Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.The synthesis of fatty acids from malonyl-CoA de novo requires several enzymatic activities (1). In most bacteria and plants the activities exist as discrete monofunctional polypeptides, whereas in animals they are integrated into a single multifunctional polypeptide (2). Partial sequences for animal fatty acid synthases have been reported (3-6). In this paper we report the complete amino acid sequencer of an animal fatty acid synthase and the ordering of the seven functional domains on the multifunctional subunit. MATERIALS AND METHODSIsolation and Sequencing of Fatty Acid Synthase cDNA Clones. Clones characterized in this study were isolated from Agtll cDNA libraries constructed from poly(A) RNA obtained from the mammary glands of lactating Long-Evans rats (7) or the livers of fasted-refed Long-Evans rats (8). To maximize the probability of including cDNA sequences corresponding to the 5' end of the fatty acid synthase in the liver library, we added a specific primer [nucleotides (nt) 3183-3200 of fatty acid synthase, antisense direction, 50 ng/ml] to the reaction mixture for first-strand synthesis.Asymmetrical adaptors with dephosphorylated EcoRI overhangs (Pharmacia) were used in the ligation reaction, eliminating the need for EcoRI methylase treatment. The liver library yielded, in Escherichia coli Y1090r-, 1.2 X 107 plaque-forming units/,ug of DNA and was amplified 7.6 x 104-fold (68% white plaques) before screening.Probes for library screening were derived from established fatty acid synthase cDNAs and were labeled with [32P]dCTP by random-priming (9). Inserts were subcloned (10) using pUC12 or pUC19 vectors and E. coli DH5a cells (Bethesda Research Laboratories). Nested deletions were constructed using BAL-31 nuclease (11). Double-stranded plasmid DNA was used directly in dideoxynucleotide sequencing reactions with purified synthetic oligonucleotide primers (3, 12).Amino Acid Sequencing. Fatty acid synthase was purified from the livers of Long-Evans rats. The thioesterase domains were removed with trypsin, and the core polypeptides...

cDNA clones coding for the medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II) from rat mammary gland were identified in a bacteriophage lambda gt11 library and their nucleotide sequences were determined. The predicted coding region spans 263 amino acid residues and includes a sequence identical with that of a peptide derived from the enzyme active site. The rat thioesterase II cDNA sequence exhibits homology with that of a thioesterase found in duck uropygial glands.

The existence of histone H1 in the yeast, Saccharomyces cerevisiae, has long been debated. In this report we describe the presence of histone H1 in yeast. YPL127c, a gene encoding a protein with a high degree of similarity to histone H1 from other species was sequenced as part of the contribution of the Montreal Yeast Genome Sequencing Group to chromosome XVI. To reflect this similarity, the gene designation has been changed to HHO1 (Histone H One). The HHO1 gene is highly expressed as poly A+ RNA in yeast. Although deletion of this gene had no detectable effect on cell growth, viability or mating, it significantly altered the expression of β‐galactosidase from a CYC1‐lacZ reporter. Fluorescence observed in cells expressing a histone H1‐GFP protein fusion indicated that histone H1 is localized to the nucleus.©1997 John Wiley & Sons, Ltd.

We have constructed an Aspergillus niger cDNA library with a yeast expression vector. The library DNA complemented a leucine auxotroph of Saccharomyces cerevisiae (strain BWG1-7a) at a frequency of 4x10(-4). Plasmids rescued from the yeast prototrophs also complemented Escherichia coli (strain MC1066) deficient in leucine biosynthesis. Sequence determination of the rescued plasmids revealed two genes for beta-isopropylmalate dehydrogenase, which we called leu2A and leu2B. Genomic-blot analysis suggested that both leu2A and leu2B were derived from single-copy genes. Northern-blot hybridization showed that in nutrient-rich medium a leu2A transcript accumulated during germination and log-phase growth while the leu2B transcript appeared late in the growth phase. In minimal medium, only leu2A expression was greatly stimulated. We examined the codon preference of these two genes. Whereas leu2A shows a bias in codon usage typical of A. niger genes, leu2B does not. These results indicate the presence in A. niger of two highly divergent, differentially regulated, isozymes for beta-isopropylmalate dehydrogenase.