| Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells. | |
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MedLine Citation:
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PMID: 1406648 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -180 and -1 bp contributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet beta cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G). Methylation of adenine and guanine residues within this sequence prevented binding of the beta-cell factor, as did mutations at positions 2, 3, and 5. Analysis of nuclear extracts from different cell lines identified UPE-binding activity in HIT M2.2.2 and beta-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in alpha-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the beta-cell type expression of this factor and showed it to be approximately 50 kDa in size. Gel mobility shift competition experiments showed that this beta-cell factor is the same that binds to similar elements, termed CT boxes, in the insulin promoter. Thus, a role for these elements (UPEs or CT boxes), and the beta-cell factor that binds to them, in determining the expression of genes in the beta cells of pancreatic islets is suggested. |
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Authors:
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K D Shelton; A J Franklin; A Khoor; J Beechem; M A Magnuson |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Molecular and cellular biology Volume: 12 ISSN: 0270-7306 ISO Abbreviation: Mol. Cell. Biol. Publication Date: 1992 Oct |
Date Detail:
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Created Date: 1992-10-26 Completed Date: 1992-10-26 Revised Date: 2010-09-07 |
Medline Journal Info:
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Nlm Unique ID: 8109087 Medline TA: Mol Cell Biol Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 4578-89 Citation Subset: IM |
Affiliation:
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Department of Molecular Physiology, Vanderbilt University Medical School, Nashville, Tennessee 37232. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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3T3 Cells Animals Base Sequence Cloning, Molecular Cricetinae DNA Gene Expression Regulation, Enzymologic Glucokinase / genetics*, metabolism Insulin / genetics Insulinoma Islets of Langerhans / enzymology* Mice Molecular Sequence Data Mutagenesis Organ Specificity / genetics Promoter Regions, Genetic* Transcription Factors / metabolism Transcription, Genetic* Transfection Tumor Cells, Cultured |
| Grant Support | |
ID/Acronym/Agency:
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DK42502/DK/NIDDK NIH HHS; DK42612/DK/NIDDK NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Transcription Factors; 11061-68-0/Insulin; 9007-49-2/DNA; EC 2.7.1.2/Glucokinase |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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