HEPATIC REGULATION OF THE HUMAN APO-B GENE

Project Details

Description

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.
StatusFinished
Effective start/end date1/09/9330/09/03