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Article: Characterization of a novel liver-specific enhancer in the human prothrombin gene

TitleCharacterization of a novel liver-specific enhancer in the human prothrombin gene
Authors
Issue Date1991
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 1991, v. 266 n. 28, p. 18927-18933 How to Cite?
AbstractThe 5'-flanking sequence of the human prothrombin gene was isolated by screening a human liver phage library with a human prothrombin cDNA as a hybridization probe. A phage was identified that contained 3 kilobase pairs of DNA upstream of the initiator methionine codon. Primer extension studies showed that the major transcription initiation sites were located 23 and 36 base pairs upstream of the initiator codon. DNA sequences in the 5'-flanking region of the human prothrombin gene were then analyzed for cis-activating transcriptional activity by a transient expression system using the human growth hormone gene as the reporter gene. The chimeric expression vector was introduced into HepG2 cells, and secreted human growth hormone was monitored by using a radioimmunoassay. These studies showed that the 3-kilobase pair fragment contained sequences that were sufficient for the initiation of transcription in HepG2 cells. Subsequent deletion studies showed that the 3-kilobase pair fragment contained two elements: a weak promoter in the region immediately upstream of the mRNA coding sequence and an enhancer located between nucleotides -860 and -940. The enhancer element was active at a distance and in either orientation. In addition, the enhancer was liver cell-specific and acted on heterologous promoters including the herpes simplex virus thymidine kinase promoter and the mouse metallothionein I promoter. Comparison of the nucleotide sequence of the enhancer with a DNA sequence data base showed the enhancer sequence to be unique. The enhancer sequence is flanked by an inverted repeat 5' CCTCCC 3' and contains a putative binding site for hepatic nuclear factor 1. Deoxyribonuclease I footprint analysis and linker scanning mutagenesis showed that the enhancer contains multiple protein binding motifs. Mutagenesis of the 3' boundary CCTCCC sequence eliminated the enhancer activity. Comparison with other liver genes showed the presence of the CCTCCC sequence in the hepatitis B virus enhancer, the α1-antitrypsin promoter, and the fibrinogen β-chain promoter, suggesting a functional role for this motif.
Persistent Identifierhttp://hdl.handle.net/10722/178508
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorChow, BKCen_US
dc.contributor.authorTing, Ven_US
dc.contributor.authorTufaro, Fen_US
dc.contributor.authorMacgillivray, RTAen_US
dc.date.accessioned2012-12-19T09:48:06Z-
dc.date.available2012-12-19T09:48:06Z-
dc.date.issued1991en_US
dc.identifier.citationJournal Of Biological Chemistry, 1991, v. 266 n. 28, p. 18927-18933en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://hdl.handle.net/10722/178508-
dc.description.abstractThe 5'-flanking sequence of the human prothrombin gene was isolated by screening a human liver phage library with a human prothrombin cDNA as a hybridization probe. A phage was identified that contained 3 kilobase pairs of DNA upstream of the initiator methionine codon. Primer extension studies showed that the major transcription initiation sites were located 23 and 36 base pairs upstream of the initiator codon. DNA sequences in the 5'-flanking region of the human prothrombin gene were then analyzed for cis-activating transcriptional activity by a transient expression system using the human growth hormone gene as the reporter gene. The chimeric expression vector was introduced into HepG2 cells, and secreted human growth hormone was monitored by using a radioimmunoassay. These studies showed that the 3-kilobase pair fragment contained sequences that were sufficient for the initiation of transcription in HepG2 cells. Subsequent deletion studies showed that the 3-kilobase pair fragment contained two elements: a weak promoter in the region immediately upstream of the mRNA coding sequence and an enhancer located between nucleotides -860 and -940. The enhancer element was active at a distance and in either orientation. In addition, the enhancer was liver cell-specific and acted on heterologous promoters including the herpes simplex virus thymidine kinase promoter and the mouse metallothionein I promoter. Comparison of the nucleotide sequence of the enhancer with a DNA sequence data base showed the enhancer sequence to be unique. The enhancer sequence is flanked by an inverted repeat 5' CCTCCC 3' and contains a putative binding site for hepatic nuclear factor 1. Deoxyribonuclease I footprint analysis and linker scanning mutagenesis showed that the enhancer contains multiple protein binding motifs. Mutagenesis of the 3' boundary CCTCCC sequence eliminated the enhancer activity. Comparison with other liver genes showed the presence of the CCTCCC sequence in the hepatitis B virus enhancer, the α1-antitrypsin promoter, and the fibrinogen β-chain promoter, suggesting a functional role for this motif.en_US
dc.languageengen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.subject.meshBase Compositionen_US
dc.subject.meshBase Sequenceen_US
dc.subject.meshCell Lineen_US
dc.subject.meshCloning, Molecularen_US
dc.subject.meshDnaen_US
dc.subject.meshDna Mutational Analysisen_US
dc.subject.meshDeoxyribonuclease Ien_US
dc.subject.meshEnhancer Elements, Geneticen_US
dc.subject.meshGene Expression Regulationen_US
dc.subject.meshGrowth Hormone - Genetics - Metabolismen_US
dc.subject.meshHumansen_US
dc.subject.meshLiver - Metabolismen_US
dc.subject.meshMolecular Sequence Dataen_US
dc.subject.meshPolymerase Chain Reactionen_US
dc.subject.meshProthrombin - Geneticsen_US
dc.subject.meshRestriction Mappingen_US
dc.subject.meshTransfectionen_US
dc.titleCharacterization of a novel liver-specific enhancer in the human prothrombin geneen_US
dc.typeArticleen_US
dc.identifier.emailChow, BKC: bkcc@hku.hken_US
dc.identifier.authorityChow, BKC=rp00681en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid1918008-
dc.identifier.scopuseid_2-s2.0-0025945326en_US
dc.identifier.volume266en_US
dc.identifier.issue28en_US
dc.identifier.spage18927en_US
dc.identifier.epage18933en_US
dc.identifier.isiWOS:A1991GJ47200080-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridChow, BKC=7102826193en_US
dc.identifier.scopusauthoridTing, V=7004316099en_US
dc.identifier.scopusauthoridTufaro, F=7004270082en_US
dc.identifier.scopusauthoridMacGillivray, RTA=7004286039en_US

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