Abstract
Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the activation of transcription of an unidentified gene which encodes a 4.9-kilobase (kb) mRNA. Several cDNAs that include the complete sequence of this mRNA were obtained and used to isolate and characterize the gene. Analysis of the nucleotide and amino acid sequences of both cDNA and genomic clones revealed that the gene encodes the mouse stearoyl-CoA desaturase (SCD), an enzyme known to be expressed upon differentiation of 3T3-L1 preadipocytes. The predicted amino acid sequence (355 residues) of the mouse 3T3-L1 adipocyte SCD exhibits 92% identity to that of the rat liver SCD. There is also a high degree of nucleotide sequence identity between the mouse and rat mRNAs in their unusually long approximately 3.5-kb 3'-untranslated regions. Mice fed a diet containing unsaturated triacylglycerides express SCD mRNA only in adipose tissue, whereas mice starved and refed a fat-free diet, express SCD mRNA in both liver and adipose tissue. The mouse gene for the desaturase spans approximately 15 kb and contains 6 exons and 5 introns with all intron-exon junctions conforming to the GT/AG splicing rule. As determined by S1 nuclease mapping and primer extension analysis, the transcriptional initiation site maps 152 nucleotides upstream from the initiation methionine codon. A canonical promoter "TATA" box is located 30 base pairs upstream of the Cap site. A typical "CCAAT" box sequence is not present in the adjacent 5'-flanking region; however, there is a GC-rich sequence (at nucleotide -215) similar to the binding site for the nuclear transcription factor Sp1. Upstream from the transcriptional initiation site are elements with homology (approximately 75%) to the putative fat-specific transcriptional element FSE2 and core consensus sequences for cAMP and glucocorticoid regulatory elements. A chimeric construct, containing 363 base pairs of 5'-flanking sequence and 30 nucleotides of 5'-untranslated sequence of the mouse SCD gene ligated to the bacterial chloramphenicol acetyltransferase gene, was transfected into 3T3-L1 cells. When cells were induced to differentiate into adipocytes, expression of the SCD chloramphenicol acetyltransferase gene increased approximately 63-fold, suggesting that the SCD promoter region contains elements that mediate the response to adipogenic agents which induce differentiation.
Highlights
From the *Departmentsof Biological Chemistry and §§MolecularBiology and Genetics, The Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland21205
When cells and genomic clones revealed that thegene encodes the were induced to differentiate into adipocytes, expresmouse stearoyl-CoA desaturase (SCD), an enzyme sion of the SCD chloramphenicolacetyltransferase known tobe expressed upon differentiation of 3T3-Ll gene increased approximately 63-fold, suggestinthgat preadipocytes
Several years ago we identified and isolated a number of cDNA clones that correspond to mRNAs which increase in abundance upon differentiationof 3T3-Ll preadipocytes [18]
Summary
From the *Departmentsof Biological Chemistry and §§MolecularBiology and Genetics, The Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland21205. Cloning of the Gene Corresponding topAL122 cDNApAL122, a cDNAclone previously isolated from a 3T3-Ll adipocyte cDNA library, contains a 1.4-kb insert which corresponds to the 3' end of a 4.9-kb mRNA that is expressed upon differentiation of 3T3-Ll preadipocytes [18].To isolate the gene encoding pAL122cDNA, the PstI-PstI insert of pAL122 [18]was used as a probe to screen a genomic library of fractionated EcoRI fragments prepared with XgtWES.b as cloning vector.
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