FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1975644972> ?p ?o ?g. }

• W1975644972 endingPage "18270" @default.
• W1975644972 startingPage "18261" @default.
• W1975644972 abstract "The ATP-sensitive potassium channels (K+ATP channels) are heteromultimeric structures formed by a member of the sulfonylurea receptor (SUR) family and a member of the inwardly rectifying potassium channel family (Kir6.x). The K+ATP channels play an essential role in nutrient-induced insulin secretion from the pancreatic β-cell. We have cloned and characterized the promoter region of the mouse SUR1 gene, and have shown that it lacks CAAT and TATA boxes or an initiator element. Studies of transcription initiation in several tissues showed that there is a common SUR1 promoter in brain, heart, and pancreas and in the pancreatic β-cell line, βTC3. The SUR1 gene uses multiple transcription start sites with the major site located 54 base pairs 5′-upstream of the translation initiation site. Transient transfection experiments in pancreatic β-cell lines showed that the proximal promoter fragment −84/+54 is sufficient for significant transcriptional activity. The proximal promoter region contains multiple SP1-binding sites, and cotransfection experiments of the SUR1 promoter-luciferase vector with SP1 expression vector inDrosophila SL2 cells demonstrated a stimulatory effect of SP1 on SUR1 transcriptional activity. The mobility shift assays confirmed the interaction of the SP1 transcription factor with the proximal promoter region of the SUR1 gene. Together, these results indicate that SP1 may mediate transcription initiation of the SUR1 gene. In addition, we have described the coordinate regulation of the gene expression of both K+ATP channel subunits by glucocorticoids. SUR1 and Kir6.2 mRNA levels are down-regulated by ∼40–50% in response to glucocorticoid treatment. Interestingly, the extent of the inhibitory effect as well as the kinetics and sensitivity are very similar for both mRNAs. Studies of mRNA turnover demonstrate that glucocorticoids most likely decrease the transcriptional activity of both SUR1 and Kir6.2 genes since glucocorticoids failed to affect the stability of each mRNA. Likewise, the reduction in mRNA levels was correlated with a decrease in SUR1 and Kir6.2 protein levels. The ATP-sensitive potassium channels (K+ATP channels) are heteromultimeric structures formed by a member of the sulfonylurea receptor (SUR) family and a member of the inwardly rectifying potassium channel family (Kir6.x). The K+ATP channels play an essential role in nutrient-induced insulin secretion from the pancreatic β-cell. We have cloned and characterized the promoter region of the mouse SUR1 gene, and have shown that it lacks CAAT and TATA boxes or an initiator element. Studies of transcription initiation in several tissues showed that there is a common SUR1 promoter in brain, heart, and pancreas and in the pancreatic β-cell line, βTC3. The SUR1 gene uses multiple transcription start sites with the major site located 54 base pairs 5′-upstream of the translation initiation site. Transient transfection experiments in pancreatic β-cell lines showed that the proximal promoter fragment −84/+54 is sufficient for significant transcriptional activity. The proximal promoter region contains multiple SP1-binding sites, and cotransfection experiments of the SUR1 promoter-luciferase vector with SP1 expression vector inDrosophila SL2 cells demonstrated a stimulatory effect of SP1 on SUR1 transcriptional activity. The mobility shift assays confirmed the interaction of the SP1 transcription factor with the proximal promoter region of the SUR1 gene. Together, these results indicate that SP1 may mediate transcription initiation of the SUR1 gene. In addition, we have described the coordinate regulation of the gene expression of both K+ATP channel subunits by glucocorticoids. SUR1 and Kir6.2 mRNA levels are down-regulated by ∼40–50% in response to glucocorticoid treatment. Interestingly, the extent of the inhibitory effect as well as the kinetics and sensitivity are very similar for both mRNAs. Studies of mRNA turnover demonstrate that glucocorticoids most likely decrease the transcriptional activity of both SUR1 and Kir6.2 genes since glucocorticoids failed to affect the stability of each mRNA. Likewise, the reduction in mRNA levels was correlated with a decrease in SUR1 and Kir6.2 protein levels. The ATP-sensitive potassium channels (K+ATPchannels) 1The abbreviations used are: K+ATP channel, ATP-sensitive potassium channel; SUR, sulfonylurea receptor; bp, base pair(s); DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; kb, kilobase(s); RACE, rapid amplification of cDNA ends; UTR, untranslated region; DRB, 5,6-dichlorobenzimidazole riboside.1The abbreviations used are: K+ATP channel, ATP-sensitive potassium channel; SUR, sulfonylurea receptor; bp, base pair(s); DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; kb, kilobase(s); RACE, rapid amplification of cDNA ends; UTR, untranslated region; DRB, 5,6-dichlorobenzimidazole riboside. couple cell metabolism to membrane potential in many tissues (see review by Ashcroft; Ref. 1Ashcroft F.M. Annu. Rev. Neurosci. 1988; 11: 97-118Crossref PubMed Scopus (768) Google Scholar). K+ATP channels are complexes of two structurally distinct subunits: an inwardly rectifying potassium channel subunit (Kir6.x) forming the pore (2Inagaki N. Gonoi T. Clement J.P. IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Google Scholar) and a regulatory subunit, the sulfonylurea receptor (SUR) belonging to the ATP-binding cassette superfamily (3Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P. IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1283) Google Scholar, 4Inagaki N. Gonoi T. Clement J.P. Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar, 5Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) Reconstitution experiments have shown that neither sulfonylurea receptor nor Kir6.x subunits are capable of channel activity when expressed alone in mammalian cells or Xenopusoocytes (2Inagaki N. Gonoi T. Clement J.P. IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Google Scholar, 3Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P. IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1283) Google Scholar, 6Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (676) Google Scholar). The subunits assemble in an octameric complex with 4:4 stoichiometry to form a functional K+ATPchannel (7Clement J.P. IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar, 8Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (246) Google Scholar, 9Shyng S. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (422) Google Scholar). Three isoforms of SURs have been cloned, SUR1 and two spliced variants of SUR2, SUR2A and SUR2B. The SUR1-Kir6.2 complex generates K+ATP channels with electrophysiological and pharmacological characteristics of pancreatic β-cell and neuronal-type channels, while SUR2A-Kir6.2 and SUR2B-Kir6.2 or Kir6.1 reconstitute the cardiac and vascular smooth muscle-type channels, respectively. The pancreatic β-cell K+ATP (SUR1-Kir6.2) channels play a critical role in the regulation of glucose-stimulated insulin secretion (10Cook D.L. Satin L.S. Ashford M.L. Hales C.N. Diabetes. 1988; 37: 495-498Crossref PubMed Scopus (207) Google Scholar). Under physiological conditions, the K+ATP channels in the β-cells are sensors of the ATP/ADP ratio. Following glucose metabolism, the increase in the intracellular ATP/ADP ratio inactivate the K+ATP channels resulting in membrane depolarization and opening of the voltage-dependentl-type Ca2+ channels with a subsequent increase in the intracellular Ca2+ concentration, initiating insulin secretion (see review by Bryan and Aguilar-Bryan; Ref. 11Bryan J. Aguilar-Bryan L. Curr. Opin. Cell Biol. 1997; 9: 553-559Crossref PubMed Scopus (107) Google Scholar). The specific functional and regulatory role of each subunit in the overall channel activity is being established by mutational analysis studies. While the Kir6.2 subunit forms the ion conducting pathway (11Bryan J. Aguilar-Bryan L. Curr. Opin. Cell Biol. 1997; 9: 553-559Crossref PubMed Scopus (107) Google Scholar, 12Shyng S. Ferrigni T. Nichols C.G. J. Gen. Physiol. 1997; 110: 141-153Crossref PubMed Scopus (128) Google Scholar), the SUR1 subunit confers sensitivity to MgADP (13Nichols C.G. Shyng S.L. Nestorowicz A. Glaser B. Clement J.P. IV Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. Science. 1996; 272: 1785-1787Crossref PubMed Scopus (468) Google Scholar, 14Shyng S. Ferrigni T. Nichols C.G. J. Gen. Physiol. 1997; 110: 643-654Crossref PubMed Scopus (246) Google Scholar) and pharmacological agents such as sulfonylureas (3Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P. IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1283) Google Scholar) and potassium channel openers (15Schwanstecher M. Sieverding C. Dorschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (196) Google Scholar). Furthermore, studies expressing a truncated form of Kir6.2 suggest that Kir6.2 may demonstrate some intrinsic sensitivity to ATP (6Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (676) Google Scholar). The essential role of both subunits in regulating insulin secretion is supported by the characterization of mutations in either subunit that abrogate channel activity and cause persistent hyperinsulinemic hypoglycemia of infancy (16Thomas P.M. Cote G.J. Wohllk N. Haddad B. Mathew P.M. Rabl W. Aguilar-Bryan L. Gagel R.F. Bryan J. Science. 1995; 268: 426-429Crossref PubMed Scopus (747) Google Scholar, 17Thomas P.M. Wohllk N. Huang E. Kuhnle U. Rabl W. Gagel R.F. Cote G.J. Am. J. Hum. Genet. 1996; 59: 510-518PubMed Google Scholar, 18Nestorowicz A. Wilson B.A. Schoor K.P. Inoue H. Glaser B. Landau H. Stanley C.A. Thornton P.S. Clement J.P. IV Bryan J. Aguilar-Bryan L. Permutt M.A. Hum. Mol. Genet. 1996; 5: 1813-1822Crossref PubMed Scopus (240) Google Scholar) characterized by constitutive insulin secretion despite severe hypoglycemia. Regulation of transcriptional activity of either SUR1 or Kir6.2 genes is important for determining the level of expression and therefore the activity of the K+ATP. In the present study, we have characterized the proximal promoter region of the mouse SUR1 gene and shown that there are no CAAT and TATA boxes or initiator element that may mediate initiation of transcription. Multiple transcription start sites exist with the major site located 54 bp 5′-upstream of the translation start site. Transient transfection experiments with different constructs of varying lengths of the SUR1 promoter region revealed that the basic elements required for significant basal transcriptional activity are located in the first 140 bp of the 5′-flanking region. In this particular region, there are multiple SP1 binding sites that are responsible for SP1 activation of SUR1 transcriptional activity. In addition, we show that glucocorticoids down-regulate the K+ATP channel levels by decreasing the SUR1 and Kir6.2 gene expression. Interestingly, the kinetics, sensitivity, and magnitude of inhibition of both genes are very similar. Moreover, dexamethasone does not affect the stability of either mRNA, suggesting an inhibition of transcriptional activity of both genes. Cell culture and reagents were purchased from Biofluids, Inc. (Rockville, MD) and Advanced Biotechnologies (Columbia, MD). LipofectAMINE and lipofectin reagents and the 5′ RACE kit were purchased from Life Technologies, Inc. RNazol reagent was purchased from Tel-Test, Inc (Friendswood, TX). The dual luciferase kit, pGL3 luciferase vectors, the pCMV Renilla construct, and the human recombinant SP1 purified protein were purchased from Promega (Madison, WI). The goat SP1 polyclonal antibody and the consensus Sp1 olionucleotide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The PacSP1-in expression vector, which contains the SP1 coding region, was a generous gift from Dr. Robert Tjian (University of California, Berkeley, CA). Insulin-free bovine serum albumin (fraction V) was obtained from Armour (Kankakee, IL). Dexamethasone, 5,6-dichlorobenzimidazole riboside (DRB), anti-β-actin antibody, and horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins were purchased from Sigma. Anti-α-tubulin antibody was purchased from Calbiochem (La Jolla, CA). βTC3 cells (19Efrat S. Linde S. Kofod H. Spector D. Delannoy M. Grant S. Hanahan D. Baekkeskov S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9037-9041Crossref PubMed Scopus (473) Google Scholar) were obtained from Dr. Shimon Efrat (New York, NY) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 25 mm glucose, supplemented with 2 mm glutamine, 15% horse serum, 2.5% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml fungizone. The mouse MIN6 cells (20Miyazaki J. Araki K. Yamato E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K. Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1053) Google Scholar) were obtained from Dr. Jun-ichi Miyazaki (Osaka, Japan) and Dr. Joel Habener (Boston, MA) and cultured in DMEM, 25 mm glucose supplemented with antibiotics, 2 mm glutamine, 10 mm Hepes, β-mercaptoethanol, and 15% heat-inactivated fetal bovine serum. βTC3 and MIN6 cells were incubated at 37 °C, in 5% CO2 and 90% relative humidity. Drosophila Schneider line 2 (SL2) cells, provided by Dr. Carl Wu (National Institutes of Health, Bethesda, MD), were grown in HyQ-CCM3 medium (HyClone, Logan, UT) at room temperature without CO2. A BAC mouse genomic library (Genome System, St. Louis, MO) was screened for the SUR1 gene with a 334-bp mouse SUR1 cDNA probe, corresponding to the 5′-region of the mouse SUR1 that includes part of the N terminus and the first two putative transmembrane domains (3Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P. IV Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1283) Google Scholar). This 334-bp cDNA fragment was obtained by reverse transcription followed by a polymerase chain reaction (PCR) and subcloned into a TA cloning vector as described previously (21Hernandez-Sanchez C. Wood T.L. LeRoith D. Endocrinology. 1997; 138: 705-711Crossref PubMed Scopus (20) Google Scholar). The positive SUR1 BAC clone, which contained ∼120 kb, was subcloned into three different sublibraries, usingBamHI, EcoRI, and HindIII restriction enzymes, with the pBluescriptII KS+ vector. Each sublibrary was screened for clones that contain the 5′ region of the SUR1 gene with the 334-bp SUR1 probe. The positive clones were characterized by restriction enzyme analysis and sequencing. The 5′ RACE procedure was performed according to manufacturer's instructions (Life Technologies, Inc.). Briefly, 1 μg of total RNA from βTC3 cells and mouse pancreas and heart were used as templates for a reverse transcriptase reaction with the SUR1 5′-ACAGAGGTGATGGCAGCCATG-3′ primer for βTC3 cells and 5′-AAGTTAGAGGTCTCAATG-3′ for the tissues. These primers are located 340 and 355 bp downstream of the translation start site, respectively. For the PCR reaction, the first and nested primers were, respectively, 5′-ATGTGTACCTTGGAGCTCTGG-3′ and 5′-GCACTACGTTGAGCGCGTCC-3′, which are located 157 and 82 bp downstream of the translation start site, respectively. The PCR products were analyzed in a 2% agarose gel and subcloned into a TA vector (Invitrogen, Carlsbad, CA). 17 clones from βTC3 cells, 4 from pancreas, and 1 from heart were analyzed by sequencing. Total RNA from βTC3 cells and mouse brain and heart was isolated using RNazol reagent according to the manufacturer's instructions. 20 μg of total RNA from βTC3 cells and 50 μg from brain and heart were hybridized with a mouse genomic SUR1 antisense riboprobe (2 × 105 dpm) that includes 605 bp of the 5′-flanking region, the first exon, and 200 bp of intron 1. The mouse genomic SUR1 riboprobe was generated by subcloning the 1000-bp BamHI to BamHI genomic fragment from the BAC mouse genomic clone into pBluescript-II KS+ atBamHI site. 32P-Labeled antisense riboprobe was synthesized by the T7 polymerase. Solution hybridization-RNase protection assays were carried out as described previously (22Werner H. Woloschak M. Adamo M. Shen-Orr Z. Roberts Jr., C.T. LeRoith D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7451-7455Crossref PubMed Scopus (327) Google Scholar). To create the −1136/+54 SUR1 promoter-luciferase construct, the 1370-bp fragment immediately upstream of the translation start site was generated by PCR using aEcoRI subclone of the BAC clone as template and 5′-TTCCATCAATGAGGAGCAGG-3′ as forward primer and 5′-AAGGCCAAGGC CATGGTGG-3′ as reverse primer. The reverse primer introduced a single mutation (underlined) immediately 5′ upstream of the ATG to create an NcoI site (bold) that allowed the subcloning of this fragment immediately upstream of the ATG of the luciferase expression vector. To minimize the possibility of introducing errors during PCR, the high fidelityPfu DNA polymerase (Stratagene, La Jolla, CA) was used and a low number of cycles (15Schwanstecher M. Sieverding C. Dorschner H. Gross I. Aguilar-Bryan L. Schwanstecher C. Bryan J. EMBO J. 1998; 17: 5529-5535Crossref PubMed Scopus (196) Google Scholar) were performed. The resulting single PCR fragment was subcloned into the pGL3 basic luciferase expression vector, between the HindIII and NcoI sites. The identity and orientation of the PCR fragment was then characterized by restriction enzyme analysis and sequencing. The −605/+54, −441/+54, and −84/+54 SUR1 constructs were generated by BamHI,NheI, and PvuI digestions of the −1136/+54 construct, respectively. The −4500/+54 construct was originated by subcloning the HindIII 3500-bp 5′-flanking fragment upstream of the −1136/+54 construct. βTC3 and MIN6 cells were transfected using the LipofectAMINE reagent according to manufacture's recommendations. βTC3 and MIN6 cells were plated in six-well plates prior transfection; each well received 1.5 and 1 μg of a given SUR1 promoter-luciferase construct, respectively, and 10 ng of the pCMVRenilla vector to correct for the variable transfection efficiencies. Firefly and Renilla luciferase activities were measured 48 h after transfection with the dual luciferase system (Promega, Madison, WI) according to manufacturer's instructions. To examine transactivation of SUR1 promoter with Sp1,Drosophila SL-2 cells were cotransfected with SUR1 promoter-luciferase and SP1 expression vector. SL2 cells were transfected using the lipofectin reagent according to manufacturer's recommendations. Twenty-four hours before transfection, SL2 cells were plated in 25-cm2 flasks, then transfected with 2 μg of SUR1 luciferase constructs mixed with different amounts of pPacSP1 expression vector. Total DNA content (up to 3 μg/flask) was normalized to pPac0 vector devoid of SP1 coding sequence. Luciferase assays were performed 48 h after transfection. The protein content of cell extracts was quantitated by the BCA protein assay reagent kit (Pierce). Nuclear extract from βTC3 cells was prepared according to Dignam et al. (23Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9153) Google Scholar). Briefly, βTC3 cells were rinsed and scraped in cold phosphate-buffered saline, resuspended in hypotonic buffer (10 mm HEPES, 1.5 mm MgCl2, 10 mm KCl, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol), and homogenized. The nuclei were removed by centrifugation and resuspended in low salt buffer (20 mm HEPES, 25% glycerol, 1.5 mmMgCl2, 20 mm KCl, 0.2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mmdithiothreitol). The salt concentration of the nuclear suspension was adjusted to 0.3 m KCl, which releases soluble nuclear proteins. The extracted nuclear proteins were collected as the supernatant after centrifugation and stored in aliquots at −70 °C. The concentration of the nuclear extract was quantitated by the BCA protein assay reagent kit (Pierce). Double-stranded oligonucleotides for mobility shift were end-labeled with [γ-32P]ATP and T4 polynucleotide kinase. Radiolabeled oligonucleotides (1 ng) were added to 4 μg of βTC3 cell nuclear extract in a final volume of 15 μl containing 1 μg of poly(dI-dC) (Amersham Pharmacia Biotech), 10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 1 mm EDTA, 0.05% Nonidet P-40, 5% glycerol, and 0.5 mm dithiothreitol. The reaction mixtures were incubated for 20 min at room temperature. Subsequently, the DNA-protein complexes were separated from the free DNA by electrophoresis through a 4% nondenaturing polyacrylamide gel in a 90 mm Tris borate, 2 mm EDTA buffer. The gels were dried and exposed to x-ray film. Reaction mixtures with Sp1 protein included 1.0 footprint unit of purified Sp1 (1 footprint unit is defined as the amount of Sp1 needed to give full protection against DNase1 digestion on the SV40 early promoter). Competition experiments included a 100-fold excess of unlabeled DNA oligonucleotides, while supershift analysis included the addition of 1 μg of anti-SP1 antibody to the mix and incubated for an additional 1 h at 4 °C. Mouse genomic P1 clone (Genome System Inc., St. Louis, MO) and mouse genomic DNA were digested with the indicated restriction enzymes. DNA digestion fragments were separated in a 0.8% agarose gel and transfer to a nylon membrane (Schleicher & Schuell). The membrane was hybridized with the mouse Kir6.2 RNA probe described below. After stripping, the membrane was re-hybridized with the mouse 3′ SUR1 antisense riboprobe described previously (21Hernandez-Sanchez C. Wood T.L. LeRoith D. Endocrinology. 1997; 138: 705-711Crossref PubMed Scopus (20) Google Scholar). βTC3 and MIN6 cells were serum-starved for 24 h prior to incubation with dexamethasone. Serum-free DMEM plus 25 mm glucose containing 0.1% bovine serum albumin, 20 mm Hepes, pH 7.5, antibiotics, and glutamine was used during serum starvation and dexamethasone stimulation. Total RNA (10–20 μg) was hybridized with a32P-labeled mouse antisense RNA Kir6.2 and SUR1 probes (2 × 105 dpm). The mouse 5′ SUR1 riboprobe was generated as described previously (21Hernandez-Sanchez C. Wood T.L. LeRoith D. Endocrinology. 1997; 138: 705-711Crossref PubMed Scopus (20) Google Scholar). The mouse Kir6.2 antisense RNA probe, corresponding to the putative pore segment and the second transmembrane domain (2Inagaki N. Gonoi T. Clement J.P. IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Google Scholar), was generated by reverse transcription followed by PCR. Briefly, the reverse transcription (Roche Molecular Biochemicals) reaction was performed with 3 μg of MIN6 cell total RNA according to the manufacturer's directions. A 217-bp PCR product was obtained and subcloned into a TA cloning vector (Invitrogen, San Diego, CA). The identity and orientation of the construct was verified by sequence analysis. After linearization of this construct withHindIII, the 32P-labeled antisense RNA probe was generated with T7 RNA polymerase. The normalization to total RNA was performed by co-hybridizing with an 18 S ribosomal RNA (rRNA) probe (Ambion, Austin, TX). A mouse β-actin antisense riboprobe (Ambion, Austin, TX) was also included. βTC3 cells were serum-starved for 24 h and then incubated with DRB (final concentration 100 μm) alone or DRB plus 10 or 100 nmdexamethasone for various times. Total RNA was isolated as described above, and the levels of SUR1 and Kir6.2 mRNA were measured by the solution hybridization/RNase protection assay. Anti-SUR1 antiserum was raised in rabbits using a synthetic rat SUR1 peptide extending from amino acids 1569–1581 (CKDSVFASFVRADK). Anti-Kir6.2 antiserum was also raised in rabbits using a synthetic mouse Kir6.2 peptide extending from amino acids 352–370 (CDRSLLDALTLASSRGPLRK). βTC3 and MIN6 cells were serum-starved for 24 h and then incubated with 100 nm dexamethasone for 16 and 24 h, respectively. Cells were lysed in the presence of 50 mmTris-HCl, pH 8, 300 mm NaCl, 10 mm EDTA, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin, and 2 mg/ml aprotinin. Cell lysates were clarified by centrifugation. Protein content was determined using the BCA protein assay reagent kit (Pierce). 30–40 μg of protein were reduced by β-mercaptoethanol and fractionated by 7.5% SDS-PAGE. Resolved proteins were electrophoretically transferred to nitrocellulose membrane (Schleicher & Schuell). For SUR1 experiments, the upper part of the membrane was incubated with anti-SUR1 antibody (1:1,000 dilution). The bottom part of the membrane was incubated with β-actin or α-tubulin antibodies (1:1,000 dilution) and detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin (1:5,000 and 1:10,000 dilution, respectively) using the ECL system. For Kir6.2 experiments, membranes were first incubated with anti-Kir6.2 antibody (1:500 dilution) detected with horseradish peroxidase-conjugated immunoglobulin 1:5,000 and then stripped and reblotted with β-actin antibody as described above. SUR1, Kir6.2, β-actin, and α-tubulin bands were scanned by Fotolook version 2.08 (Agfa) and quantify by MacBas version 2.31 (Fuji Photo Film Co). To characterize the promoter region of the mouse SUR1 gene and study the possible regulatory elements that control SUR1 transcriptional activity, we cloned the 5′-flanking region of the mouse SUR1 gene. One genomic clone from a mouse BAC library was isolated and subcloned as described under “Experimental Procedures.” Computer analysis using the MatInspector program (24Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2423) Google Scholar) of the nucleotide sequence of the 5′-flanking region of the mouse SUR1 revealed that there is no TATA box, CAAT box, or initiator element (Fig.1; GenBank accession number AF037274), items that commonly specify the transcription start site. However, the proximal 5′-flanking region (−86 to +24) contains several GC boxes (GGCG) that predict multiple SP1 binding sites. SUR1 gene is expressed in the pancreatic islets as well as other tissues (21Hernandez-Sanchez C. Wood T.L. LeRoith D. Endocrinology. 1997; 138: 705-711Crossref PubMed Scopus (20) Google Scholar): mainly brain, heart, and skeletal muscle. To study whether these tissues utilize the same promoter or there are distinct promoter regions, we examined the transcription start site in mouse pancreas, brain, and heart as well as in the mouse pancreatic β-cell line βTC3, using the 5′ RACE and solution hybridization RNase protection assay methods. The 5′ RACE procedure is a sensitive method for cloning the 5′ end of a particular mRNA. We performed this procedure on RNA from the βTC3 cells and mouse pancreas and heart RNA. As shown in Fig.2 A, a 270–280-bp initial PCR product was obtained in βTC3 cells and pancreas. The subsequent nested PCR displayed an ∼194-bp product in the three cases. All of these PCR products were cloned and subsequently sequenced. Identical sequences were obtained from the βTC3 cells and pancreatic and cardiac tissues, although some diversity in the 5′-untranslated region (5′-UTR) length was noted, extending between 82 and 21 bp of the 5′-flanking region. We found the variability mainly in the βTC3 cells. To determine the exact transcription start site of the SUR1 gene, we hybridized 20 and 50 μg of total RNA from βTC3 cells and brain and heart, respectively, with a mouse genomic antisense riboprobe corresponding to 200 bp of intron 1, the complete first exon, and 605-bp of the 5′-flanking region, in a solution hybridization/RNase protection assay. As shown in Fig. 2 B, a major 204-bp protected band was obtained in all cases. In the case of the βTC3 cells, other signals were seen, suggesting the presence of other minor protected bands. When other genomic probes extending further upstream, up to 2800 bp, were used, no additional protected bands were seen (data not shown). These data suggest that there is a common promoter and similar transcription start sites in βTC3 cells, mouse pancreas, heart, and brain tissues. It is likely that the 5′-UTR of SUR1 is similar in the other tissues expressing SUR1 (21Hernandez-Sanchez C. Wood T.L. LeRoith D. Endocrinology. 1997; 138: 705-711Crossref PubMed Scopus (20) Google Scholar). The major transcription start site is located approximately 54-bp 5′ to the ATG translational codon. Other minor start sites for transcription were mapped between 82 and 21 nucleotides 5′ to the ATG translational codon. The presence of multiple transcription start sites is characteristic of TATA-less GC-rich promoters (25Reynolds G.A. Basu S.K. Osborne T.F. Chin D.J. Gil G. Brown M.S. Goldstein J.L. Luskey K.L. Cell. 1984; 38: 275-285Abstract Full Text PDF PubMed Scopus (266) Google Scholar, 26McKeon C. Moncada V. Pham T. Salvatore P. Kadowaki T. Accili D. Taylor S.I. Mol. Endocrinol. 1990; 4: 647-656Crossref PubMed Scopus (39) Google Scholar). The functional role of the 5′-" @default.
• W1975644972 created "2016-06-24" @default.
• W1975644972 creator A5013489365 @default.
• W1975644972 creator A5026684297 @default.
• W1975644972 creator A5062031732 @default.
• W1975644972 creator A5069366555 @default.
• W1975644972 creator A5070276458 @default.
• W1975644972 date "1999-06-01" @default.
• W1975644972 modified "2023-10-14" @default.
• W1975644972 title "Characterization of the Mouse Sulfonylurea Receptor 1 Promoter and Its Regulation" @default.
• W1975644972 cites W1545776085 @default.
• W1975644972 cites W1577890972 @default.
• W1975644972 cites W1667331074 @default.
• W1975644972 cites W1982558048 @default.
• W1975644972 cites W1986381180 @default.
• W1975644972 cites W1986804446 @default.
• W1975644972 cites W1993073314 @default.
• W1975644972 cites W1993243592 @default.
• W1975644972 cites W1995589371 @default.
• W1975644972 cites W2012181178 @default.
• W1975644972 cites W2022446120 @default.
• W1975644972 cites W2037104430 @default.
• W1975644972 cites W2038862314 @default.
• W1975644972 cites W2059328843 @default.
• W1975644972 cites W2061787346 @default.
• W1975644972 cites W2063564339 @default.
• W1975644972 cites W2063574029 @default.
• W1975644972 cites W2064879068 @default.
• W1975644972 cites W2074351361 @default.
• W1975644972 cites W2074874980 @default.
• W1975644972 cites W2075931967 @default.
• W1975644972 cites W2083584165 @default.
• W1975644972 cites W2092249523 @default.
• W1975644972 cites W2127897718 @default.
• W1975644972 cites W2129668416 @default.
• W1975644972 cites W2141063928 @default.
• W1975644972 cites W2147706045 @default.
• W1975644972 cites W2148255642 @default.
• W1975644972 cites W2148959338 @default.
• W1975644972 cites W2159947557 @default.
• W1975644972 cites W2160289208 @default.
• W1975644972 cites W2168187284 @default.
• W1975644972 cites W2287333882 @default.
• W1975644972 doi "https://doi.org/10.1074/jbc.274.26.18261" @default.
• W1975644972 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10373428" @default.
• W1975644972 hasPublicationYear "1999" @default.
• W1975644972 type Work @default.
• W1975644972 sameAs 1975644972 @default.
• W1975644972 citedByCount "34" @default.
• W1975644972 countsByYear W19756449722012 @default.
• W1975644972 countsByYear W19756449722015 @default.
• W1975644972 countsByYear W19756449722016 @default.
• W1975644972 countsByYear W19756449722017 @default.
• W1975644972 countsByYear W19756449722018 @default.
• W1975644972 countsByYear W19756449722020 @default.
• W1975644972 countsByYear W19756449722021 @default.
• W1975644972 crossrefType "journal-article" @default.
• W1975644972 hasAuthorship W1975644972A5013489365 @default.
• W1975644972 hasAuthorship W1975644972A5026684297 @default.
• W1975644972 hasAuthorship W1975644972A5062031732 @default.
• W1975644972 hasAuthorship W1975644972A5069366555 @default.
• W1975644972 hasAuthorship W1975644972A5070276458 @default.
• W1975644972 hasBestOaLocation W19756449721 @default.
• W1975644972 hasConcept C102942909 @default.
• W1975644972 hasConcept C104292427 @default.
• W1975644972 hasConcept C104317684 @default.
• W1975644972 hasConcept C134018914 @default.
• W1975644972 hasConcept C170493617 @default.
• W1975644972 hasConcept C185592680 @default.
• W1975644972 hasConcept C2778854520 @default.
• W1975644972 hasConcept C55493867 @default.
• W1975644972 hasConcept C555293320 @default.
• W1975644972 hasConcept C86803240 @default.
• W1975644972 hasConcept C95444343 @default.
• W1975644972 hasConceptScore W1975644972C102942909 @default.
• W1975644972 hasConceptScore W1975644972C104292427 @default.
• W1975644972 hasConceptScore W1975644972C104317684 @default.
• W1975644972 hasConceptScore W1975644972C134018914 @default.
• W1975644972 hasConceptScore W1975644972C170493617 @default.
• W1975644972 hasConceptScore W1975644972C185592680 @default.
• W1975644972 hasConceptScore W1975644972C2778854520 @default.
• W1975644972 hasConceptScore W1975644972C55493867 @default.
• W1975644972 hasConceptScore W1975644972C555293320 @default.
• W1975644972 hasConceptScore W1975644972C86803240 @default.
• W1975644972 hasConceptScore W1975644972C95444343 @default.
• W1975644972 hasIssue "26" @default.
• W1975644972 hasLocation W19756449721 @default.
• W1975644972 hasOpenAccess W1975644972 @default.
• W1975644972 hasPrimaryLocation W19756449721 @default.
• W1975644972 hasRelatedWork W1171717213 @default.
• W1975644972 hasRelatedWork W2054847105 @default.
• W1975644972 hasRelatedWork W2073901267 @default.
• W1975644972 hasRelatedWork W2113676079 @default.
• W1975644972 hasRelatedWork W2379669210 @default.
• W1975644972 hasRelatedWork W2438518849 @default.
• W1975644972 hasRelatedWork W2748952813 @default.
• W1975644972 hasRelatedWork W2917502842 @default.
• W1975644972 hasRelatedWork W3098409675 @default.

• next