding region (5′-GCGCAGAGCTTAGAACTCGGATCCTCCGGT-3′) and reverse primer in the 3′-noncoding region (5′-CTTTGGACGCAGTCTTGCAGGAGATGGCC-3′) using Taq polymerase (Takara Shuzo, Kyoto, Japan). The resultant PCR product was radiolabeled by random oligonucleotide primer extension using [α-32P]dCTP and used as a probe(14Matsubara H. Liman E.R. Hess P. Koren G. J. Biol. Chem. 1991; 266: 13324-13328Abstract Full Text PDF PubMed Google Scholar). Positive clones were purified and characterized by restriction endonuclease mapping and Southern blotting. The specific restriction fragments subcloned into the pBluescript II vector (Stratagene) were sequenced by dideoxy chain termination. In addition to T3 and T7 primers, sequence-specific oligonucleotides were synthesized using a DNA synthesizer 381A (Applied Biosystems, Inc.).RNA AnalysisPrimer extension reactions and RNase protection were carried out with 40 μg and 10 μg of total RNA, respectively, as described(11Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocol in Molecular Biology. Greene Publishing Associates, New York1983Google Scholar). For analysis of V1a receptor mRNA, 30-nucleotide (nt)-long primers (nt −68 to −98 and +21 to +51 relative to the first ATG codon) were synthesized. For analysis of chloramphenicol acetyltransferase (CAT) mRNA, a 30-nt-long primer corresponding to nt +2 to +32 downstream from the HindIII site of the CAT gene was used(15Murasawa S. Matsubara H. Urakami M. Inada M. J. Biol. Chem. 1993; 268: 26996-27003Abstract Full Text PDF PubMed Google Scholar). For RNase protection analysis, a HincII-digested EcoRI and BamHI genomic sequence (−480 to −72 relative to the ATG codon) was used for the probe. A RNA probe was generated using [α-32P]CTP and T7 RNA polymerase (Takara Shuzo, Kyoto, Japan). After the probe was hybridized with RNA at 52°C overnight, it was digested with RNase T1 (0.21 unit/ml, U. S. Biochemical Corp.) and RNase A (2.1 units/ml, U. S. Biochemical Corp.) and analyzed on a 6% sequence gel, as described previously(18Mori Y. Matsubara H. Folco E. Siegel A. Koren G. J. Biol. Chem. 1993; 268: 26482-26493Abstract Full Text PDF PubMed Google Scholar).The Sequence of the Rapid Amplification Product of the 5′-End of Rat V1a Receptor cDNATwo micrograms of total cellular RNA from liver and A10 cells were reverse-transcribed for 45 min at 42°C using murine Moloney leukemia virus reverse transcriptase (2.5 units/μl, Life Technologies, Inc.). The primer was −264 to −294 relative to the ATG codon (Fig. 2). After removing excess primer with a spin column (Chroma spin-100, Clontech), the first strand reaction products were tailed with dATP using 10 units of terminal deoxytransferase as reported(18Mori Y. Matsubara H. Folco E. Siegel A. Koren G. J. Biol. Chem. 1993; 268: 26482-26493Abstract Full Text PDF PubMed Google Scholar). Amplification proceeded using a primer consisting of a T7 primer sequence and (dT)20 (TAATACGACTCACCTATAGGGCGAATTGAAGCTTTTTTTTTTTTTTTTTTTT) and a nested primer complementary to the nucleotides from position −316 to −336 relative to the ATG codon. The PCR product was subcloned into pGEM-T vector (Promega) and sequenced using the nested primer(16Nio Y. Matsubara H. Murasawa S. Kanasaki M. Inada M. J. Clin. Invest. 1995; 95: 46-54Crossref PubMed Google Scholar). The product was analyzed on a 6% denaturing polyacrylamide gel. After fixing and drying, the gel was autoradiographed at −70°C for 4 days.The Sequence of the Rapid Amplification Product of the 3′-End of Rat V1a Receptor cDNATotal cellular RNA (2 μg) from liver and A10 cells was reverse-transcribed for 45 min at 42°C using murine Moloney leukemia virus reverse transcriptase. The primer was the same (dT)20 sequence as that used to determine the 5′-end of the cDNA. After removing excess primer, amplification proceeded with the (dT)20 primer and oligonucleotides designed from the 3′-noncoding region of the rat V1a cDNA sequences reported by Morel et al.(4Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (458) Google Scholar) (+1280 to +1300 relative to the ATG initiation codon). The PCR product was subcloned into the pGEM-T vector (Promega) and sequenced using M13 primers. The product was analyzed on a 6% denaturing polyacrylamide gel, which was autoradiographed at −70°C for 4 days.Construction of V1a Receptor CAT Expression VectorsThe 2.2-kb EcoRI-ApaI fragment was fused 5′ to the chloramphenicol acetyltransferase (CAT)-pBluescript KS(-) construct (V1a 2208, Fig. 5). Sequential deletion mutants of the 5′-flanking region (Fig. 5) were prepared by using restriction enzyme sites of XhoI (V1a 1605) and SacI (V1a 221), or Exonuclease III (double-stranded Nested Deletion Kit, Pharmacia). In brief, the V1a 2208-CAT construct was digested with KpnI and EcoRV (linker sites of pBs vector). The linearized DNA was incubated with exonuclease III, and samples were removed at timed intervals, then treated with S1 nuclease to remove single-stranded regions generated by the exonuclease III. Deletion mutants of desired sizes were recircularized with T4 DNA ligase. The 5′-end of deletion mutant was determined by reading the sequence. The ΔV1a 2208-CAT that lacks the sequence between −348 and −222 from V1a 2208-CAT construct was constructed by digestion with PstI and SacI followed by treatment with S1 nuclease and T4 DNA ligase.Figure 5:Expression of CAT fusion genes containing 5′-deleted V1a receptor gene sequences. The 5′-deletion mutants were transiently expressed in A10 and NIH3T3 cells. The cell extracts were prepared 48 h after transfection and assayed. The names of these deletion mutants indicate the 5′-end points of the promoter sequence. ΔV1a 2208 indicates the CAT construct lacking the sequence between −348 and −222 from V1a 2208. The CAT activity levels, normalized with β-galactosidase activities and protein contents, are expressed relative to those in promoterless pBs-CAT, assigned a value of 1.0. The mean activities and S.E. from four separate assays are presented. ∗, p < 0.05;∗∗, p < 0.01 compared with the V1a 1006- and V1a 296-CAT construct, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DNA Transfection and CAT AssayPlasmids were banded in CsCl before transfection. Transfections were performed by the calcium phosphate/DNA precipitate method(15Murasawa S. Matsubara H. Urakami M. Inada M. J. Biol. Chem. 1993; 268: 26996-27003Abstract Full Text PDF PubMed Google Scholar). Cells were incubated for 4 h with 30 μg of plasmid DNA, treated with 15% glycerol for 1 min (glycerol shock), and supplemented with the growth medium. After a 48-h incubation, cells were harvested and extracted in 0.25 M Tris-HCl, pH 7.8, by freezing-thawing. Using cell extracts, CAT activity was determined by a dual-phase diffusion assay that relies on the direct diffusion of the 14C-labeled acetylchloramphenicol into liquid scintillation counting fluid, as previously reported(15Murasawa S. Matsubara H. Urakami M. Inada M. J. Biol. Chem. 1993; 268: 26996-27003Abstract Full Text PDF PubMed Google Scholar, 18Mori Y. Matsubara H. Folco E. Siegel A. Koren G. J. Biol. Chem. 1993; 268: 26482-26493Abstract Full Text PDF PubMed Google Scholar). During this period, each vial was counted at 30-min intervals (at least six times). The relationship between incubation time and radioactivity was linear, and the slope (calculated by linear regression) was proportional to CAT activity. In each transfection, 5 μg of Rous sarcoma virus β-galactosidase was co-transfected. CAT activity was normalized for transfection efficiency by β-galactosidase activity and for cell density by protein concentration. The β-galactosidase assay was performed as previously reported(15Murasawa S. Matsubara H. Urakami M. Inada M. J. Biol. Chem. 1993; 268: 26996-27003Abstract Full Text PDF PubMed Google Scholar, 18Mori Y. Matsubara H. Folco E. Siegel A. Koren G. J. Biol. Chem. 1993; 268: 26482-26493Abstract Full Text PDF PubMed Google Scholar). Protein assay was performed by the Bradford method (Bio-Rad). CAT activity was expressed relative to the activity of a promoterless CAT construct. Most transfection experiments were repeated a minimum of four separate times.Gel Retardation AssaysThe gel retardation assay was performed and nuclear extracts from culture cells were prepared as described(11Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocol in Molecular Biology. Greene Publishing Associates, New York1983Google Scholar). The DNA fragment between −296 and −221 bp relative to the ATG codon was obtained from V1a 296-CAT construct (Fig. 5) by BssHII (linker site of pBs vector) and SacI digestion and labeled with [α-32P]dCTP using Klenow fragment (Takara Shuzo) and purified as reported(11Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocol in Molecular Biology. Greene Publishing Associates, New York1983Google Scholar). Nuclear extracts were incubated for 15 min on ice in a 30-μl reaction mixture containing 12 mM Hepes, pH 7.9, 60 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 12% glycerol, and 2 μg of double-stranded poly(dI-dC) in the presence or absence of excess competitor DNA. A radiolabeled DNA probe was added (0.1 ∼ 0.5 ng; 15,000 cpm), and the incubation was continued for 30 min at room temperature. Thereafter, the mixture was loaded on a 6% polyacrylamide gel in 1 × TBE (90 mM Tris-HCl, pH 8.0, 89 mM boric acid, 2 mM EDTA) and electrophoresed at 140 V for 3 h followed by autoradiography.Southwestern (DNA-Protein) BlottingSouthwestern blotting proceeded as follows. Crude nuclear extracts (80 μg of protein each) mixed with 2.5% SDS, 2.5 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 10% glycerol, 0.025% pyronin for 5 min at room temperature were separated by SDS-PAGE (10% polyacrylamide) and blotted onto a nitrocellulose membrane. The membrane was incubated in binding buffer (10 mM Hepes, pH 8.0, 100 mM KCl, 10 mM MgCl2, 0.1 mM EDTA) containing 5% nonfat dried milk for 30 min at room temperature, and then in the same buffer containing salmon sperm DNA (100 μg/ml), 0.25% nonfat dried milk, and 32P-labeled probe (nt −296 and −221 relative to the ATG codon) for 1 h at room temperature. After washing three times for 10 min at room temperature in the binding buffer, the membrane was exposed to x-ray film.Stable Transfectants of the Promoter-CAT Chimeric Plasmid and Cell CultureFifty micrograms of the V1a 2208-CAT construct were cotransfected with 5 μg of pcDNAneo (Invitrogen) into A10 cells. Two days after transfection, 400 μg/ml G418 (Life Technologies, Inc.) was added to the medium. Three weeks after transfection, G418-resistant colonies were pooled and used for the experiments. The rat vascular smooth muscle (A10) cells (Dainippon Pharmaceutical, Osaka, Japan), NIH3T3 (RIKEN Cell Bank, Tsukuba, Japan), and rat skeletal muscle (Sol 8) cells (a kind gift of Dr. Koren, Harvard University) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. All culture medium contained 100 units/ml penicillin G and 100 μg/ml streptomycin.Genomic Southern BlotsTen micrograms of rat genomic DNA prepared from the spleen was digested with the restriction endonucleases, EcoRI, BamHI, and XbaI, resolved by electrophoresis on a 0.9% agarose gel, and blotted onto a nylon membrane. The membrane was hybridized with the 32P-labeled 708-bp BamHI-KpnI cDNA fragment as described (14Matsubara H. Liman E.R. Hess P. Koren G. J. Biol. Chem. 1991; 266: 13324-13328Abstract Full Text PDF PubMed Google Scholar) and washed in 0.1 × SSC + 0.1% SDS at 60°C.Reagents and Statistical MethodsAll reagents were purchased from Sigma unless otherwise indicated below. Results are expressed as mean ± S.E. Analyses of variance and the Newmann-Keuls test were used for multigroup comparisons. Values of p < 0.05 were considered statistically significant.RESULTSIsolation and Characterization of Rat V1a Receptor Genomic ClonesA total of ∼106 clones from the genomic DNA library were screened with a 32P-labeled rat V1a cDNA probe. Four positive clones were isolated and analyzed further by means of restriction enzyme mapping and Southern blotting. The results indicated that these could be segregated into two overlapping clones, of which two (RGV1 and RGV2) harbored the entire rat V1a receptor cDNA sequence as reported by Morel et al.(4Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (458) Google Scholar). Fig. 1 shows a map of these clones with recognition sites for the restriction endonucleases EcoRI and BamHI.Figure 1:Schematic representation of the rat V1a receptor gene. A, the gene locus is shown with the recognition sites for EcoRI (R) and BamHI (B) marked. Open and hatched boxes indicate nonexpressed and expressed exons, respectively. B, the structure of the rat V1a cDNA is presented at the bottom. Encoded transmembrane domains (I-VII) and other domains are displayed as hatched and open boxes, respectively. Exons in the genomic DNA and their corresponding regions of the cDNA are connected by solid lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Exon-Intron Structure of Rat V1a Receptor GeneA 3.7-kb BamHI-BamHI DNA fragment (Fig. 1) was subcloned into pBs KS(-) and sequenced bidirectionally with oligonucleotide primers designed from the V1a cDNA sequences(4Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (458) Google Scholar). A comparison of the V1a receptor genomic sequence with that of its cDNA indicated that one intron (∼1.8 kb) was located between the sixth and seventh putative transmembrane domains. The sequence of the splice site conformed to the GT-AG rule (Table 1)(17Breathnach R. Benoist C. O'Hare K. Cannon F. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4853-4857Crossref PubMed Scopus (866) Google Scholar). PCR using the primers designed from the intron sequence showed that the intron is about 1.8 kb in length (data not shown).Table I Open table in a new tab Sequence Analysis of Upstream 5′-Flanking RegionTo identify the DNA elements that control and direct rat V1a receptor gene transcription, we sequenced the 2208-bp region upstream from the ATG initiation codon. As shown in Fig. 2, neither TATA nor CCAAT promoter elements were found within the region containing the proximal major transcriptional initiation sites and the promoter activity, although sequences homologous to these motifs could be identified more than 800 bp upstream of the translational initiation site. It was, however, noted that the G + C content was 62% in the 400 bp immediately 5′ to the ATG initiation codon. This is higher than that of the average mammalian genome (40%); however, a GC box motif was not found in this region, suggesting that the promoter of the V1a receptor gene has features typical of a housekeeping gene(19Dynan W.S. Trends. Genet. 1986; 2: 196-197Abstract Full Text PDF Scopus (370) Google Scholar).Further analysis of the 5′-flanking region using a computer program (TFD 7.2) revealed potential TATA-like (−616 and −447)(20Breathnach R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Crossref PubMed Scopus (3287) Google Scholar), Sp1 binding (−557 and −1449)(21Kadonaga J.T. Jones K.A. Tjian R. Trends Biochem. Sci. 1986; 11: 20-23Abstract Full Text PDF Scopus (875) Google Scholar), CCAAT(−1530)(22Maniatis T. Goodbourn S. Fischer J.A. Science. 1987; 236: 1237-1245Crossref PubMed Scopus (908) Google Scholar), and AP-1(−1705) and AP-2 binding (−1688 and −605) (23Faisst S. Meyer S. Nucleic Acids Res. 1992; 20: 3-26Crossref PubMed Scopus (938) Google Scholar) sequences (Fig. 2). The TATA-like sequence(−447) is located at the position more than 200 bp upstream of the major transcriptional initiation sites and 48 bp upstream of the minor initiation site. The rat oxytocin receptor gene also has a promoter region and transcriptional initiation sites at positions more than 500 bp downstream of the TATA sequence(10Rosen F. Russo C. Banville D. Zingg H.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 200-204Crossref PubMed Scopus (197) Google Scholar). Although NF-κB (−716 and −677), PEA3 (−1122 and −683), and GATA (−1713) binding sites (23Faisst S. Meyer S. Nucleic Acids Res. 1992; 20: 3-26Crossref PubMed Scopus (938) Google Scholar) are located in the 5′-flanking region, the regulation of the rat V1a receptor expression by these factors has not been reported.Determination of Transcriptional Initiation Sites by Primer Extension, RNase Protection, and 5′-RACEResults of primer extension experiments using total RNA isolated from rat liver and A10 (rat vascular smooth muscle) cells are shown in Fig. 3A. Two primers were designed from the sequences from nt −68 to −98 and +21 to +51 relative to the ATG initiation codon. Three extended bands were detected by both primer extension analyses, corresponding to positions −405, −243, and −236 relative to the ATG codon (the results obtained using nt −68 to −98 primer are shown in Fig. 3A). No extended signals were observed when yeast tRNA or total RNA prepared from NIH3T3 or Sol 8 skeletal muscle cells, in which no V1a receptor mRNA was detected by reverse transcription-PCR analyses, was used as a control. To confirm the results obtained by primer extension analyses, we performed RNase protection analyses. The riboprobe used in this assay was an antisense RNA runoff from a HincII-digested EcoRI-BamHI genomic DNA fragment subcloned into pBs KS(-). This HincII site is located at position −480 from the first ATG codon. If transcriptions begin at position −405, −243, and −236, as the result of primer extension analyses suggested, this probe would protect fragments 334, 171, and 164 nucleotides in length, respectively. Fig. 3B shows the results of RNase protection assays showing two major bands migrating at positions corresponding to approximately 171 nt and 164 nt, and one faint band of approximately 334 nt following RNase protection of total RNA prepared from liver and A10 cells. Protected bands were not evident when yeast tRNA or total RNA from NIH3T3 or Sol 8 skeletal muscle cells were used as controls. Thus, RNase protection analyses confirmed the presence of the three transcriptional start sites indicated by primer extension analyses.Figure 3:Primer extension (A) and RNase protection (B) analyses of the transcriptional initiation sites of the rat V1a receptor gene. In A, the end-labeled 30-nt primer (see Fig. 2) was hybridized at 42°C