Studies of acetyl-CoA carboxylase gene expression
Abstract
Acetyl-Coenzyme A carboxylase (ACC; acetyl-CoA:carbon dioxide ligase (ADP forming), EC 6.4.1.2) catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The construction of ribozyme genes that are designed to hydrolyze acetyl-CoA carboxylase mRNAs and the effects of their expression on long chain fatty acid synthesis are described. These ribozymes not only precisely cleaved ACC mRNA at the expected sites in a cell free system, but also specifically suppressed ACC gene expression in the cells. The ribozyme-expressing cells show a substantial reduction in the amount of ACC mRNA as compared to non-ribozyme expressing cells. The ACC enzyme activity of the ribozyme-expressing cells fell to about 30 to 70% of the control, and the decrease in the ACC enzyme activity was directly reflected in the rate of fatty acid synthesis. This indicates that ACC is the rate-limiting enzyme in the pathway of fatty acid synthesis. cDNA clones for human acetyl-CoA carboxylase have been isolated and sequenced. Human ACC mRNA contains an open reading frame 7020 nucleotides long; it encodes a polypeptide of 2340 amino acids having a calculated molecular weight of 264,575. The human ACC shows approximately 85% identity in nucleotide sequence with previously cloned rat ACC, and 90% identity in the amino acid sequence. Two human ACC mRNA species which differ solely in the 5$\sp\prime$-end untranslated region were isolated, and these mRNAs were expressed in a tissue specific manner. The construction of full length cDNA containing the complete coding region of rat ACC and expression of the recombinant ACC are described. The recombinant ACC contains all the properties of the endogenous ACC with respect to the molecular weight, cross-activity with antiserum raised against purified rat ACC, and inactivation pattern by 5$\sp\prime$ AMP-dependent and cAMP-dependent protein kinase. The success in expressing the recombinant ACC will provide an opportunity to reveal the true functional sites as well as phosphorylation site(s) critical for the inactivation of ACC.
Degree
Ph.D.
Advisors
Kim, Purdue University.
Subject Area
Biochemistry|Molecular biology
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