• Das, Hriday (PI)

Project Details


Heart disease is a lethal disease in the United States. Atherosclerosis develops as a result of high serum cholesterol. Cholesterol is transported through the circulation as lipoprotein particles and the protein components of these particles are called apolipoproteins. ApoB, the only protein of LDL, is synthesized primarily in the liver, and plasma levels of LDL cholesterol and apoB correlate directly with atherosclerosis susceptibility. Therefore, factors regulating apoB gene expression should participate in the regulation of plasma cholesterol level, and may be involved in the development of hyperlipidemia and heart diseases. Since traps-acting protein factors interact with cis-acting DNA elements and with themselves to mediate liver specific expression of the human apoB gene, it is necessary to purify and characterize these factors and clone their structural genes to understand the mechanism of hyperlipidemia. To achieve this goal, two such apoB gene regulatory factors BRF-1 and BRF-2, which bind to the apoB gene regulatory elements (-84 to -60) and (-128 to -85) respectively have been purified to apparent homogeneity by DNA- specific affinity chromatography. The molecular weights of these proteins have been determined to be 68 kDa and 120 kDa respectively by SDS/PAGE. The NH2- terminal sequence of BRF-l has been determined to be NH2-Gly-Arg- Ser-Ala-Gly-Ala-Phe-Gly-Arg-Val-Arg-Ile-Glu. A polyclonal anti serum against this peptide has also been raised in rabbit to clone cDNA encoding BRF-1. Factor(s) BRF-3/BRF-4 which produced footprints~(+30 to +41) and (+44 to +53) on the apoB promoter, have also been partially purified. Factors BRF-3/BRF-4 will be purified by DNA-specific affinity chromatography and characterized by SDS/PAGE, DNaseI footprinting and in vitro transcription. Genes encoding these factors will be cloned using antibody probes. Full or partial length cDNAs will be used to synthesize full size or truncated forms of proteins in E. Coli or in an in vitro transcription-translation system. Recombinant proteins could be used to study the role of these factors in apoB gene transcription. Truncated proteins synthesized in vitro could be used to map their domains for DNA- protein, and protein-protein interactions as well as to delineate domains needed for the formation of oligomeric structure. The possible role of these factors in the transcription of other apolipoprotein genes will also be studied.
Effective start/end date1/09/9330/09/03


  • National Heart, Lung, and Blood Institute


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