Document Detail

Characterization of the murine mitochondrial glycerol-3-phosphate acyltransferase promoter.
MedLine Citation:
PMID:  7836409     Owner:  NLM     Status:  MEDLINE    
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate, a committed step in triacylglycerol and phospholipid biosynthesis. We have previously reported the cDNA cloning and transcriptional regulation of the murine mitochondrial GPAT (mGPAT). We now report the cloning of the 5'-flanking region of the murine mitochondrial GPAT. The transcription start site was identified by primer extension and RNase protection assays. A TATA box-like motif (TTATTAT) was located between -34 and -29 and a reverse CCAAT box (ATTGG) was located between -78 and -74, relative to the transcription start site. To begin studying mechanisms underlying transcriptional regulation of the mGPAT gene, chimeric luciferase (LUC) plasmids containing serial deletions, from -1447 to -38, of the 5'-flanking region of the murine mGPAT gene were prepared and transfected into 3T3-L1 cells. The fusion construct -1447 GPAT.LUC showed high promoter activity and deletions to -1353, -747, -322, and -86 did not markedly change the promoter activity. With all constructs, luciferase activity was 2-fold higher when plasmids were transfected into 3T3-L1 adipocytes. However, deletion of sequences between -86 and -55 resulted in a 9-fold decrease in LUC activity in both preadipocytes and adipocytes. Deletion of sequences between -55 and -38 did not alter promoter activity. DNase I footprint analysis revealed a protected region between -95 and -65 which included the putative CTF/NF1 binding site. Electrophoretic mobility shift assays demonstrated a single protein-DNA complex formation. Oligonucleotides synthesized according to the CTF/NF1 consensus sequence or the adenovirus NF-1 site showed a different and more complex pattern of protein-DNA interaction and were not able to compete away the mGPAT promoter-protein complex, indicating that a distinct protein was bound to -86/-55, a region important for the basal promoter activity in 3T3-L1 cells. Luciferase activity was increased 2.8- and 8-fold when adipocytes stably transfected with -322 GPAT.LUC were treated with 5 and 25 mM glucose, respectively, in the presence of 10 nM insulin. These results indicate that carbohydrate-responsive sequences are located within -322 base pairs of the mGPAT promoter.
A A Jerkins; W R Liu; S Lee; H S Sul
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  270     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1995 Jan 
Date Detail:
Created Date:  1995-02-24     Completed Date:  1995-02-24     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1416-21     Citation Subset:  IM    
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115.
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MeSH Terms
3T3 Cells
Base Sequence
DNA Primers
Glycerol-3-Phosphate O-Acyltransferase / genetics*,  metabolism
Molecular Sequence Data
Nuclear Proteins / metabolism
Promoter Regions, Genetic*
Sequence Deletion
Grant Support
Reg. No./Substance:
0/DNA Primers; 0/Nuclear Proteins; EC O-Acyltransferase

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