NORMAL GENOMIC

• Al-Feel W, Chirala SS, Wakil SJ
• Cloning of the yeast FAS3 gene and primary structure of yeast acetyl-CoA carboxylase.
• Proc Natl Acad Sci U S A. 1992; 89: 4534-8
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• We have isolated and determined the nucleotide sequence of the yeast FAS3 gene, which encodes acetyl-CoA carboxylase (EC 6.4.1.2). The sequence has an open reading frame of 6711 bases coding for a protein of 2237 amino acids with a calculated molecular weight of 250,593. The presence of the unique biotin-binding site, Met-Lys-Met, and the known CNBr peptide and COOH-terminal sequences confirmed the nucleotide-derived amino acid sequence. The yeast, chicken, and rat carboxylases have an overall sequence identity of 34%, suggesting that the eukaryotic carboxylase evolved from a single ancestral gene. The amino acid sequences of yeast fatty acid synthase subunits are least homologous with the animal synthase sequences, whereas carboxylase sequences are highly conserved. The sequences of the ATP, HCO3-, and CoA binding sites of the carboxylases are also well conserved (approximately 50% identical). The sequences surrounding the biotin binding site are poorly conserved, suggesting that this sequence may not be critical as long as the biotin is available for carboxylase reactions. On the basis of this sequence identity, we have defined the putative biotin carboxylase and transcarboxylase domains.

• Fisher RP, Lisowsky T, Breen GA, Clayton DA
• A rapid, efficient method for purifying DNA-binding proteins. Denaturation-renaturation chromatography of human and yeast mitochondrial extracts.
• J Biol Chem. 1991; 266: 9153-60
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• We describe a novel method for the purification of DNA-binding proteins. Isolated mitochondria were lysed in boiling sodium dodecyl sulfate-containing buffer, the extracts were chromatographed on hydroxylapatite in the presence of sodium dodecyl sulfate, and DNA-binding activities were identified after adding a large excess of nonionic detergent (Triton X-100) and assaying fractions by a gel retardation procedure. Fractions containing DNA-binding activity were bulk renatured and chromatographed on phosphocellulose in the presence of Triton X-100. When applied to human mitochondria, the technique resulted in the purification to homogeneity of fully functional mitochondrial transcription factor 1 (mtTF1), the major activator of mammalian mitochondrial transcription. Moreover, the yield of mtTF1 purified by this method was at least 25 times higher than that obtained by conventional nondenaturing chromatographies. When yeast mitochondria were subjected to the same protein isolation scheme, a 19-kilodalton putative yeast homologue of mtTF1 was purified to homogeneity. These results suggest that the denaturation-renaturation approach may be a valuable general method for the identification and high yield purification of DNA-binding proteins.