Characterization of a genomic and cDNA clone coding for the thioesterase domain and 3' noncoding region of the chicken liver fatty acid synthase gene

Abstract

The fatty acid synthase (FAS) of animal tissue is a dimer of two identical subunits, each with a Mr of 260,000. The subunit is a single multifunctional protein having seven catalytic activities and a site for binding of the prosthetic groiup 4''-phosphopantetheine. The mRNA coding for the subunit has an estimated size of 10-16 kb, which is about twice the number of nucleotides needed to code for the estimated 2300 amino acids. We have isolated a positive clone, .lambda. CFAS, containing FAS gene sequences by screening a chicken genomic library with a segment of a 3'' untranslated region of goose fatty acid synthase cDNA clone, pGFAS3, as a hybridization probe. The DNA insert in .lambda.CFAS hybridizes with synthetic oligonucleotide probes prepared according to the known amino acid sequence of the thioesterase component of the chicken liver fatty acid synthase [Yang, C.-Y., Huang, W.-Y., Chirala, S., and Wakil, S. J. (1988) Biochemistry (preceding paper in this issue)]. Further characterization of the DNA insert shows that the .lambda.CFAS clone contains about a 4.7-kbp segment from the 3''end of the chicken FASgene that codes for a portion of the thioesterase domain. Complete sequence analyses of this segment including S1 nuclease mapping, showed that the .lambda.CFAS clone contains the entire 3'' untranslated region of the chicken FAS gene and three exons that code for 162 amino acids of the thioesterase domain from the COOH-terminal end of the fatty acid synthase. Using the exon region of the genomic clone, we were able to isolate a cDNA clone that codes for the entire thioesterase domain of chicken liver fatty acid synthase. The cDNA clone is sequenced, and the deduced amino acid sequence is consistent with amino acid sequence studies of this domain, except the deduced sequence extends the reported COOH-terminal end of the thioesterase and synthase of chicken liver by four amino acids -Val-Arg-Glu-Gly.