Document Detail


Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.
MedLine Citation:
PMID:  2850495     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.
Authors:
P G Quinn; T W Wong; M A Magnuson; J B Shabb; D K Granner
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Molecular and cellular biology     Volume:  8     ISSN:  0270-7306     ISO Abbreviation:  Mol. Cell. Biol.     Publication Date:  1988 Aug 
Date Detail:
Created Date:  1989-02-23     Completed Date:  1989-02-23     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  8109087     Medline TA:  Mol Cell Biol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  3467-75     Citation Subset:  IM    
Affiliation:
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232.
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MeSH Terms
Descriptor/Qualifier:
Animals
Base Sequence
Cell Line
Chromosome Deletion
Cloning, Molecular
Cyclic AMP / pharmacology*
Genes*
Molecular Sequence Data
Mutation
Phosphoenolpyruvate Carboxykinase (GTP) / genetics*
Promoter Regions, Genetic* / drug effects
Regulatory Sequences, Nucleic Acid* / drug effects
Transfection
Grant Support
ID/Acronym/Agency:
DK 07061/DK/NIDDK NIH HHS; DK 35107/DK/NIDDK NIH HHS; F32 DK07870/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
60-92-4/Cyclic AMP; EC 4.1.1.32/Phosphoenolpyruvate Carboxykinase (GTP)
Comments/Corrections

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