Abstract
Apolipoprotein CIII (apoCIII) is a major protein constituent of triglyceride-rich lipoproteins and is synthesized primarily in the liver. Cis-acting DNA elements required for liver-specific apoCIII gene transcription were identified with transient expression assays in the human hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines. In liver cells, 821 nucleotides of the human apoCIII gene 5'-flanking sequence were required for maximum levels of gene expression, while the proximal 110 nucleotides alone were sufficient. No expression was observed in similar studies with HeLa cells. The level of expression was modulated by a combination of positive and negative cis-acting sequences, which interact with distinct sets of proteins from liver and HeLa cell nuclear extracts. The proximal positive regulatory region shares homology with similarly located sequences of other genes strongly expressed in the liver, including alpha 1-antitrypsin and other apolipoprotein genes. The negative regulatory region is strikingly homologous to the human beta-interferon gene regulatory element. The distal positive region shares homology with some viral enhancers and has properties of a tissue-specific enhancer. The regulation of the apoCIII gene is complex but shares features with other genes, suggesting shuffling of regulatory elements as a common mechanism for cell type-specific gene expression.
Highlights
ApolipoproteinCIIIis a major protein con- Apolipoprotein CIII’ is a major protein constitustituent oftriglyceride-richlipoproteins andis synthe- ent of the triglyceride-rich lipoproteins and is present in sized primarilyin the liver
To facilitate the study of apoCIII gene expression, we extended the previously reported sequence to include 821base pairs upstream of the transcription initiation site (Fig. 1).A portion of the apoCIII gene from approximately -2200 to +24 was cloned immediately upstream of the chloramphenicol acetyltransferase (CAT) gene coding sequences in the plasmid pKT.To identify DNA sequence elementsimportant for transcription,a series of deletion mutants extending from -2200 toward the start site of transcription was constructed (Fig. 2B)
These mutant constructions were introduced into both HepG2 and HepG2 cells epithelial carcinoma (HeLa) cells by the calcium phosphate co-precipitation method andtransient expression of CAT enzymatic activity measured
Summary
To facilitate the study of apoCIII gene expression, we extended the previously reported sequence to include 821base pairs upstream of the transcription initiation site (Fig. 1).A portion of the apoCIII gene from approximately -2200 to +24 (relative to the startsite of transcription) was cloned immediately upstream of the CAT gene coding sequences in the plasmid pKT (pKTCIII, Fig. 2A).To identify DNA sequence elementsimportant for transcription,a series of deletion mutants extending from -2200 toward the start site of transcription was constructed (Fig. 2B).
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